GB2497719A - Inhibitors of lipoprotein receptor degradation - Google Patents

Abstract

Agents which interact with the IDOL (MYLIP / Myosin Regulatory Light Chain Interacting Protein) protein may be used to inhibit degradation of the Low-Density Lipoprotein receptor (LDLR), the Very Low Density Lipoprotein Receptor (VLDLR) and/or the Low Density Lipoprotein Receptor-Related Protein 8 (apoER2), promote lipoprotein uptake, or prevent, treat or ameliorate hypercholesterolaemia and/or cardiovascular disease. The agent may inhibit interaction of IDOL (and in particular its FERM domain â residues 183-344) with LDLR, VLDLR and/or apoER2, inhibit interaction of IDOL with a UBE2D ubiquitin-conjugating enzyme, inhibit binding of iron ions with IDOL, or inhibit the dimerisation of IDOL. The agent which inhibits the interaction of IDOL with a lipoprotein receptor may be, for example, a peptide corresponding to the conserved cytoplasmic tail of LDLR, VLDLR or apoER2.

Description

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LIPID METABOLISM

The present invention relates to lipid metabolism, and in particular to the uptake of lipoproteins via the Low-Density Lipoprotein receptor GDLR, the Ve Low Density Lipoprotein Receptor VLDLR) and/or the Low density lipoprotein receptor-related protein B (apoER2) receptor. The invention provides novel biological targets associated with the degradati)n of these receptors, and pharmaceutical compositions, medicaments and methods of treatment for use in preventing, ameliorating or treating symptoms of hypercholesterolaeniia and cardiovascular disease.

Plasma Low-Density Lipoprotein (LDL) cholesterol levels are strongly linked to cardiovascular disease risk. The TAM. receptor (LDLR) is a cell membrane protein that mediates uptake of LDL cholesterol and is a niaor determinant of plasma lipoprotein levels. Loss of function LDLR mutations in humans reduce LDL clearance, elevate plasma LDI. levels and are associated with accelerated atherosclerosis. The most effective drugs for lowering circulating cholesterol are called HMG-CoA reductase inhibitors or "statins", which cause the liver to produce more LDL receptors, which bring the cholesterol into the liver cells where it is metaholised. However, although statins are often very effective, in 1 (YVo of patients they are associated with undesirable side effects, such asrhahdomyolysis. Thus, despite the widespread use of statin drugs, there still remains an ui-gent need for additional therapeutic strategies to modulate human lipid levels, which avoid these side effects. Understanding molecular mechanisms involved in the control of EDT.

uptake and processing will have important implications for treatment of human cardiovascular disease.

In eukaryotic cells, the degradation of many proteins is carried out by the uhiquitin system. In this pathway, proteins are targeted for degradation through the covalent conjugation of the 76-amino acid polypeptide uhiquitin. Conjugation proceeds via a J0 three-step mechanism involving three enzymes, El, E2 and L3. To initiate the process, a ubiquitin molecule is activated by the ubicuitin-activating enzyme, El, to form a high-energy intermediate with El. The activated ubiquitin molecule is then

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transferred to a ubiquitin-conjugating enzyme, E2, to form an intermediate with the E2. Finally, association of this ubiquitin-charged E2 with an P3 ligase facilitates the conjugation of the ubiquitin molecule to the target protein. Specificity in uhiquitinaüon pathways derives from the ability of individual [3 ligases to recognize a discreet set of target proteins.

There are two major categories of E3 ligases: HRCT domain and RING domain [3 ligases. I1LCT domain E3 ligases mediate the conjugation of uhiquitin by formation of a HECT-uhiquitin intermediate, whereas RING domain E3 ligases facilitate the direct transfer of ubiquinn from the E2 to the substrate. The ubiquitin system is organized into a hierarchical structure: a single El can transfer uhiquitin to several species of P2 enzymes, and each F2 acts in concert with either one or several E3 enzymes. Upon the completion of uhiquitin conjugation, the proteolysis of ubiquitinated proteins can he conducted in either the proteasome or the lysosome.

As discussed above, the IJ)L receptor (LDLR) is a cell membrane protein essential for the uptake of L1)L cholesterol and the regulation of plasma lipoprotein levels.

Loss of function LDLR mutations in humans reduce hepatic LDL clearance, elevate plasma 11)1. levels and accelerate atherosclerosis. The abundance of the IDER is regulated by both transcriptional and post-transcriptional mechanisms in response to cellular cholesterol levels. [he primary transcriptional regulator for I D1 R is the SREBP-2 transcription factor. A reduction in the cholesterol levels in the endoplasmic reticulum (ER) triggers the processing of SREBPs to their mature nuclear forms and consequentl activates the expression of genes important for the synthesis and uptake of cholesterol.

The RING domain E3 uhiquitin ligase, IDOL, has recently been identified as an additional post-transcriptional regulator of the LDLR pathway (Zelcer et al. 2009).

Expression of the TOOl. gene is induced by the sterol-activated transcription factors io LXRa and LXRf3. Increased IDOL expression triggers the ubiquitination of the LDLR, leading to its internalization and degradation, thereby increasing plasma cholesterol levels. Although it is clear that increased expression of the [3 ligase, IDOL, leads to ubiquitination of the LDLR on its cvtoplasmic domain and subsequent degradation, the mechanism by which this is accomplished still remains to be elucidated. Tn particular, IDOL is unusual among F3 ligases in that it appeals to affect the degradation of a ver small number of proteins. Furthermore, although it is postulated that IDOL acts directly on the LDLR itself, this has also never been formally established. Since the expression of the IDOL gene is not regulated by SREBPs, the L)CR-IDOI. pathway represents an independent mechanism for feedback inhibition of the LDLR by cellular cholesterol levels.

Finally, the uhiquitinarion and subsequent degradation of the LDLR is presumed to depend on a cascade of ubiquitin transfer reactions carried out by El, E2, and E3 enzymes. However, although 11)01. has been identified as the E3 ligase, the identity of the specific R2 that is involved in the cascade has remained elusive.

Tt is therefote an aim of embodiments of the present invention to target the mechanisms of activity of IDOL in order to upregulate levels of the LDLR, and hence lo\ver cholesterol concentrations circulating in the plasma. Such improved therapeutics and methods which result in the upregulation of LDLR can he used for treating symptoms of hypercholesterolaemia and cardiovascular disease.

To effectively target the ability of IDE)] to degrade the LDI R, the inventors conducted a series of structural, biophysical and cell-based assays to understand, in molecular detail, the interaction between IDOL and the LDLR. As a result of these studies, the inventors now have a detailed understanding of the mechanism by which T1)0T degrades not only the L1)TR, hut also two closely-related receptors, the Very Low Density Tipoprotein Receptor (VLDLR) and the low density lipoprotein receptor-related protein 8 (apoER2), both of which can also be targeted by TOOL.

Accordingly, the inventors have demonstrated that agents, which are capable of preventing the interaction between specific reons of TOOL, the target receptor (i.e. o LDLR, VLDLR and/or apoER2) and/or the specific E2 enzyme that is involved in ubiquitination, can he effectively used to inhibit receptor degradation, and thereby promote lipoprotein uptake, resulting in i-educed plasma cholesterol levels.

Hence, according to a first aspect of the invention, there is provided an agent capable ot: (a) inhibiting binding or interaction between a sub-domain of the FERtvI domain of IDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ Ti) No:1, or a functional fragment or variant thereof, and: a Low-Density Lipoprotein receptor (LDLR, (ii) a Very Low Density Lipoprotein Receptor (VTflLR and/or (iii) a Low density lipoprotein receptor-related protein 8 (apoER2); b) inhibiting binding or interaction between IDOL and a K/RN WXXKNXXST/VDCF motif present in the LDLR, \/LDLR and/or apoER2; (c inhibiting interaction or binding between IDOL and a member of the ubiquitin-conjugaung enzyme (IJBE2I)) family; (d) inhibiting or preventing binding of iron ions with IDOL; or (e inhibiting or preventing the dimerisation of IDOL, for use in inhibiting LDIR, VLDLR and/or apoER2 degradation and/or promoting lipoprotein uptake.

According to a second aspect, there is provided an agent capable of: (a) inhibiting binding or interaction between a sub-domain of the FERN domain of IDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ TD No:1, or a functional fragment or variant thereof, and: (i) a Low-Density 1 ipoprotein receptor (1 1]] jR), (ii) a Very Low Density Lipoprotein Receptor (VLDLR) and/or (iii) a Low density lipoprotein receptor-related protein 8 (apoER2); (b) inhibiting binding or interaction between IDOL and a K/RNWXXKNXXST/MXr motif present in the L1)LR, VLDLR and/or apoER2; (c inhibiting interaction or binding between IDOL and a member of the ubiquitin-conjugating enzyme (UBL2D) family; d) inhibiting or preventing binding of iron ions with T1)OT; or (e inhibiting or preventing tile dimerisadon of IDOL, for use in die treatment, prevention or amelioration of hvperchoiesteroiaemia or cardiovascular disease.

According to a third aspect, there is provided a method of inhibiting LDLR, \QDLR and/or apoER2 degradation and/or promoting lipoprotein uptake in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable ot (a) inhibiting binding or intel-action between a sub-domain of the FERM domain of TDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ ID No: 1, or a functional fragment or variant thereof, and: (i) a Low-Density Tipoprotein receptor (JA)LR), (ii) a Very Low Density Iipoprotein Receptor (VLDLR) and/or (iii) a Low density lipoprotein receptor-related protein 8 (apoER2); b) inhibiting binding or interaction between IDOL and a K/RNWXXIKNXXST/MXF motif present in the LDTA{, VLDLR and/or apoER2; or (c inhibiting interaction or binding between IDOL and a member of the uhiquitin-conjugating enzyme (UBE2D) family; (d) inhibiting or preventing binding of iron ions with IDOL; or (e inhibiting or preventing the dimerisation of IDOL, to inhibit TflLR, VLDLR or apoER2 degradation and/or promote lipoprotein uptake in the subject.

According to a fourth aspect, there is provided a method of treating, preventing or ameliorating hypercholesterolaemia or cardiovascular disease in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable of: (a) inhibiting binding or interaction between a sub-domain of the PERM domain of IDOL, the sub-domain being represented by amino acid residnes 183-344 of SEQ ID No:I, or a functional fragment or variant thereof, and: (i) a Low-Density Lipopt-otein receptor (LDLR, (ii) a Very Low Density Lipoprotein Receptor (VLDLR and/or (iii) a Low density ilpoprotein receptor-related protein 8 (apoER2); b) inhibiting binding or intel-action between IDOL and a K/RN WXXKNXXSI/DCL motif present in the LDLR, \/TflTR and/or apoER2; or (c inhibiting interaction or binding between IDOL and a member of the uhiquitin-conjugating enzyme (IJBE2D) family; (d) inhibiting or preventing binding of iron ions with IDOL; or (e) inhibiting (-)r preventing the dimerisation of IDOl to treat, prevent or ameliorate hypercholesterolaemia or cardiovascular disease in the subject.

Advantageously, blocking the action of IDOL by inhibiting or preventing binding or interaction between TDOT. and either one of the above-mentioned receptors or a UBE2D protein presents a novel strategy for increasing levels of the LDLR, \/LDLR and/ot-apoFR2 receptors, and hence lowering circulating cholesterol in the subject being treated. Such an approach should provide an alternative and/or complementary J0 therapy to treatment with statins.

The inventors have determined the structure of IDOL, and its interactions with the 11)1. receptor and L2 ligase, using NMR and/or X-ray crystallographic approaches, and a range of biochemical and cell-based interaction assays. Together these experiments have provided a detailed understanding of the structure and function of IDOL, and its mechanism of interaction with both the LDLR, VLDLR and/or apoRR2 receptors, as well as with the uhiquitin-conjugating en2ynie UBFID family of proteins (i.e. U B K2D 1, UBE2D2, UBE2D3 and U B K2D4, as the E2 enzymes that collaborate with IDOL in receptor uhiquitination. 1 lie inventors have also successfully obtained the crystal structure of the IDOL RING domain-IJI3L2D complex. Based on the information provided by this structure, the inventors have surprisingly demonstrated that disruption of the interaction interface between IDOL and UBF2I) prevents LDLR, \/L1)LR and/or apoER2 receptors from being degraded by TOOL. These results provide a much better understanding of the molecular mechanism underlying the sterol-depenclent regulation of protein levels of the L1)LR, VLDLR and/or apoER2 receptors.

Surprisingly, the data also suggest that the closely-related family members IA)LR, VLDLR and apoLR2 are the only proteins targeted by IDOL. The basis for this remarkable specificity was previously unknown, and so the inventors have now defined the molecular basis for IDOL target recognition, and provide clear evidence that specific targeting of membrane receptors by binding of the IDOL FERM domain underlies an evolutionarily conserved, post-translational mechanism for the regulation of lipoprotein uptake.

The agent may he used for the treatment, amelloration or prevention of a cardiovascular disease selected from a group consisting of disorders of the heart and vascular system, such as congestive heart failure; myocardial infarction; ischemic diseases of the heart; ischemic cardiomyopathy; myocardial disease; all kinds of atrial and ventricular arrhythniias; hypertensive vascular diseases; peripheral vascular diseases; atherosclerotic coronary artery disease; heart failure; hvpertrophic cardioniyopathy; restrictive cardiomyopathv; congestive heart failure; cardiogenic shock; and hypertension.

As illustrated in Figure 13\, IDOL is 445 amino acids in length, and is divided into an N-terminal FERN! domain (344 amino acids in length), a 22 amino acid linker region aM a C-terminal RING domain 7 amino acids in length). The amino acid sequence of human IDOL is provided herein as SEQ ID No:l, as follows: )

MLCYVTRPDAVLMEVEVEAKAHGEEJCLNQVCRRLGT TEVDYFGLQFTGSKGESLWLNLRH

RTSQQMDGLAPYRLKLRVKFFVEPHLI LQEQTRHIFFLHIKEALLAGHLLCSPEQAVELS

ALLAQTKFGDYNQNTAECYNYEELCAKELSSA:LNSIVAKHKELEGTSQASAEYQVLQIVS

AMENYGIEWHSVRDSEGQKLLIGVGPEGIS ICKDDFSPINRIAYPVVQMATQSGKNVYLT

VTKESGNS IVLLFKMI STRAASGLYRAIIEIHAFYRCDTVTSAVMMQYSRDLKGHLASLF

LNENINLGKKYVFDIKRTSKEVYDHARRALYNAGVVDLVSRNNQSPSHSPLKSSESSMNC

S S CEGL SC QQT RVL QEKLRKLKEAMLCMVCCEEE INS TFCPCGHTVCCE S OAAQLQS CPV

CRSRVEHVQHVYLPTHTSLLNLTVI

SEQ ID No: 1 The FERNI domain itself is sub-divided into three discrete sub-domains, denoted herein as "El", "F2" and "P3". Sub-domain Fl is defined by amino acid residues 1-of SEQ Ti) No:l, sub-domain P2 is defined by amino acid residues 86-182 of SEQ ID No:i, and sub-domain F3 is defined by amino acid residues 183-344 of SEQ TI) No: I Accordingly, the agent is preferably capable of inhibiting binding or interaction between the receptor (i.e. LDLR, VLDLR and/or apoER2 and the P3 sub-domain of the FERM domain of IDOL, wherein the F3 sub-domain is defined by amino acid residues 183-344 of SEQ TI) No:l, or a functional fragment or variant thereof.

As shown in Figure i3\, sub-domain P3 of the FER±\1 domain is itself subdivided in three sub-domains denoted herein as "F3a", "P3h", and "F3c". Sub-domain F3a is defined by amino acid residues 183-214 of SEQ TI) No:1, sub-domain F3h is defined by amino acid residues 21 5-272 of SEQ ID No:1, and sub-domain P3c is defined by amino acid residues 273-344 of SEQ TI] No: I. Accordingly, the agent may be capable of inhibiting binding or interaction between the receptor @.e. LDLR, VLDLR and/or apoER2) and an F3a, F3h or F3c sub-domain of the FERM domain of TDOL, wherein sub-domain F3a is defined by amino acid residues 183-214 of SEQ Ti) No:l, or a functional fragment or variant thereof, sub-domain F3b is 31 defined by amino acid residues 215-272 of SEQ U) No:1, or a functional fragment or variant thereof, and sub-domain F3c is defined by amino acid residues 272-344 of SEQ Il] No: I, or a functional fragment or variant thereof.

In one preferred embodiment, however, the agent may he capable of inhibiting binding or interaction between an F3b sub-domain of the FERN domain of TDOT.

and LDLR, VLDLR and/or apoER2, die F3b sub-domain being represented by amino acid residues 21 5-272 of SEQ ID No:1, or a functional fragment or variant thereof. Preferred amino acid residues in the F3h sub-domain of TOOL, which may be targeted by the agent to prevent binding or interaction with the receptor, may be selected from a group of residues consisting of residues: 232; 265; and 269 of SEQ II) No:l.

In another preferred embodiment, the agent may be capable of inhibiting binding or interaction between an F3c sub-domain of the FERM domain of 11)01 and LDLR, V]DLR and/or apoER2, the F3c sub-domain being represented by amino acid residues 273-344 of SEQ ID No:1, or a functional fragment or variant thereof.

Preferred amino acid residues in the 173c sub-domain of IDOT, which may he targeted by the agent to prevent binding or interaction with the receptor, may he selected from a group of residues consisting of residues: 285, 323, 327 and 342 of SEQ ID No:1.

The agent may he capable of inhibiting binding or interaction between a sub-domain of the FRRMI domain of IDOI. and amino acid residues conserved between (i) the LDLR, (ii) the \LI)TR, and (iii) the apoER2 receptors. Figure 151' shows a sequence alignment of the tail portion of human LDLR, VLII)LR and apoER2 receptors. The amino acid sequence of the tati portion of human LDLR @.e. residues 810-860) is provided herein as SEQ ID No:2, as fbllows: iCwKNwRLKNiNsINFDNpvYQKIIEDEvHIcHNQDGYsYpsRQLwsLEDDvAO SEQ ID No:2 As can be seen, the alignment illustrates die presence of a conserved motif of 811IK/RN\Y,/\)(JJ4X\5I/y1XFS23 between amino acids 811 and 823 of SEQ ID No:2, to which IDOL hinds, where K is lysine, R is arginine, N is asparagine, W is trvptophan, S is serine, MI is methionine, I is isoleucine, F is phenvialanine and X may -10 -he any amino acid. For example, the X amino acid at residue 14 may he arginine (R, glutamine (Q) or lysine (K), and the X amino acid at residue 15 may he lysine (L), histidine (H) or arginine (R). The X amino acid at residue 18 may be isoleucine (I), methionine or threonine cf), die X amino acid at residue 19 may he asparagine (N) or lysine (K), and the X amino acid at residue 22 may he asparane (N).

The agent may he capable of inhibiting binding or interaction between IDC)I. and a Sl/MXF motif present in the LDLR, VLDLR and/or apoER2, as represented in SEQ TI) No:2. The motif may he represented by amino acid residues 820 and 823 of SEQ ID No:2, i.e. 820S1/1V1XF823, to which TOOL hinds. Inspection of the alignment shown in Figure iSA highlights other conserved regions between LDLR, VLDLR and/or apoTiR2 to which iDOl. may bind. Therefore, in another embodiment, the agent may he also capable of inhibiting binding or interaction between IDOl Sand a 810WKNW813 motif represented in SEQ TD No:2, the motif being present in the is TJ)LR, VTA)LR and/or apoER2.

In addition, the agent may be capable of inhibiting binding or interaction between IDOL and a 816J{J817 motif represented in SEQ TD No:2, the motif being present in the TA)LR, \TLDLR and/or apoER2.

Also, the agent may be capable of inhibiting binding or interaction between IDOL and a 824DNPVY828 motif represented in SEQ II) No:2, the motif being present in the LDLR, \/TJ)TR and/or apoER2.

Tn a preferred embodiment, the agent may he capable of inhibiting binding or interaction between 11)01. and one or more of the binding motifs described herein, which are present in TA)LR, VEDER and/or apoRR2, and preferably all of these motifs.

Jo Accordingly, in embodiments of the invention, the agent preferably prevents binding or interaction between the FFR\1 domain of IDOL and one or each motif represented in SEQ ID No:2. In a preferred embodiment, the agent may prevent -Il -binding or interaction between one or each motif and the F3 sub-domain of the IThRM domain of IDOL, wherein the F3 sub-domain is defined by amino acid residues 183-344 of SEQ TD No:1, or a functional fragment or variant thereof. In another preferred embodiment, the agent may prevent binding or interaction between one or each motif and the 131i sub-domain of the FEB14 domain of IDOL, wherein the F3h sub-domain is defined by amino acid residues 215-272 of SEQ ID No:i, or a functional fragment or variant thereof. In another preferred embodiment, the agent may prevent binding or interaction between one or each motif and the F3c sub-domain of the FERli domain of IDOL, wherein the F3h sub-domain is defined by amino acid residues 273-344 of SEQ ID No:1, or a functional fragment or variant thereof.

The agent maybe capable of inhibiting interaction or binding between die RING domain of IDOL and a member of the uhiquitin-conjugating en2yme (UBE2D) family, which member may he UIW2I) 1, UBE2D2, UBE2D3 or UBE2I)4. The agent may be capable of inhibiting interaction of binding between the member of the ubiquitin-conjugating en7yme (UBE2D) family and one or more amino acid residues of the RING domain of IDOL selected from the group of residues consisting of: G1u383; \a1389; Leu4lS and Pro419 of SEQ ID No:l.

In one embodiment, an amino acid sequence of human uhiquitin-conjugating enzyme (UBE2D1) is provided herein as SEQ ID No:3, as follows:

MALKRIQKELSDLQRDPPAHCSAGPVGDDLFHWQAIIMGPPDSAYQGGVFFLTVHFPTDY

PFKPPKTAFTTKTYHPNTNSHGSIOLDILRSQWSPALTVSKVLLSIOSLLCDPNPDDPLV

PDTAQIYKSDKEKYNRHAREWTQKYM1 SEQ ID No:3 J0 The agent may be capahie of inhibiting interaction of binding between IDOL and one or more amino acid residues of the member of the uhiquitin-conjugating enzyme (UBE2D) family selected from the group of residues consisting of: LysS; Argl5; Pro6l; Phe62 and Pro9S of SEQ ID No:3.

-12 -Preferably, the agent is capable of inhibiting interaction of binding one or more amino acid residues of the RING domain of IDOL selected from the group of residues consisting of: G1u383; Va1389; Leu4l 5 and Pro4l 9 of SEQ ID No:1 and one or more amino acid residues of the member of the ubiquitin-conjugaung enzyme (UBE2D) fantily selected from the group of residues consisting of: Lys8; ArglS; Proól; Phe62 and Pro9S of SEQ Ti) No:3.

As described in Example 7, and as shown in Figure 7, the inventors were very surprised to observe that IDOL is an iron-binding protein. They have determined at least three cysteine residues at the N-terminal end of the RING domain of TOOT., which together define a pocket into which iron ions can bind. Titus, the inventors believe that blocking binding of iron ions with IDOL can prevent ubiquitinanon of the LDLR, \T.i)LR and/or apoER2 receptor, and thus he used to reduce plasma cholesterol levels. Hence, the agent may he capable of inhibiting or preventing binding of iron ions with IDOL, preferably the RING domain thereof, and most preferably at the N-terminal of the RING domain. The agent may be capable of inhibiting or preventing binding of iron ions with amino acid residue C360, C363 and/or C383 of SEQ ID No:1.

As described in Examples 4 and 6, and as illustrated in Figures 6E and 6G. the inventors have denionstrated for the first time that IDOL dimer formation scents to be important for its biolog*al function, because the dimer-defective mutant V431R/L433R was unable to promote LDLR degradation and was resistant to auto-catalyzed degradation. They therefore believe that any agent, which can prevent or inhibit IDOL dimerisation, would he very useful for treating the diseases described herein.

As described in Example 14 and Figure 18, the inventors have also surprisingly shown that the cell membrane is involved in IDOL-dependent degradation of the LDLR, \TDT.R and/or apoER2 receptors. Thus, the inventors believe that target recognition by IDOL involves a tripartite interaction between (i) the FERl'1 domain of IDOL, cii) the lipoprotein receptor tail, and (iii) phospholipids present in the cell -13 -membrane. Accordingly, the agent may also be capable of inhibiting or preventing binding of membrane phospholipids with iDOL, preferably the IFERM domain thereof.

Membrane-facing amino acid residues in the FER1v[ domain of IDOL which have been shown to he involved in the interaction with membrane phospholipids, and which may also be targeted by the agent, to prevent binding or interaction with membrane phospholipids, may he selected from the group of residues including 73; 75; 193; 199; 259; 137; and 146 of SEQ 11) No:l.

It wjll be appreciated that once the skilled person has knowledge of the target amino acid residues present @) in 11)01. (either the FERM or RiNG domain), (ii) in the member of the uhiquitin-conugating enzyme (UBF2D) family.e. UBE2I)1, UBE2D2, TJBE2D3 or UBE2D4) and/or ciii) in the receptor @.e. LDLR, \LDLR and/or apoTLR2 receptor), then it will be a straightforward task to design a suitable agent, which is capable of inhibiting binding or interaction between IDOL (IFERM or ifiNG domain) and receptor; or IDOL FLRM or RTNG domain) and the member of the uhiquitin-conjugating enzyme (UBE2D) family @.e. UBE2D1, LBE2D2, IJBR2D3 or UBE2D4 and/or TOOT. FFRM or RING domain) and membrane phospholipids.

As described in the examples, the residues conserved between LDLR, VLI)LR and apoER2 are important for IDOL recognition. Tt has been determined that the lF3b and E3c sub-domains of the FERM domain of IDOL are especially important for interaction with the tail ends of these receptors at the 811K/RNWXXKNXXST/MXF823 motif, as well as other conserved motifs.

Furthermore, the inventors have shown that specific residues in the FL]IUv[ domain are required for an interaction with membrane phospholipids, and that certain residues in the RING domain are required for binding to the UBE2D1-4 protein, and $0 for binding iron ions. Disruption of any of these interactions using the agent would inactivate the IDOL-receptor degradation pathway via uhiquitination. -14-

By way of example, the agent may comprise a competitive polypeptide, or a derivative or analogue thereof, or a peptide-like molecule or a small molecule. For example, the agent may be an antibody or a fragment thereof.

The term "derivative or analogue thereof" can mean a polpeptide within which amino acids residues are replaced by residues whether natural amino acids, non-natural amino acids or amino acid mimics) with similar side chains or peptide backbone properties. Additionally, either one or both terminals of such peptides may be protected by N-and C-terminal protecting groups, for example groups with similar properties to acetyl or amide groups. Tt will be appreciated that the amino acid sequenced may be varied, truncated or modified once the final polypeptide is formed or during the development of the peptide.

Design of such peptide inhibitors, based on the sequence of the natural protein partners has been successfully used previously. In the case of BCL6, peptides based on the BCOR protein bind BCT6 and blocks SMRT from interacting at the same site and in doing so blocks BCL6-mediated transcriptional repression and kills lymphoma cells (Ghetu et al 2008). Likewise, the design of a synthetic, cell-permeable, stahilised peptide that targets the protein-protein interface in the NOTCH transactivation complex has been successfully used in leukaemic cells in culture. In a similar way, a wildtype peptide corresponding to the LDLR tail sequence, SEQ ID No:2, would compete for binding to the 11)01. FERM and prevent degradation of the LDLR Receptor. Modifications/optimisation of the peptide seciuence could he made to increase the affinity so that it is tighter than the wildtvpe vhich is naturally a relatively weak and short-lived interaction.

The inclusion of a ubiquitination motif, as in the LDLR tail, in the synthetic peptide would serve a secondary function; as well as binding the FERJ'vI domain it could he possible for it to he uhic1uitinated reducing the available pool of active R2 for uhiquitinating end ogenous LD LR -15 -It will he appreciated that such inhibitory peptides, pepude mimics or small molecules will exploit the inventor's knowledge of the LDLR FLR\1 interaction and he based upon the sequences that have been identified as being important to that interaction.

The agent would bind tighth and specifically to the FERM domain preventing interacuon and hence degradation of the EDt receptor.

In the sections below, the IDOL-LDLR interaction is used as an example as to how an agent may he developed, though it will be appreciated that similar methods may he used to develop agents that are capable of inhibiting any of the other interactions (i.e. the interaction between the ELRM domain and membrane phospholipids, the interaction between the RING domain and UBL2DI-4 protein, and the ability of the RING domain to bind iron ions) described herein.

The IDOL-LDLR recognition sequence can be used as the basis for screens aimed at identifying small molecules that specifically disrupt IDOL-L1)LR interaction, e.g. by targeting this region of LDLR. Accordingly, in certain embodiments, screening systems are contemplated that screen for the ability of test agents to bind the F3bc sub-domains of the FLRM domain of IDOL and/or to bind/interact with the region of LDLR that interacts with IDOL (e.g.. the SI/MXIF motif) and/or that inhibit the interaction of IDOL and LDLR. Methods of screening for agents that bind the F3bc sub-domains of the FERM domain of IDOL or that bind to the IA)LR region (e.g. the SI/vDCF motif) that interacts with IDOL are readily available to die skilled technician (Colas 2008).

For example, in one embodiment, the F3bc sub-domains of the IDOL FERM domain and/or the IDIR domains are imniohiliied and probed with test agents.

Detection of the test agent (e.g., via a label attached to the test agent) indicates that the agent binds to the target moiety and is a good candidate modulator of IDOL/LDLR interaction. In another embodiment, the association of LDLR and Jo IDOI. or a FLRM domain of IDOL in the presence of one or more test agents is assayed. This can he accomplished using, for example, a fluorescence resonance energy transfer system (FRET) comprising a donor fluorophore on one moieP (e.g., -16-LDLR and an acceptor fluorophore on the IDOL molecule. The donor and acceptor quench each other when brought into proximity by the interaction of LDLR and IDOL. When association is reduced or prevented bya test agent, the FRET signal decreases indicating that the test agent inhibits interaction of LDLR and IDOL.

These assays are illustrative and not limiting. Using the teaching provided herein, numerous binding and/or L1)TR/TI)OT. interaction assays will he available to the skilled technician.

In certain embodiments, cells, tissues, and/or animals are provided that are transfected with an TOOL-encoding construct so they overexpress IDOL. Tn other embodiments, cells, tissues, and/or animals in which IDOL is "knocked out" are provided. It will be appreciated that one or both of these constructs may he used in screens for suitable agents of the invention for inhibiting an\ of the IDOL interactions.

For example, in certain embodiments, test agent(s) (e.g., small molecules) may he screened for their effect on the TOOL pathway based on the T1)OT-/-cells, tissues or animals. WT and TI)OT-/-are screened for response to candidate small molecules.

The effect of IDOL-specific small molecules will be lost in the IDOL-/-cells. These screening methods can he used, for example, in conjunction with the cell-based reporter screens described herein. In certain embodiments, knockout IDOT animals may he used in screens for suitable agents.

The assays of this invention have immediate utility in screening for agents that inhibit IDOL activity in a cell, tissue or organism. The assays of this invention can he optimized for use in particular contexts, depending, for example, on the source and/or nature of the biological sample and/or the particular test agents, and/or the analytic facilities available. Thus, for example, optimization can involve determining optimal conditions for binding assays, optimum sample processing conditions (e.g. preferred PCR conditions), hybridization ct-)nditions that maximi7e signal to noise, protocols that improve throughput, etc. In addition, assay formats can be selected and/or optimized according to the availability of equipment and/or reagents. Thus, -17 -for example, where commercial antibodies or ELISA kits are available it may be desired to assay protein concentrabon. Conversely, where it is desired to screen for modulators that alter transcription of IDOl gene, nucleic acid based assays are preferred. )

It will he appreciated that agents according to die invention may be used in a medicament which may be used in a monotherapy (i.e. use of only an agent which inhibits binding between IDOL, the target receptor and/or the UBE2D), for treating, ameliorating, or preventing hypercholesterolaemia or cardiovascular disease.

/0 Alternatively, modulators according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing hypercholesterolaemia or cardiovascular disease. For example, agents of die invention may be used in combination with known agents for treating hypercholesterolaemia or cardiovascular disease, such as statins.

The agents according to the invention maybe combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to he used. Thus, for example, the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar soluUon, transdermal patch, liposome suspension or any other suitable form that maybe administered to a person or animal in need of treatment. It will he appreciated that the vehicle of medicaments according to the invention should he one which is well-tolerated by the subject to whom it is given.

Medicaments comprising agents according to the invention may he used in a number of ways. For instance, oral administration may he required, in which case the agents may be contained \vithmn a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents of the invention may he administered by inhalation (e.g. intranasally). Compositions may also he formulated for topical use. For instance, cl-cams or ointments may he applied to the skin, for example, adjacent the heart.

-18 -Agents according to the invention may also be incorporated within a siow-or delayed-release device. Such devices may, for example, he inserted on or under die skin, and the medicament may be released over weeks or even months. The device may he located at least adjacent the treatment site, e.g. the heart. Sucl devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).

In a preferred embodiment, agents and compositions according to the invention may he administered to a subject by injection into the blood stream or directly into a site requiring treatment. For example, the medicament may be injected at least adjacent heart. Injections may he intravenous (holus or infusion) or subcutaneous bolus or infusion), or intradermal bolus or infusion).

It will be appreciated that the amount of the agent that is required is determined by its biological activity and bioavailabilit, which in turn depends on the mode of administration, the physiochemical properties of the modulator and whether it is being used as a monotherapy or in a combined therapy. The frequency of administration will also he influenced by the half-life of the agent within the subject being treated. Optimal dosages to he adnunistered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the hvpercholesterolaemia or cardiovascular disease, Addition factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.

Generally, a daily dose of between 0.Olgg/kg of body xveight and 500mg/kg of body \veight of the agent according to the invention may he used for ti-eating, ameliorating, or preventing hypercholesterolaemia or cardiovascular disease, depending upon Xi which agent is used. More preferably, the daily dose is between 0.01mg/kg of body weight and 400mg/kg of body weight, more preferably between 0.1mg/kg and -19 - 200mg/kg body weight, and most preferably between approximately 1mg/kg and 100mg/kg body weight.

The agent flay be administered before, during or after onset of hypercholesterolaemia or cardiovascular disease. Daily doses may be given as a single administration (e.g. a single daily injection). Alternatively, the agent may require administration twice or more times during a day. As an example, agents may be administered as two (or more depending upon the severity of the hypercholesterolaemia or cardiovascular disease being treated) daily doses of between 25mg and 7000 mg (i.e. assuming a body weight of 70 kgi.A patient receiving treatment may take a first dose upon waking and then a second dose in the evening on a two dose regime) or at 3-or 4-hourly intervals thereafter. Alternatively, a slow release device may he used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses.

Known procedures, such as those conventionally employed by the pharmaceutical industry (e.g. in viva experimentation, clinical trials, etc.), may be used to form specific formulations comprising the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration). The inventors believe that they are the first to suggest an anti-cardiovascular disease composition or an anti-hypercholesteroaernia composiflon, based on the use of the agents of the invention.

I lence, in a fifth aspect of the invention, there is provided an anti-hypercholesteroaemia or anti-cardiovascular disease composition comprising a therapeutically effective amount of an agent capable ofi (a) inhihiting binding or interaction between a sub-domain of the FERNI domain of IDOl, the sub-domain being represented by amino acid residues 183-344 of SEQ ID No: 1, or a functional fragment or variant thereof, and: (i) a Low-Density lipoprotein receptor (IflLR), -20 - (ii) a Very Low Density Lipoprotein Receptor (VLDLR and/or (iii) a Low density lipoprotein receptor-related protein 8 (apoLR2); i) inhibiting binding or interaction between IDOL and a K/RNVVXXIKNXXST/MXF mouf present in the TJ)TR, VLDLR and/or apohR2; (c inhibiting interaction or binding between IDOL and a member of the uhiquitin-conjugating enzyme UBE2D) family; /0 (d) inhibiting or preventing binding of iron ions with IDOL; or (e) inhibiting or preventing the dimerisation of IDOL, and optionally a pharmaceutically acceptable vehicle.

F he term "anti-cholesterolaemia composition" can means a pharmaceutical fbrmulation used in the therapeutic amelioration, prevention or treatment of hypercholesterolaemia in a subject. The term "anti-cardiovascular disease composition" can mean a pharmaceutical formulation used in the therapeutic amelioration, prevention or treatment of a cardiovascular disorder in a subject, such as myocardial infarction.

The invention also provides in a sixth aspect, a process for making the composition according to the flfth aspect, the process comprising contacting a therapeutically effective amount of an agent capable of: (a) inhibiting binding or interaction between a sub-domain of the FERN domain of IDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ ID No:1, or a functional fragment or variant thereof, and: (i) a Low-Density 1 ipoprotein receptor (1 iDI jR), (ii) a Very Low Density Lipoprotein Receptor (VLDLR) and/or (iii) a Low density lipoprotein receptor-related protein 8 (apoER2); -21 - (b) inhibiting binding or interaction between IDOL and a K/RNWXXIKNXXST/MXF motif present in the L1)LR, VLDLR and/or apoER2; (c inhibiting interaction or binding between IDOL and a member of the ubiquitin-conjugating enzyme (UBL2D) family; d) inhibiting or preventing binding of iron ions with T1)OT; or (e inhibiting or preventing tile dimerisadon of IDOL, with a pharmaceutically acceptable vehicle.

The agent may he a polypeptide, peptide or peptide-like molecule, for example an antibody.

A "subject" may he a vertebrate, mammal, or domestic animal. hence, compositions and ntedicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.

Most preferably, however, the subject is a human being.

A "therapeutically effective amount" of agent is any amount which, when administered to a subject, is the amount of drug that is needed to treat the cardiovascular disorder or hypercholestero]aemia disorder, or produce the desired effect.

For example, the therapeutically effective amount of modulator used may he from about 0.01 mg to about BOO mg, and preferably from about 0.01 mg to about 500 mg. Tt is preferred that the amount of modulator is an amount from about 0.1 mg to about 250 mg, and most preferably from about 0.1 mg to about 20 mg.

A "pharmaceutically acceptable vehicle" as referred to herein, is any known compound or combination of known compounds that are known to those skilled in tile art to be useful in formulating pharmaceutical compositions.

-22 -In one embodiment, the pharmaceutically acceptable vehicle may he a solid, and the composition may he in the form of a powder or tablet. A solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, soluhilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, s\veeteners, preservatives, dyes, coatings, or tablet-disintegrating agents. The vehicle may also he an encapsulating material. 1n powders, the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention. In tablets, the active agent (e.g. the peptide or antibody) may he mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 999/b of the active agents. Suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins. In another embodiment, the pharmaceutical vehicle may be a gel and the composition may he in the form of a cream or the like.

However, the pharmaceutical vehicle maybe a liquid, and the pharmaceutical composition is in the form of a solution. Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, eixirs and pressurized compositions. The active agent according to the invention may he dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats. The liquid vehicle can contain other suitable pharmaceutical additives such as soluhilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators. Suitable examples of liquid vehicles for oral and parenteral administration include water partia]ly containing additives as above, e.g. cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil). For parenteral administration, the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate. Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration. The liquid -23 -vehicle for pressurized compositions can he a halogenated hydrocarbon or other pharmaceutically acceptable propellant.

Liquid pharmaceutical compositions, which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection. The agent may he prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.

The agents and compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorhitan monoleate, polysorhate 80 (oleate esters of sorhitol and its anhydrides copolymerized with ethylene oxide) and the like. The agents used according to the invention can also be administered orally either in liquid or solid composition form. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, eixirs, and suspensions. Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.

It will be appreciated that the invention extends to any nucleic acid or peptide or variant, derivative or analogue thereof, which comptises substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including functional variants or functional fragments thereof. The terms "substantially the amino acid/nucleotide/peptide sequence", "functional variant" and "functional fragment", can he a sequence that has at least 40°/h sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No:l (i.e. 11)01.

J0 protein) or its encoding nucleotide, or 40°/h identity with the polypeptide identified as SEQ H) No:2 (i.e. the tail portion of human LDLR) or its encoding nucleotide, and so on.

-24 -Amino acid/polynucleotide/polvpeptide sequences with a sequence identity which is greater than 50°/h, more preferably greater than 65°/h, 70°/h, 75°/b, and still more preferably greater than 809/a sequence identity to any of the sequences referred to are also envisaged. Preferably, the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferab'y at least 901⁄4, 921⁄4, 95°/h, 97°/h, 98?/, and most preferably at least 991⁄4 identity with any of the sequences referred to herein.

The skilled technician will appreciate how to calculate the percentage identity between two amino acid/polvnucleotide/polpeptide sequences. In order to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences, an alignment of the two sequences must flrst he prepared, followed by calculation of the sequence identity value. The percentage identity for two sequences may take different values depending on:-(i) the method used to align the sequences, for example, ClustalW, I3TAST, FASTA, Smith-Waterman (implemented in different programs), or structural alignment from 3D comparison; and (ii) the parameters used by the alignment method, for example, local vs global alignment, the pair-score matrix used (e.g. BLOSUM62, PAM25O, Gonnet etc.), and gap-penalty, e.g. functional form and constants.

Having made the alignment, there are many different ways of calculating percentage identity between the two sequences. For example, one may divide the number of identities by: @) the length of shortest sequence; (ii) the length of alignment; @ii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.

1 lence, it will be appreciated that the accurate alignment of protein or DNA sequences is a complex process. The popular multiple alignment program ClustaIW (Thompson et a!., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson cliii., -25 - 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred way for generating multiple alignments of proteins or DNA in accordance with the n\rendn.Suitable parameters for ClustalW may be as follows: For DNA alignments: Gap Open Penalty = 15.0, Gap Extension Penalt = 6.66, and Matrix = Identit. For protein alignments: Gap Open Penalty = 10.0, Gap Extension Penalty = 0.2, and Matrix = Gonnet. For DNA and Protein alignments: ENI)GAP = -1, and GAPDTST = 4. Those skilled in the art will be aware that it may be necessary to vary these and other parameters for optimal sequence alignment.

Preferably, calculation of percentage identities between two amino acid/polynucleotide/polypeptide sequences may then be calculated front such an alignment as /T)*l00, where N is the number of positions at which the sequences share an identical residue, and F is the total number of positions compared including gaps hut excluding overhangs. I lence, a most preferred method for calculating percentage identity between two sequences comprises 0) preparing a sequence alignment using the ClustalW program using a suitable set of parameters, for example, as set out above; and (ii) inserting the values of N and T into the following forntula:-Sequence Identity = (N/T)*100.

Alternative methods for identifying similar sequences will be known to those skilled in the art. For example, a substantially similar nucleodde sequence will he encoded by a sequence which hybridizes to any sequences referred to herein or their complements under stringent conditions. By stringent conditions, we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°C followed by at least one wash in 0.2x SSC/0.1°/b SDS at approximately 20-63°C. Alternatively, a substantially similar polypeptide may differ by at least I, hut less than 5, 10, 20, 50 or 100 amino acids from the sequences shown in SEQ ID No: 1-3.

J0 Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein could he varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.

-26 -Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change. Other suitable variants are those having homologous nucleotide sequences hut comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophvsical properties to the amino acid it substitutes, to Produce a conservative change. For example small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large non-polar, hydrophobic antino acids include phenylalanine, tryptophan and tyrosine. The polar neutral amino acids include scrine, threonine, cysteine, asparagine and glutamine. The positively charged (basic) amino acids include lysine, arginine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore he appreciated which amino acids may he replaced with an amino acid having similar biophysical properties, and the skilled technician will known the nucleotide sequences encoding these amino acids.

All of the features described herein (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined with any of the above aspects in any combinati n, except combinations where at least some of such features and/or steps are mutually exclusive.

For a better understanding of the invention, and to show how embodiments of the same may be carried into effect, reference will now he made, by way of example, to the accompanying diagrammatic drawings, in which:-Figure 1 shows that embodiments of ubiquitin-conjugating enzyme family members (UBE2Ds) of the invention are specific partners for IDOL autoubiquitination. A) Immunoblot of a panel of 19 human Ll3R2 enzymes that were expressed in B. co/i as fusion proteins with 6xHis tags on their N-tei-mini. B) Autoubiquiunauon of TI)OT o induced by LTBE2D family proteins in an in vitro autoubiquitination assay.

Immunoprecipitated TAP-IDOL was incubated with UBEI, HA-ubiquitin and the indicated UBE2 proteins. IDOl. uhiquitination was detected by immunoblotting for -27 -I IA-tagged ubiquitin associated with IDOL. C) Autoubiquitination of IDOL induced by UBE21) redluires an active RING domain. Immunoprecipitated TAP-iDOL and TAP-IDOL C387A were incubated with UI3FA, HA-ubiquitin and the indicated L1BE2 proteins. IDOL uhiquitinaflon was detected by immunohiotting for hA-tagged uhiquitin associated with IDOL. D) Autoubiquitination induced by UBE2D is specific to IDOL. Immunoprecipitated TAP-EGFP, TAP-T1)OT. and TAP-IDOL C387A were incubated with UBEI, HA-ubiquitin and TJBE2D proteins. IDOL uhic1uitination was detected by immunoblotting for HA-tagged uhk1uitin associated with IDOL. The amounts of TAP-tagged proteins in the in vit,v autoubiquitinanon assay were determined by immunoblotting using anti-FLAG antibody. E) UIW2I) family proteins have similar capacity for inducing IDOL autouhiquitination.

UBEI) 1-4 protein levels used in the IDOl. autouhiquitination assay were normalized by Coomassie staining of SDS-PAGL gels. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with liBEl, l-[A-ubiquitin and the indicated IJBE2I) family proteins. IDOL ubiquitinaflon was detected by immunoblotting for HA-tagged ubiquitin associated with IDOL. F) 11)01. autoubiquitination is not exclusively dependent on the LysI 1, Lys4S or the 1xs63 linkage of ubiquitin.

Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with IJBFA, UBE2D2 and HA-tagged ubiquitin with indicated lysine mutations. IDOL uhiquitination was detected by immunoblotting; Figure 2 shows that UBE2D family proteins are the F2 enzymes for LDLR ubiquitination. A) Ubiquitination of the LDLR by UBE2I) and IDOL in an in vitro uhiquitination assay. Membrane preparation of 293 cells expressing LDLR-GFP or GFP alone were incubated with UBFA, UBE2D2, tandem affinity purified IDOL or IDOL C387A, and HA-uhiquitin. LDLR was then immunoprecipitated with an anti-GFP antibody. The ubiquitination of LDLR was detected by intmunoblotting for HA-tagged ubiquitin associated with I DLR. B) Expression of dominant negative UBE2D2 inhibits the degradation of LDLR. Immunohiot analysis of protein levels in o 293 cells transfected with \VT or dominant negative UBE2D or UBL2I l, in addition to L[)IJ( and IDOL. C). lmmunohlot analysis of IDOL and LDLR expression in response to inhibition of proteasomal or lysosomal degradation pathways. 293 cells -28 -were transfected for 24 h with expression vectors for WT or C387A mutant IDOL and L1)LR. Cells were treated with MG-I 32 (25 uM) or hafilomycin (I3IJL, 101) nM) as indicated 4 h prior to harvest; Figure 3 shows NI'vW chemical shift mapping of the IDOL RING domain with UBE2I) 1. A FI,'5N J-ISQC spectra of 150 iM 15N-lahelled TI)OT. RING domain in the absence (blue) and presence of UBE2IJ I (green) at an equimolar ratio. B) Weighted shift map obtained from the [1 1,1 5N-HSQC spectra of IDOL RING domain with the addition of U13F21) 1. C) Ribbon representation of the crystal structure of the 11)01. RING domain. D) Surface representation of the H)OL RING domain with the most significant shifts (>0.O5ppm) shown in orange and smaller perturbations (>0.O2Sppm) shown in yellow; Figure 4 shows the crystal structure of the IDOL RING domain dimer complexed with UI3R2D1. A) Cartoon representation with the IDOL RING shown in purple and the TJBE2I)i in grey. B) The RING domain dimer interface. C) Close up of the RING domain dimer interface. D) The interface of the IDOL RING domain with UBE2D1. B) Close up of the IDOL RING-UBL2DI complex interface; Figure 5 shows specificity determinants for the II)OI-RING:UBL2D interaction.

A) Klectrostatic potential of the interface between the IDOl. RING domain (1eft and UBE2D I (right). Note that the main interaction surface on the E2 is highly basic and the complementary surface on the E3 is acidic. ArgI 5 in UBE2D1 provides a basic pocket to accommodate G1u383 froni IDOL. In non-complementary E2s such as UBB2F3 (insert) the residue in this position is neutral or acidic and disfavors interaction. B) Some R2s that are non-complementary with IDOL have a basic residue in position is, hut an important serine at the interface (Ser94 in UBB2I)i) is substituted with other amino acids, such as lysine in LJBL2I 3. The serine makes an important backbone contact that could not he formed by the alternative residues. C) Jo Alignment of key regions of various L2 Jigases. Only members of UBL2D family have both a basic residue and a serine to support appropriate interactions with the 11)01. RING; -29 -Figure 6 shows disruption of the 11)OL-UBL2D interaction blocks TJDI.R degradation. A) Mutations in the IDOL RJNG domain-UBE2D interaction interface inhibit LDLR degradation. Immunoblot analysis of protein levels in 293 cells fransfected with LDLR and \VT or mutant IDOL expression vectors. B) UBE2D is unable to catalyze the autoubiquitination of mutant IDOL with a disrupted IDOL MNG domain-U B K2D interaction. Immunoprecipitated TAP-IDOL, TAP-IDC)1 C387A and TAP-IDOL V389R were incubated with UBEI, UBL2D2 and HA-ubiquitin. T1)OL ubiquitination was detected by Western blot for HA-tagged ubiquitin associated \Vith IDOL. C) LTBE2I)2 mutated at the interface with T1)0I. is unable to catalyze IDOL autoubiquitination. Immunoprecipitated TAP-IDOL and TAP-IDOl. C387A were incubated with liBEl, WT or P61 A/FG2R UBE2D2 and IIA-ubiquitin. T1)OI. uhiquitination was detected by Western blot for I IA-tagged uhiquitin associated with IDOL. D) Mutation of UBE2D2 residues predicted to be involved in 1DOL specificity determination reduces the ability of UIW2I)2 to support IDOL autoubiquitination. Immunoprecipitated TAP-IDOL and TAP-IDOL C387A were incubated with UBEI, WI or RiSE, S94T<, or KBE UBE2D2 and HA-uhiquitin. IDOL ubiquitination was detected by Western blot for HA-tagged uhiquitin associated with IDOL. E) IDOL forms a dimer in vivo. 293 cells were transfected with vectors expressing TAP-IDOL and VS-IDOL. VS-IDOI. in the cell lysate was inimunoprecipitated with an anti-VS antibody. The TAP-IDOL that co- immunoprecipitated with VS-IDOL was detected by immunoblotting using an anti-FLAG antibody. 17) Structure-based mutations predicted to disrupt dimer formation prevent the co-immunoprecipitated of TAP-IDOL and VS-IDOL. 293 cells were transfected with indicated combination of expression vectors. VS-IDOI. and VS-mutant IDOL in the cell lysate were immunoprecipitated with an anti-VS antibody.

The co-imniunoprecipitated TAP-IDOL was detected by immunoblotting using anti- 171 AG. G) A dimer-defective IDO1. mutant is unable to induce 1 D1 M degradation.

Immunoblot analysis of protein levels in 293 cells transfected with LDLR and WT or mutant IDOL expression vectors. Ii) IDOL harboring a mutation in the IDOL RING domain-UBE2D interaction interface functions as a dominant negative in LDLR degradation assays. Immunoblot analysis of protein levels in 293 cells -30 -transfected with increasing amount of VS-tagged \VF or mutant IDOL, in addition to constant levels of LDLR and TAP-T1)OL; Figure 7 shows that IDOL is an iron-binding protein. A) Schematic diagram of the domain structure of IDOL. B) Alignment of IDOL sequences, hs horno sapiens, cf canisftimila.th, mm sims muscu/us, xl xenopus Ia.evis, gggallusgallus. The three conserved Cys residues N-terminal to the UNG domain are highlighted in yellow. The Cys zinc ligands are highlighted in orange and the Ills zinc ligand is highlighted in blue. C) Results from Atomic Absorption Spectroscopy. D) Photogi-aph of protein samples of IDOL constructs eluted from Glutathione Sepharose by cleavage with TEN protease. E) Coomassie-stained SDS-PAGE gel of IDOL constructs eluted from (Jlutathione Sepharose by cleavage with TEV protease. F) and U) Disruption of the putative iron-binding cysteine residues alters T1)OT. stability and LDLR degradation.

Immunoblot analysis of protein levels in 293 cells transfected with LDLR and WT or mutant TDOL expression vectors. H) Effect of IDOL intel-action mutants on the ability of IDOL to inhibit IDE uptake. 293 cells were transfected with LDLR and \Vf or mutant T1)OT. expression vectors and then incubated for 4h with DiT-labeled LDL. Cells were washed and cellular LDL associated quantified by fluorescence.

Results are presented as % WT T1)OT. inhibitoty activity in L[)T. uptake assays. The inhibitory activity of WI TI)OL was defined as 100% and that of the inactive RTNG mutant (C387A) was defined as 0. *1) <0.05; Figure 8 shows degradation of LDLR by TAP-tagged IDOL. (A) Imntunohlot analysis of protein levels in 293 cells transfected with vectors encoding LDLR and indicated TAP-tagged proteins. (B) Autouhiquitination of 11)01. induced by UBE2D requires an active RTNG domain. Purified TAP-TI)OT and TAP-IDOL C387' were incubated with UBFA, IJBE2D2 and JIA-ubiquitin. IJbiquitinated IDOL was detected by Western blot for IDOL associated with ILk-tagged ubiquitin; o Figure 9 is a schematic diagram of the in vittv autoubiquidnation assay of IDOL; Figure lOis a schematic diagram of the in ui/tv uhiquitination assay of Ll)LR; -31 -Figure 11 shows the structure of the IDOL RING-LBE21)1 complex. A) IDOL RING complexed with UBE2D1 colored by B-factors. This shows that the interface ofDBL2D1 with the IDOL RING domain is well ordered and that the N-terminal helix of the IDOL RING domain is not as well ordered. B) Superposition of IDOL RTNG complexed with LBF21)1 (cyan) with the structure of the IDOL RING alone (magenta). This shows that there are some small conformational changes in the IDOL RING domain upon binding UBE2DI. F he first ioop and the first zinc are less well-ordered in the structure of the IDOL RING domain alone. (C) The two Pro434 residues within the RING dimer move 4.3 A towards each other, tightening the homodirneric interface, in the complex with UBE2D1 compared to the RING domain alone; Figure 12 shows that the IDOL FLRM domain directly interacts with Epoprotein receptor cytoplasmic tail. (A) Immunoblot analysis of l1LK293T whole cell lysates following overnight co-transfection with LDLR and FLAG-IDOL WI, RING mutant (C389A) or mutants deleted after the indicated residue. (B) Fluorescence polarization assay of the binding of BODIPY-labeled YLDLR 820-842 ot control peptide to Hist-tagged IDOL constructs 1-273. The binding curves were analyzed using GraphPad Prism. Dissociation constants were I SuM and >25OuM; Figure 13 shows that the FERAl 3b subdomain of 11)01. is critical for LDLR recognition. (A) Domain structure of IDOl and potential configurations of FFRM F3 domain; residue numbers indicate domain boundaries (top); Computer generated 31) modeling of IDOL denoting surface residues available for target interaction in either conformation based on Talln interaction with integrin (midd1e. (B) Imntunoblot of HF1K293T whole cell lysates following overnight co-transfection with 1 D1 M and IDOL W'I or F3h or F3c subdontain domain mutants as indicated.

The ratio of IDOL:LDLR expression plasmid was varied while keeping the total amount of DNA transfected constant; -32 -Figure 14 shows the key residues in the E3b subdornain required for IDOL regulation of the LDLR pathway. (A) Immunoblot analysis of HEK293T cell surface protein isolated by biotinvlation following overnight co-transfection with L1)TR and IDOL \VT or F3b subdomain mutant constructs as indicated. (B) IDOL-dependent inhibition of DiI-LDL uptake following overnight co-transfection of HEK293T cells with TA)TR and IDOL \VT, ring mutant (C387A or F3h subdomain mutants as indlicated. Cells were maintained in 10% LPDS overnight prior to incubation with DII-LDL 4Jtg/mT for I hour at 37°C. Data represented as % inhibition and expressed as mean ± SEvl, performed in triplicate. The inhibitory activity of WT U) IDOL was assigned a value of i00% and the inactive RING MUT was defined as 0% activity. p<O.OS, p<O.Ol vs VT IDOL. (C) Immunoblot analysis of whole cell lysates from T1)OL-/-mouse embryonic fibroblasts (MFTs) stably expressing retroviral IDOL \VT or F3h subdomain mutant constructs as indicated. (Tells were cultured in 10% lipoprotein deficient serum (LPI)S) overnight. D) IDOL-dependent inhibition of DiI-LDL uptake in T1)OT. -/-MLEs stably expressing retroviral IDOL WT or F3h subdomain mutant constructs as indicated. Cells were maintained in 10% T131)S overnight prior to incubation with 1)il-LDL 4g/mT) for 1 hour at 37°C.

Data represented as % iniuhition and expressed as mean ± SENI, performed in triplicate. The inhibitory activity of WT IDOL was assigned a value of 100% and the 2U inactive RiNG MET was defined as 0% activity. <ttp<0.00i vs \VC IDOl. (h) Analysis of ubiquitinated LDLR in I 1LK293T cell lysates following o\ernight co-transfection with GFP-LDLR, HA-uhiquitin and IDOL expression plasmids as indicated. Proteins xvere immunoprecipitated overnight with anti-GEP antibody followed by immunoblotting for HA-uhiquitin. (F) Immunoblot analysis of HF1K293T whole cell lysates following overnight co-transfection with TA)LR and TAP-TDOL constructs with mutations in the F3b subdomain as indicated; Figure 15 shows that IDOL recognizes a conserved "SI/l'vlxF" motif in its lipoprotein receptor targets. (A) Sequence alignment of the cytoplasmic tail of the 36 three IDOL targets with key residues for IDOL recognition and ubiquitination (Ub) highlighted; homologous residues are also shaded in grey. (B) 3-dimensional model of -ii -IDOL/LDLR interaction highlighting critical residues in the LDLR tail; IDOL is colored by element with red: acidic residue -blue: basic residue and yellow: sulphur (C). 3-dimensional model of IDOL/LDLR interaction highlighting critical residues in the IDOL F3b domain; green residues indicate those predicted to be most important; orange residues indicate those predicted to be somewhat important; IDOL is colored by element with red: acidic residue -blue: basic residue and yellow: sulphur. D) Immunoblot analysis of H EK293T whole cell lysates following overnight co-transfection with IDOL and LDLR \VT or cytoplasmic tail mutants as indicated; * denotes mutations that affect IDOL-mediated degradation of the TA)LR. (L) IDOL- 16 dependent inhibition of 1)iI-LDL uptake in HETK293T cells transfected with 11)01.

and LDLR \V'f or cytoplasmic domain mutants overnight as indicated prior to Dii-LDL (4 g/mL) uptake for 1 hour at 37°C. Data represented as % inhibition and expressed as mean ± SEM, performed in triplicate. The inhibitory activity of WT IDOL on \VT LDLR was assigned a value of lOO9/o. p<O.OOl vs WT LDLR. (F) is Analysis uhiquitinated LDLR in l-1LK293T cell lysates following overnight co-transfection with HA-uhiquitin, FLAG-IDE)]. and GIFP-LDLR WE or cytoplasmic domain mutants. Proteins were immunoprecipitated overnight with anti-GF] antibody or IgG as indicated, followed by immunoblotting for I 1A-uhiquitin. (G) Immunoblot analysis of I11LK293T whole cell lysates following overnight co-transfection with 11)01. and V5-VLDLR WT or cytoplasrnic tail mutants as indicated. (H) Immunoblot analysis of HEK293T whole cell lysates following overnight co-ansfection with 11)01. and V5-ApoER2 WT or cytoplasmic tail mutants as indicated; Figure 16 shows that IDOL-1A)LR structure function relationships are conserved in insect orthologs. (A) Sequence alignment of the F3h domain of IDOL and its insect homolog, DNR1, demonstrating conservation of key residues across species; homologous residues are shaded in gray. B) Immunoblot analysis of HLIC93T whole cell lysates following overnight co-transfection with L[)LR and WT, ring mutant (C387A) or F3 mutant FLAG-DNR1 constructs as indicated. (C) IDOL-dependent inhibition of Dii-L1)L uptake in 11LK293T cells transfected with LDLR and DNR1 constructs as indicated. Cells were maintained in lO% LPDS overnight -34 -prior to incubation with DiI-TA)1. (4 g/mL for 1 hour at 37°C. Data represented as % inhibition and expressed as mean ± SRNI, performed in triplicate. The inhibitory activity of WT DNRI on WI LDLR was assigned a value of 100%. *p<0.OS, vs WI DNRI. D) Sequence alignment of the cytoplasmic tails of the IDTR and its insect homolog, lipophorin LpR, with key residues fbr T1)OL recognition and uhiquitination highlighted; homologous residues are shaded in gray.

(F) Tmmunohh)t of HEK293T whole cell lysates 48 h following co-transfection with IDOL and FLAG-LpR WI or cytoplasmic tail mutants as indicated; lo Figure 17 shows that the FERAl 3c subdornain of 11)01. is required for autouhiquitination. (A) Sequence alignment of the F3c sub domain of IDOL/DNR1 demonstrating conservation of key lysine residues across species; homologous residues are shaded in grey. (B) Immunoblot analysis of HEK293T whole cell lysates following overrnght co-transfection with LDLR and TAP-IDOL \VT, ring mutant (C387A or F3c single lysine mutants as indicated. (C) Irnmunohk)t analysis of HFJK293T whole cell lysates following overnight co-transfection with LDI R and TAP-IDOL W],ring mutant (C387A or F3c multiple lysine mutants as indicated.

D) Analysis of IDOL autouhiquitination in HEK293T cell lysates following overnight transfection with TAP-IDOL WT, ring mutant C387A) or F3c multiple lysine mutants and HA-uhiquitin. Cells were incubated with MG-132 for 5 hours prior to harvest. TAP-IDOL was immunoprecipitated overnight with septactin beads followed by immunoblotting for HA-uhiquitin. (F) Immunoblot analysis of I 111K293T whole cell lysates following co-transfection with LDLR and FLAG-DNRI WT, ring mutant (C579A) or F3c lysine mutants as indicated; Figure 18 shows that the membrane is required for T1)OL-dependent LDLR degradation. (A) Assay of TDOL association with the L1)T.R in membrane fractions.

J-1EK293T cells were transfected overnight with vector or LDLR and TAP-IDOL WT or F3c suhdomain mutants as indicated. Membrane fractions were obtained following permeabili2ation with digitonin (0.05%). Immunohlot analysis of whole cell lysate inputs is shown at top. Analysis of proteins in membrane pellets is shown at bottom. (B) 3-dimensional modeling of the electrostatic surface of the 11)01.

-35 -FLRM domain denoting key residues of the F3 domain involved in membrane interaction; the basic surfaces are shown in blue and the red indicates acidic surfaces.

(C) The IDOL FFMM domain interacts with negatively charged membrane phospholipids. IDOL 1-273 (0.15mg / nil) was mixed with vesicles (0.5 mg / ml) consisting of phosphatidylcholine (PC), phosphatidylserine (PS), or a 4:1 ratio of PC:PS and then cennifuged. Talin 1 96-400 which binds tightly to negatively charged lipids (Thtltis et aI. 2009 EMBOJ) was used as a positive control and Talin 1655-1822 which does not bind lipids was used as a negative control. D) Mutations of the basic surface on 173 abolish the interaction of the IDOL FERtvI domain with these vesicles.

10) Immunoblot analysis of HEK293T whole cell lysates following overnight co-transfection with LDLR and TAP-IDOL \V'F, ring mutant C387A) or IDOL mutants in which basic residues on membrane-facing surfaces of the 171 (R73/K75), F2 Kl37/ 146) or F3 (RI 93/K199/R259) subdomains were mutated to glutamic acid; Figure 19 A) shows a sequence alignment of human IDOL with the FERM domain of Talin. denotes lOO% homology; : denotes conserved substitutions; . denotes semi-conserved substitutions. (B) shows a sequence alignment of human IDOL with the FERM domain of Macsin, and Figure 19C shows a sequence alignment of human IDOL with the FERM domain Radixin; Figure 20 (A) shows imniunoblot analysis of HFK293T whole cell lysates following overnight co-transfection with IDOL and IA)IR \VT or cvtoplasmic tail mutant constructs as indicated; C839A indicates deletion of residues downstream of cysteine 839. (B) shows immunoblot analysis of IILTK293T whole cell lysates following co-transfection with IDOL and WT LDLR or truncated LDLR constructs; A indicates deletion of cytoplasmic tail downstream of denoted residue. Figure 20C shows IDOL-dependent inhibition of DiI-LDL uptake in LDLR-/-MEFs transfected with WT and mutant LDLR constructs as indicated. Cells were maintained in lO?/o LPDS overnight prior to incubation with DiT-LDL (4 tg/mL) for 1 hour at 37°C. Data represented as /i inhibition and expressed as mean ± SEM, performed in triplicate.

-36 -The degree inhibition of WE LDLR uptake by WT IDOL was assigned a value of 100%. p<O.OS vs WT LDLR; Figure 21 shows immunoblot analysis of HLK293T whole cell lysates following overnight co-transfecuon with LDLR and TAP-IDOL WT or F3c multiple lysine mutants. Cells were incubated with the proteasomal inhibitor MG-132 (25 tM for 5 hours prior to harvest as indicated; Figure 22 shows initnunoblot of 11EK293T whole cell lysate following overnight /0 transfection with TAP-IDOL and WT LDLR, V5-LGFP or \T5EGFPLDLR (C-terminal domain) chimera expression plasmids as indicated; and Figure 23 is a schematic dra\ving showing the postulated interactions between IDOL, E2 ligase, LI)LR and the phospholipid membrane. /5

Materials and Methods Plastijicis and consinicis pSA2-N-TAP plasmid that contains the 3xFLAG-Sep tag and the pcl)NA-V5-Dest plasmid were kindily provided by Dr. tLnric1ue Saez (Scripps). pDONR22I and pET300N-Dest plasniids were purchased from Invitrogen. The DNA sequence of the human Idol gene was amplified from a pcDNA-\/5::hTdol construct as previously reported (Zelcer et al. 2009), and was then subcloned into pSA2-N-TAP plasmid.

The IDOL mutations for the pcl)NA-V5::hldol and the pSA2-N-TAP::hldol constructs were introduced by site-directed mutagenesis. The human E2 genes were cloned from HF1K293 cell eDNA and were then sequentially suhcloned into pI)0NR221 and pET300N-Dest using the Gateway technology (Invitrogen. In addition, the human Dhe2d2 and IJhe2h genes in the pDONR22I::hUhe2d2 and pDONR22I::hlJhe2h constructs were subcloned into pcl)NA-V5-Dest plasmid using the Gateway technology, and the LBE2D2 CBSA and the UBL2J 1 C87A XI mutations for the pcDNA-V5::hLhe2d2 and the pcDNA-V5::hUhe2h constructs, respectively, were introduced by site-directed mutagenesis.

--

An//bottles Rabbit anti-hLDLR antibody was purchased from Cayman Chemicals. Rabbit anti- actin and mouse anti-FLAG M2 antibodies were purchased from Sigma. Mouse anti-VS antibody, I IRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were purchased from Invitrogen. Rabbit anti-VS antibody was purchased from Abcam.

Rabbit anti-CUP antibody was purchase from Clontech. Mouse anti-HA antibody was purchased from Covance. All commercially available antibodies were used according to the manufacturers' instructions.

CIi culture and trans,/ection (1) HEK293 cells wet e maintained in D-MEM nvitrogen) supplemented with 109/b fetal bovine serum (FBS; Omega), 2 mM L-giutamine (Invitrogen), 50 U/mi penicillin nvitrogen), and 50 j.ig/ml streptomycin (Invitrogen). Cells were grown in a humidified incubator at 37 °C and 5°/b CO2 atmosphere. HEK293 cells were transfected using FuGliNti 6 reagents (Roche) according to the manufacturer's instructions. Clonal stable cell lines expressing TDOT. were established by serial dilution selection with 2 tg/ml puromycin (Clontech).

(2) TI)OL-/-and LDLR-/-mouse embryonic fibroblasts (MEFs) were immortalized by stable expression of the SV4O Large T antigen retrovirus and subsequent selection with hygromycin B. Stable expression of control retrovirus (pliabe) or wild type or mutant IDOL or TJ)LR constructs was performed as previously described (Zelcer, Science) and selected with puromycin. Cells were maintained in DMEM supplemented with 10% FBS and MILM non-essential amino adds (Gihco) unless otherwise specified.

limminoblo//ing Proteins were resolved on 4°/h-129/o gradient SDS-PAGE (Invitrogen) using standard protocols. The protein was electrophoretically transferred to nitrocellulose Jo membranes (Amersham Biosciences) and blocked with millc solution (150 miM NaCI, mM Tris, 59/b niilk, 0.2% Tween, pF-I 7.5) to quench nonspecific protein binding.

The blocked membranes were probed sequentialiy with priman-and secondary -38 -antibodies diluted in the milk solution, and the bands were visualized with the ECL kit (Arnersham Biosciences).

IDOL autoubiquitincition assay To prepare the B. co/i lysates containing human TJBL2 proteins, the BL21 DL3) strain (N cxv England Biolabs) of fl. co/i containing various pET300N::hube2 constructs were cultured in LB broth (Sigma) at 37 degree overnight. F lie bacteria cultures were then diluted 1:10 in LB broth and cultured at 37 degree for another 1 to 2 h until 0D600 reached approximately 0.8, at which point a final concentration of 1 mIVI IPTG was added to induce the expression of the 1J13E2 proteins. 2 h after the addition of IP'f C, bacteria were collected in eppendorf tubes, washed with PBS and then sonicated using a thin-tip sonicater (Misonix). Crude iysate was cleared by centrifugation at 12,000 g for 10 mm and the supernatant was collected for the in vitro autouhiquitination assays.

3xFLAG-Strep tagged human IDOL, DOT. C387A, TDOL V389R and LGIFP were expressed in HFX293 cells. Cells were lysed in RTPA buffer (Boston BioProducts, Inc.) supplemented with tile Complete protease inhibitor cocktail (Roche). Cell lysate was cleared by cennifugation at 12,000 g for 10 mm and the supernatant was then incubated with Streptactin beads (IBA GmbH) at 4 degree for 2 h. The beads were then extensively washed with RIPA buffer before the in vitro autoubiquinnation as says.

For each in vitro autoubiquitination assay, 25 pi of IDOL or EGFP bound Streptactin beads were mixed with 5 jil H. co/i lysate containing UBE2, 50 ng recombinant rabbit liBEl (Calbiochem) and 10 ptg recombinant HA-uhiquitin (Boston Biochem).

Reaction buffer contains 50 mM Tris-ITCI (pH 7.4, 5 mM MgCl2, 2 mM ATP and 25 tN1 1MG132 (Sigma). The reaction mixture was incubated at 37 °C for I h. After the reaction, the Streptacin beads were separated and then extensively washed xvith o RIPA buffer before the proteins on the heads were eluted by heated protein loading buffer (Invitrogen). Uhiquitination status was analyzed by immunobloting using an anti-HA antibody.

-39 -I DI i< abiquitination assay HEK293 cells expressing LDLR-GFP or GEP control were permeabilized and the cytosolic proteins were removed according to a protocol previously published (Song and DeBose-Boyd 2004). IDOL and IDOL C387A stably expressed in f1EK293 cells were purified using a tandem affinity purification protocol (Gloeckner et al. 2009).

For each in vitro ubiquinnation assay, 25 d of pelleted permeabilized cells were mixed with 2 pl purified IDOL, 2 pJ B. coil lysate containing TJBE2, 50 ng recombinant rabbit UBFA and 10 ig recombinant HA-uhiquitin. Reaction buffer contains 50mM Tris-HC1 (pl-l 7.4), 5 mM MgCI2, 2 mM VIP and 25 jiM MG132 (Sigma). The reaction mixture was incubated at 37 degree for 1 h. After the reaction, the permeabilized cells were separated and lysed in RIPA buffer supplemented with the Complete protease inhibitor cocktail. The cell lysate was then cleared by centrifugaflon at 12,000 g for 10 mi LDLR in the lysate was immunoprecipitated with a rabbit anti-GFP antibody and Protein G beads (Santa Cru7), and the ubic1uitination status of LDLR was analyzed by immunoblotting using an anti-HA antibody.

NMR Spectroscopy For the NvW experiments 5N 13C his-tagged T1)0I. 369-445 was purified on Ni-NTA (Qiagen) and after TEV-cleavage of the tag purified further on a Resource-Q column (GE Healthcare). The protein was transferred into 20 mM sodium phosphate, 150 mM NaC1 and 0.25mM TCFA using a P1)10 (GE Hea1thcare and concentrated to 0.6mM immediately prior to collection of NMR spectra. NMR experiments for the resonance assignment of IDOL 369-445 were carried out with 0.6 mv1 protein in 20 mM sodium phosphate, pH 6.5, 100 mv1 NaC1, 10% v/v 2J 120. Nl'vffl spectra of all the proteins were obtained at 298 K using Bruker AVANCR DItX 600 or A\TANCV DRX 800 spectrometers both equipped with CryoProbes. Proton chemical shifts were referenced to external 2,2-dirnethyl-2-Jo silapentane-5-sulfonic acid, and 5N and 13C chemical shifts were referenced indirectly using recomniended gyromagnetic ratios Vishart etal. 1995). Spectra were processed with TopSpin (Bruker Corp.) and analyzed using Analysis (Vranken et al. -40 - 2005). Three-dimensional HNCO, 1 IN(CA)CO, HNCA, F1NCO)CA, I 1NCACB, and HN(CO)CACB experiments were used for the sequential assignment of the backbone NH, N, CO, CA, and CB resonances.

(7iysta11iatthn and X-ray structure determination For the crystaThsation experiments GST-tagged IDOL 369-445 was purified using glutathione-sepharose resin (GE Healthcare, eluted by THY cleavage and purified further on a Resource-Q (GL Ilealthcare). His-tagged UBE2DI was purified on Ni-NTA (Qiagen) and, after TEY-cleavage of the tag, on a Supei-dex-75 column (GE 1-Iealthcare). TOOT. 369-445 alone was concentrated to 7.5 mg/ml in a buffer containing 50 mM Tris ph 8, 100 mM NaC1 and 0.5 mM TCEP. IDOL RING domain alone was crystallized from DIM sodium acetate pH 7-8, 16-20% MPD in the spacegroup I 1 2 1. For the complex crystals the two proteins were concentrated independently, mixed at equimolar concentrations and crystallized from 0.1 M sodium citrate p'1 5.5, 0.2 M sodium acetate, 10% PEG 4000 in the space group p 1 21 1. Data was collected at the synchrotron at ESRF on TD23-1 to 3.0 A for the ifiNG domain alone and at Diamond on 104 (to 2.1 A) for the complex. The data was processed using MOSFL.M (Leslie 2006) and both structures were solved by molecular replacement using Phaser (McCoy et al. 2007). The model for the RING domain alone was taken from the clAP UNG structure (3LB5) (Mace et al. 2008).

The complex was solved by using the 3A IDOL 369-445 domain structure and the UBE2D2 structure from 3EB6 (Mace et aT. 2008). The UBE2D2 complexed with clAP (3EB6) was not a successful search model as the two proteins have moved with respect to each other in the IDOL 369-445 UBE2DI structure. Model building and retinement were performed using Coot, RFLEMAC and Phenix (CCP4 1 994; Adams et al. 2010; Emsley et al. 2010). The crystallographic statistics are shown in Table 1.

tomic absoiplion.rpecirosco,hy For the atomic absorption spectroscopy C-ST tagged TOOL 358-445 and C-ST tagged o IDOL 369-445 were purified as described above. Zinc and iron standards were used.

The zinc concentration for the brown TOOL 358-445 was 0.3 mM and the iron -41 -concentration was 0.06 mM. The zinc concentration for the clear IDOL 369-445 \\Tas 0.47 mM and the iron concentraaon was 0.012 mM.

_4ccession nuuihers Coordinates and structure factors for the IDOL RING domain and the IDOL RING domain-UBE2DI complex crystal structures have been deposited in the Protein Data Bank D codes 2YHN and 2Y1-lO respectively), and the 1H, I5N, & 13C NMR chemical shifts for the IDOL RING domain have been deposited in the BioMagResl3ank database (accession code 17550).

Eptviion (7onstiwcts and Tranjèciions Human ApoER2, drosophila melanogaster DNRI and LpR (Open Biosystems) and human \7LDLR (Hong, JBC) were cloned into the gateway plasmid, pDONR221 nvitrogen). Constructs were sub-cloned into tagged destination vectors using gateway technology (Invitrogen). The FLAG destination plasmid was a kind gift of Dr James Wohlschlegel (UCLA) and the VS destination plasmid was a kind gift of Dr Tom Vallim (UCLA). pSA2-N-TAP plasmid that contains the 3xFLAG-Strep tag was kind gift of Dr Enqricue Saez (Scripps). Truncated LDLR and IDOL constructs were amplified from appropriate wild type constructs using Platinum pfx ([nvitrogen and introduced into pDONR221. The LGFP-LDLR chimera was made using traditional cloning techniques and was made up of an N-terminal EGFP, a 10 amino acid linker, and the cytoplasmic tail ofLDLR (amino acids 811-860). All other constructs were obtained as previously described (Zelcer, Science). Mutations were introduced using the Quickchange site-directed mutagenesis kit (Stratagenc. DNA sequencing was used to verify mutant constructs. Transfections were performed using Fugene (Roche Diagnostics) according to the manufacturers instructions with an LDLR/VLDLR/ApoRR2/LpR:IDOL/DNR1 ratio of 4:1 or 2:1 unless otherwise stated. Cells were harvested approximately 24-48 hours following transfection. \Vhen indicated, the proteasomal inhibitor, MG-132 (25 I1M, was added approximately 5 XI hours Prior tO harvest.

JiivweniohIotti;g, Biolinylalion, Imninnoprecipitation and Fractional/on -42 - 11LK293T cells were washed with PBS then harvested in RIPA buffer (Boston l3ioproducts; Tris-1 10 50mM, pH 7.4, NaCI I 50mM, NP-40 1%, Sodium cleoxycholate 0.5°/h, SDS 0.1°/b) supplemented with protease inhibitors (Roche Diagnostics). Lysates were clarified by centrifugation then quantified using the Bradford assay (Biorad) with BSA as a reference. Proteins were separated on Nupage Bis-Tris gels then transferred to VVI)F (GE Osmonics). Membranes were probed with antibodies against the following, LDLR (Cavman Chemical Company), VS (Invitrogen), FLAG (Sigma, Hi\ (Covance), a-tubulin (Caihiochem), 13-actin (Sigma) and pan-cadherin (Santa Cruz). Appropriate secondary F1RP-con5ugated antibodies /0 were used (Invitrogen, Biorad) and visualized with cheniiluminescence Amersham).

To assess cell surface expression, samples were hiorinylated (thermo Scientific; 250g/mT) for 30 minutes at 4°C then subsequently quenched and washed with PBS prior to harvesting in RTPI\ buffer with protease inhibitors. Equal amounts of clarified lysate were incubated with neutravidin agarose resin (Pierce) overtught with rotation then washed and heated to 95C with 2x sample buffer for 5 minutes. For LDLR-GFP immunoprecipitation, equal amounts of claiThed lysate were incubated with anti-GI1 antibody (Abcam) or IgG control overnight with rotation followed by the addition of protein G beads (Santa Cruz). For TAP-IDOL immunoprecipitation, equal amounts of clarified lysate, which had previously been treated with MG-l32 (25 RNi) for 5 hours prior to harvest, was incubated with streptactin beads CIBA Gnibl I) overnight with rotation. Samples were washed then heated to 70°C with 2x sample buffer for 20 minutes pnor to immunoblotting. Cell membrane fractionation was performed by incubating cells with digitonin (0.05°/o) at 4°C for 1 hour with rotation and subsequent pelleting by centrifugation at 3000g for 1 minute prior to immunohlotting.

DiI-LDL [Piake 11)1. uptake was performed cells following overnight transfection with LDLR and IDOL constrncts (HEK2Y3f cells), overnight treatment with lipoprotein deficient serum (LPDS; IDOL -/-MEEs) or overnight treatment with LPI)S in the absence or presence of GW3965 (1kM; LDLR-/-MEFs). Cells were incubated with DiI-LDL qnvitrogen; 4g/mL) for 1 hour at 37°C then washed with PBS and harvested in -43 -RIPA buffer supplemented with protease inhibitors. Samples were clarified by centrifugadon then moved to a 384-well plate in triplicate and measured on a fyphoon apparatus (Amersham).

Aiouleliig The amino acid sequence of human TOOL (Q8WY64) was compared with the FERM domains of mouse talin-i (P26039) residues 86-405 (delta 139-168), human radixin (P35241) residues 1-295 and human moesin (P26038) residues 1-295.

Sequence alignments were carried out using T-coffee (Notredame et al. 2000).

Structural homology models were generated using PRYRE (Kelley and Steinberg 2009)) using the IDOL sequences 1-276 and 1-344 (A2i5-272). Docking of the L1)LR peptide was achieved using comparison of talin in complex with layillin, PIPFJ-y and integrin-fill) (PDB IDs: 21c00, 2(135 and 3(19W respectively) and DABI in complex with the apoER2 cytoplasmic tail (PDB ID: lNfV). Electrostatic surfaces were calculated using the APBS pvmol plugin (Baker et al. 2001).

Phospliol4bid cosedimentation atcuys Large multilamellar vesicles were prepared essentially as described earlier (Anthis et al 2(09). Briefly, films of dried phospholipids (Sigma) were swollen at 5 mg/mI in 20 mIVI llepes pH 7.4, 0.2 mM EG'FA for 3h at 42°C. The vesicles were then centrifuged (20,000 g for 20 mm at 4°C), and the pellet was resuspended in the same buffer at 5 mg/mi. Protein samples were diluted into 20 mM Tris/ HCI (pH 7.4), 0.1 mM EDTA, 15 mM -mercaptoethanol. After centrifugadon (20,000 g for 20 mm at 4°C) proteins (0.15 mg/mI) were incubated (30mm, 25°C) in the absence or presence of phospholipid vesicles (0.5 mg/mI), 200 tl total volume, followed by centrifugation (25,000 g for 20 mm at 4°C). Pellet and supernatant fractions were analyzed on a 10- 20% gradient gel Lxpedeon) and proteins detected by Coomassie-blue staining.

Fluorescence Polc'n'ation Assays xi VTflLR peptides with an amino-terminal cysteine residue were synthesized by BioMatik. Peptide stock solutions were made in PBS containing imM TCEP and then coupled via the amino-terminal cysteine to the thiol-reactive BOPIDY IMR dye -44 - (Invitrogen) in accordance with the manufacturers instructions. Unreacted dye was removed by gel filtration using a P1)-I 0 c&umn (GE I 1ealthcare and the labeled peptide was concentrated to a final concentration of I mM using a centricon with 3K MWCO. Fluorescence polarization experiments were performed in a black 96 well assay plate (Corning). Multiple titrations were performed using a fixed concentration of TJ)Tr peptide of 51iM with increasing concentrations of TOOT, protein, in a final volume of iOOiil of assay buffer (PBS, 0.01% v/v Triton X-100, 0.1mg/mi BSA).

The plate was mixed by shaking for I ntin, and measurements were taken using a Victor X5 plate reader (Perkin Elmer) at room temperature with an excitation wavelength of 531 nm and an emission wavelength of 595 nm. Experiments were performed in triplicate and data were analyzed using GraphPad Prism (version 4.0, GraphPad Software, Inc.). lKd values were calculated by nonlinear curve fitting using a one site binding (hyperbola) model (Y = Bmax *x/(Kd + X).

Results Example 1 -Tdentification of the E2 for IDOL autoubiquitination Because it was not known whether IDOL could directly ubiquitinate the LDT.R, the inventors established an in vitro IDOL autoubiquitination assay in order to identify I1)OT.-interacting E2 enzymes. They hypothesized that, similar to other E3 ligases, IDOL might employ the same E2 partner for both autoubiquitination and target (1 Dl R) uhiquitination. Autouhiquitination is characteristic of RING-type E3 ligases (Yang et al. 2000), and can be evaluated in vitro by incubating the E3 with its cognate E2 and the other factors required for ubiquitination. To establish the autouhiquitination assay, the inventors first immunoprecipitated IDOL from i-1F1K293 celis stably expressing an 11)01. protein taed with 3xFIAG and Strep on its N-terniinal end (TAP-IDOL). The efficacy of TAP-IDOL at degrading the LDLR was confirmed in cotransfection assays (see Figure BA).

According to the HUGO Gene Nomenclature Committee, 38 E2 genes have been Jo documented in the human genonie (Brufotd et al. 2008). Recent systematic studies have defined a subgroup of these P2 enzymes that preferentially participate in uhiquitination mediated by RING-type E3 ligases Markson etal. 2009; van Wijk et -45 -al. 2009). The inventors therefore screened a representative panel of the 19 E2 proteins belonging ti this categon-. They expressed I us-tagged E2 proteins in B. co/i (see Figure IA), and then combined the crude lysates with immunoprecipitated IDOL, recombinant human LiBEl, recombinant I IA-tagged uhiquitin and the ATP-generating system (see Figure 9). Polvuhiquitinated IDOL was detected in the presence of the four closely related members of the UBE21) family (UBE2DI-4), hut not in the presence of any other E2 protein screened (see Figure 1B). This reaction was dependent on IDOL E3 activity, because UBE2D proteins failed to ubiquitinate an IDOL mutant harboring a cysteine mutation in the RING domain C387A (see Figure 1C and Figure SB). This point mutation disrupts the function of the RTNG domain and abolishes the ahihty of IDOL to degrade itself or the LDLR (Zelcer et al. 2009). Furthermore, the uhiquitination reaction specifically targeted IDOL, because UBE2D proteins failed to induce the polyuhiquitination of a FAP-EGFP protein immunoprecipitated in parallel with IDOL (see Figure ID).

T he inventors then addressed the relative efficacy of the individual members of the UBE2D family in supporting IDOL autoubiquitination. They expressed L'IW2D1, LBE2D2, UBE2D3 and UBE2D4 proteins in the same batch of B. co/i cells, aM used the same amount of each protein in autouhiquitinanon assays. They found that individual members of the UBE2D family members exhibited similar capacity for forming polyuhiquitinated IDOl. (see Figure IL). Polyuhiquitin chains ate usually formed via linkage on Lys4S, Lysli or Lys63 residues of ubiquitin Pickart 2001).

They sought to determine whether the autouhiquitination of IDOL was dependent on the Lys4S or Lys63 linkage. They therefore provided exclusively wild-type uhiquitin, 1<1 1R uhiquitin, 1<48K uhiquitin, or K63R uhiquitin in the IDOL autoubiquitination assays. Interestingly, none of the mutant ubiquitins inhibited the autoubiquitination of TDOL (see Figure IF), suggesting that the IDOL autoubiquitination catalyzed by the UBK2D enzymes (lid not exclusively utilize either the Lys4S, Lys63 or Lys Ii linkage. Xi

Example 2 -UBL2D family proteins are the E2 enzymes Rn I.DLR uhiquitination -46 -In order to test the ability of IDOL to uhiquitinate the LDLR in a cell-free system and to determine whether the U]3E21) family proteins are the E2 enzymes for LDLR ubiquitination, the inventors sought to reconsfltute an in i'll/v system where T1)OL, together with UBE1 and UBE2D enzymes, could mediate the transfer of ubicjuitin to the LDLR. To this end, they expressed an LDLR-GFP fusion or GFP control in F-1EK293 cells and prepared membrane fractions by permeabilizing the plasma membrane and removing cytosolic proteins (Song and DeBose-Boyd 2004). They then mixed the membrane preparation with recombinant UBE1, crude lysate of B. co/i expressing UBE2D2, tandem affinity purified IDOL, recombinant HA-tagged ubiquitin and the ATP-generating system (see Figure 10). After the in vitm ubiquitination reaction, the membrane preparation was disrupted and the LDLR vas immunoprecipitated. Ubiquitination was then assayed by immunoblotting.

Remarkably, the inventors found that polvubiqnitinated LDLR was formed in the presence of LTBE2D2 and IDOL, hut not in the absence of UBE2D2, or in the presence of RING domain mutant TDOT. (C387A; see Figure 2A).

In order to demonstrate that the UBE2D family proteins are the E2 enzymes that catalyze LDLR ubiquitinadon in ivo, the inventors employed a dominant negative version of L1BE2D2 lacking a critical cysteine residue within its catalytic domain (C85A (Gonen et al. 1999), Expression of the dominant negative LTBE2D2 in H FtK293 cells markedly inhibited IDOl -dependent LDLR degradation (see Figure 2B). By contrast, the expression of a dominant negative mutant of an nnrelated E2, UBE2H (CS7A), did not inhibit LDLR degradation. Taken together, these results indicate that the UBE2D family proteins participate in the endogenous IDOL-LDLR ubiquitination cascade.

To provide further insight into the functional effects of IDOL and LDLR ubiquitination in cells, the inventors treated cells expressing \VT or RING MET IDOL and L1)TA( with inhibitors of protein degradation. Transfection of RTNG 3o MUT IDOL expression vector gave rise to markedly increased protein levels compared to \VT IDOL expression vector, consistent with loss of autouhiquitination and degradation (see Figure 2(i). Addition of the proteasomal inhibitor MG-I 32, hut -47 -not the lysosomal inhibitor hafilomycin, stabili2ed \VF IDOL protein levels, consistent with the hypothesis that ubiquitinated IDOL is degraded in the proteasome. By contrast, TDOL-dependent TI)LR degradation was blocked by hafilomycin, but not by MG-132. These results strongly suggest that IDOL-dependent LDLR ubiquitination and IDOL autoubiquitination have distinct functional consequences and lead to distinct degradation pathways.

Example 3 -The IDOL RING domain interacts directly with UBL2D1 To further investigate IDOL-1J13R21) interaction, the inventors employed NvlR spectroscopy. The TOOT. RING domain protein (residues 369-445) was expressed in E. co/i and readily purified. The H,"N HSQC NMR spectrum indicated a stable and well-defined protein fold. However, the line widths suggested a molecular weight higher than would he expected for a 9 kDa protein. The interaction between the IDOL RING domain and UBE2D1 was studied by collecting JI,1N JISQC spectra RING domain in the presence of increasing concentrations of unlabeled UBE2I) I (see Figure 3A. A number of resonances showed progressive changes in chemical shift indicative of a direct interaction. To further analyze this, chemical shift data we used 13_S labeled protein IDOL RING domain (residues 369-445) to complete the backbone assignment. The weighted H, 3N chemical shifts in IDOL RING induced by UBE2DI \vere plotted as a function of residue number (see Figure SB) and this analysis showed that the interaction is specific and involves residues M388, \7389, C390, C391 and C411 of the 11)01. RING domain. with chemical shift perturbations greater than OJJ5ppm 6A ppm (see Figure 3B). Thus, NMR analysis confirmed the direct interaction between IDOL and UBL2D1 suggested by the inventors' in vittv screen.

Example 4-Structure of the IDOL R1[NG-UBE2DI complex In addition to the NIV[R chemical shift titratlons, the inventors determined the crystal structure of the 11)01. RING domain (residues 369-445), both alone (see Figure 3C Jo and D) and in complex with UBE2DI (see Figure 4A-E, Figure 11, and Table I).

-48 -Tahie I -Data collection and refinement statistics TOOT. (369-445) IDOl. (369-445) UBE2D 1 Data collection Spacegroup 1121 P1211 Cell dimensions a, b, c A) 79.26, 22.77 87.84 37.69, 137.87, 63.73 , , y °) 90.00, 116.82, 90.00 90.00, 106.39, 90.00 Resolution 39.19-3.00 (3.16-55.91 -2.1(3(2.21-2.1) 3.00) Ran or Rrnergc 13.8 (34.4) 8.9 (35.5) I / of 12.2 (7.4) 9.0 (3.2) Completeness (°/o) 99.2 (100.0) 99.6 (99.8) Redundancy 3.9 (4.0) 3.3 (3.6) Refinement Resolution (A) 3.0(3 2.1 No. reflections 2,928 53,163 R.,1k / 22.84 (29.7) 18.56 (23.33) No. atoms Protein 880 6766 Water 406 Acetate 36 B-factors Protein and acetate 21.88 31.26 Water 33.30 R.m.s. deviations Bond lengths (A) 0.019 0.008 n&°)0.842L158 It was not immediately apparent from the protein sequence which residues of the IDOL RING domain would chelate zinc, due to the vesence of multiple cysteine and histidine residues in addition to those normally observed in RING domains.

However, the structure reveals that the IDOL RING domain employs seven cysteines and one histidine to coordinate two zinc ions in a conventional pattern (Barlow et aI. 1 994, with the protein structure interleaved around the zinc ions (see Figure 3C) .An amino-terminal helix precedes the 11)01. RING domain. The whole /0 structure forms a homodimer in the crystal lattice. This is mediated, in part, by the amino-terminal helix, but mainly through a tight interface between the RING domains that have a highly complimentary shape, such that the honed surface area of the dimenization interface is 1862 A2 (see Figure 4B). The 11)01. RING dimerization interface is one of the most ordered parts of the IDOl. UBE2DI complex structure -49 - (see Figure hA), with multiple non-polar amino-acids (Val431, Leu433, 11e395, Pro434) at the interface (see Figure 4(i). In addition to the hydrophobic interactions, there arc backbone interactions between fyr432 and G1y403'. Tyr432 is also involved in a stacldng interaction with the histidine ring of His 404' (see Figure 4C).

The side chain of G1n429 makes a hydrogen bond to the backbone of Pro4Oi'.

Three leucine residues (Leu374, Leu378, Leu381) in the helix preceding the iinc-binding domain also appear to contribute to dimerization, but this part of the structure is less well ordered (see Figure 1 lA.

In the structure of the complex, the IDOL-Li W21) 1 interface is well ordered and, like the dimerization interface, is predominantly hydrophobic (see Figures 4D and E).

The core of the interface consists of amino acids Va1389, Leu4l 5 and Pro419 of IDOL packing on Phe62, Pro6l and Pro95 on LBF2DI. UBE2D1 side chains Ixs4, ArgS and Ser94 make hydrogen bonds to the side chain of G1u392 and the backbone ofMet388 and Pro419 of IDOL respectively. Arg422 of 11)01. makes hydrogen bonds to the backbone of Gln92. The interface observed in the crystal structure is consistent with the NMR chemical shift mapping. Interestingly, the RING:RING dimer interface is somewhat rearranged in the complex with UBL2D1 so as to form a tighter interface compared with the RING dimer alone (see Figure 1 11-3). The two Pro434 residues within the RING dimer move 4.3 A towards each other, tightening the homodimeric interface, in the complex with UBE2DI compared to the RING domain alone (see Figure 1 1C). The inventors believe that there may be some cooperative rearrangement on binding the E2, though the influence of crystal packing cannot he ruled out.

The IDOL RING' UBF2DI structure is similar to the structure of TJBL2D2 in complex with the cTAP2 RING (Mace et al. 2008) and explains why IDOL can interact with all members of the UBE2D family ofE2 enzymes. The two structures vary however, in the orientation of the helix preceding the zinc-binding RTNC'r o domain. The interface between the IDOL RING and UBL2DI is not as extensive as the RING:RING dimer interface and buries only 1140 A2, which would suggest that the complex may he rather transient in nature (see Figures 41) and B).

-50 -Example 3 -Stereocheniica] basis of the specificity of 1DOI. for UBE2Ds To understand why TDOT. requires UBE2Ds and does not degrade the LDTJ{ in combination with other E2 ligases, the inventors carefully examined residues at the interface of the complex. It appears that residues in two positions in the interface play an important role in determining specificity. ArglS is conserved in UBE2D1-4.

Together with I xs8, Argi 5 provides a basic pocket that accommodates the acidic sidechain of G1u383 in the iDOL RING (see Figure 3A. in nearly all the E2s that do not support IDOL-mediated degradation of the LDLR, the residue at this position is either uncharged or acidic. Due to the close proximity of Aspl6, either a neutral or acidic residue at position 13 results in an acidic surface that would perturb interaction of the E2 with the TOOT. RING domain, as is the case for UBE2E3 (see inset in Figure 5A). All of the E2 ligases that do have an Arginine or Lysine equivalent to Argi 5 in UBE2D 1, are lacking a key serine residue (Ser94 in UBE2DI) at the other end of the interface (see Figures SB and C). This serine sidechain makes a critical hydrogen bond to the backbone carhonyl oxygen of Pro419 in IDOT. and this in turn brings about a tight stacking of the rings of Pro93 in UBE2DI and Pro419 in IDOL. In UBL2L3, UBE2G1, and LTBE2T, the serine is substituted by much larger sidechains (Lys, Leu and Arg respectively), which could not he accommodated at the interface with IDOL.

Example 6 -Disruption of the IDOL RING domain-UBE2D interaction inhibits LDLR denradation Based on their structural data, the inventors generated targeted mutations to further interrogate the IDOL-UBED2 interaction. The structure suggested that Val389 and Leu4l 5 were potentially critical IDOL residues mediating hydrophobic interactions with TJBE2I) 1 (see Figure 4E). In order to validate these predictions, they expressed LDLR together with native or tagged IDOL mutants in 111K1K293 cells. Compared to WI IDOL, the IDOL mutants V389R and L4I5E exhibited reduced capacity for o LDLR degradation (see Figure 6A). Interestingly, the auto-degradation of IDOL was also clearly inhibited by the introduction of the VSS9R and L4ISE mutations. The inventors also performed an in i'itv autoubiquitination assay using 11)01. V389R.

-51 -Consistent \vidI the cellular results, LBE2D2 was unable to efficiently catalyze the polyubiquitination of the IDOL \389R mutant (see Figure 613). Furthermore, introduction of mutations in ProGl and Phe62 in U]3E2D2 P61A, F62R, residues which are buried at the hydrophobic E2-E3 interface, also inhibited the ability of UBE2D2 to support IDOL autouhiquitination (see Figure 6C).

Mutational analyses also validated our model for IDOl -U 8E213 specificity outlined in Figure 5. Residue Glu383 in IDOL interacts with a basic pocket formed by ArglS and Txs8 0fTJBtL2D. Mutation of these basic residues in UI3F21)2 (RISE, KBE) reduced the ability of UBE2D2 to support T1)0T autoubiquitination in vitro (see Figure 6D). The model further predicts that Ser94 of UBE2D, which makes an important hydrogen bond with the IDOL backbone, is a key determinant of specificity. UBE2L3, which is unable to pair with TDOL, has a lysine residue in this position (see Figures SB and C). In support of the model, a UBE2D2 S94K mutant exhibited markedly reduced ability to catalyze IDOL autoubiquitination.

As the crystal structure of the IDOL RING domain-U13E2D complex revealed that IDOL could form a dimer via the residues within the RING domain, the inventors performed co-immunoprecipitation experiments to validate the physiological relevance of this finding. They found that when co-expressed with a VS-tagged IDOL in HLK293 cells, TAP-IDOL could he co-immunoprecipitated with the \75-tagged IDOL (see Figure 6E). These data indicated that the two different tagged versions of IDOT. could form a complex in the cell. Furthermore, introduction of structure-guided mutations predicted to disrupt dimer formation V43lR/L433R abolished the ability of TAP-I1)OT and VS-IDOL to he co-immunoprecipitated from cells (see Figure 6F). Moreover, IDOL dimer formation appears to he essential for its biological function, because the dimer-defective mutant V43 1R/L433R was unable to promote LDLR degradation and was resistant to auto-catalyzed degradation (see Figure 6G). Xi

Based on these results, the inventors reasoned that overexpression of IDOL mutants not capable of interacting with its cognate tL2 should interact with and sequester wild- -52 -type IDOL molecules, thereby preventing them from participating in uhiciuitin transfer. Such mutant IDOL proteins should therefore function as dominant negatives. fo test this hypothesis, they co-expressed increasing amounts of TOOT.

V389R with a predetermined amount of WI IDOL and LDLR in J-1LK293 cells.

Indeed, expression of IDOL V389R inhibited die degradation of LDLR by the WI TOOT. in a dose-dependent manner (see Fijure 6H). Furthermore, the auto-degradation of WT IDOl was also inhibited by the expression the V389R mutant.

Example 7 -IDOL is an iron-binding protein Immediately amino-terminal to the crystallized TOOT. RING construct there are three cysteine residues (Cys360, Cys363 and Cys368; see Figures 7A and B).

Expression and purification of an extended RiNG domain containing residues 358- 445 yields a brown protein (see Figures 7G. 1) and F. Other constructs of 11)01.

containing this region, including the full-length protein, are also brown (data not shown). Atomic absorption spectroscopy was performed (in both TDOI. RING constructs to measure the metal content in comparison to zinc and iron standards.

For the shorter 369-445 construct, a 0.24 mlvi protein sample gave a zinc concentration of 0.47 mId and an iron concentration of 0.012 mM; for the longer 358-443 construct a 0.15 mlvi protein sample gave a zinc concentration of 0.3 mlvi and an iron concentration of 0.06 mlvi (see Figure 7C). These concentrations correspond to two zinc ions per Protein molecule and one iron ion er dimer for tile longer protein. Protein disorder prediction of 11)01. using RONN (Yang et al. 2005) suggests that most of TDOL is ordered but that amino-acids 332-371 is inherently disordered. It is therefore possible that this region contributes to the dimerization interface when folded around an iron ion.

In order to address the potential functional signiflcance of this iron-binding region, the inventors introduced niutations into each of die 3 cysteine residues, alone or in combination (see Figure 717). Unexpectedly, these mutations led to increased TA)LR 0 degradation activity compared to WT IDOL. Analysis of TAP-IDOL expression revealed that the increased L[)LR degradation in these experiments likely resulted from increased IDOL stahilin-. Levels of TDOL protein expression were increased in -Di -an additive manner with mutation of C360, C363 and C368 (see Figure 7F),with the most prominent effects observed with the triple mutant (Il)OT. AAA) (see Figure 7G). The data suggest that the structure of the TI)OI. protein in the presence of iron may he more conducive to autouhiquitination and degradation. )

Finally, TflL uptake studies confirmed the functional consequences of the dirnerization, R2-interaction and iron-binding mutations. The E2-interaction mutants (VIS9R and L415E) and the dimerization mutant L433R/V431R reduced TOOT, activity, whereas the iron-binding mutant (IDOL AAA) actually increased IDOL's ability to block JUL uptake (see Figure 7H). Thus, the combination of biochemical and structural analyses has defined molecular interactions critical for the IXR-T1)OIrLDLR sterol regulatory pathway.

Discussion Tn Examples 1 to 7, the inventors have identified the uhk1uitin-conjugating enzyme E2D family proteins (U13E2D1-4 as the E2 uhiquitin carrier proteins involved in IDOL-dependent TA)T.R ubiquitination. The results provide strong evidence that IDOL directly facilitates the transfer of uhiquitin to LDLR by acting in a complex with UBE2I). The inventors also successfully carried out a biochemical and structural characterization of the E2-L3 complex and demonstrated that disruption of U 13 h2f) activity or the interaction interface between UB K2D and IDOL inhibits the degradation of the TJ)LR. These results provide a better understanding of the molecular n]echanism underlying the sterol-dependent regulation of LDLR protein levels.

Since the LDLR is a membrane protein, it is challenging to study IDOL-L1)LR interaction in a cell free system. The available assays for IDOL-dependent T.DTJ{ ubiquitination were not amenable to the screening of potential R2 enzymes. They therefore employed an alternative approach that assayed the auto-ubiquitination of o IDOL in vittv. Auto-ubic1uitination is characteristic of RING-type tL3 ligases. It is achieved via the same chemical reaction as the uhic1uitin-substrate ligation and mediated by the same L2 protein (Yang et al. 2000). The inventors screened 19 -54 -candidate E2 enzymes previously identified as preferentially interacting with the RING-type tL3 ligases Markson eta]. 20(39; van Wijk et al. 2009). Of these, proteins in the UBE2I) family were the only ones that were able to catalyze IDOL auto-ubiquitination in vihv. )

Although the inventors initially identified the IDOL-UBE2I) interaction based on IDOL autoubiquidnadon, several lines of evidence indicate that the UBE2D family enaymes also mediate the uhiquitination and degradation of the LDLR. The inventors showed that UBE2I)2, together with recombinant El and purified IDOL, was able to induce uhiquitination of LDLR in cell-free membrane preparations in vi/m. In addition, they demonstrated that the inhibition of UBE2D activity by over-expressing a dominant negative UBE2I) enzyme inhibited the ability of TDOL to degrade the TA)TR in cells. These dominant negative enzymes arc postulated to function by interacting with the ES enzyme and consequently prevent it from associating with endogenous E2 (Gonen et al. 1999).

The inventors also successfully obtained the crystal structure of the IDOL RING domain-LBE2D complex. The E2 uhiquitin-conjugating enzymes are structurally related and they share a conserved core domain with about 1 50 amino acids harboring the cysteine residues required for the formation of the ubiquitin-E2 thioester intermediate (Zheng eta!. 2000). Binding of an E2 to a RING-type ES is dependent on the E3 RTNG finger domain, which contains one histidine and seven cysteine residues that coordinate with two zinc ions Qoazeiro and Weissman 2000).

The RING-based E3s share many structural similarities in their RING domains, as do different E2s in their E2 core domains. Consequently, the biophysical basis for the specific functional pairings between E2s and E3s in ES auto-ubiquitination as well as the ubiquitination of substrates has been a long-standing puzzle. Careful examination of the structure of the complex and comparison of the sequences of the E2s that do and do not support IDOL activity has enabled the inventors to identify residues at t\vo key positions at the interface that appear to determine specificity.

-55 -I lowever, it is important to note that specificity may not only he dependent upon the stereochemistry of die interface, but may also require optimal dynamics of association /dissociation. This is important because it is well-established that the El and P3 binding surfaces on the E2 are oerlapping and that binding is mutually exclusive.

This fits well with die observation that the interface between the IDOL RING and UBE2I) I is relatively small (1140 A2), which is consistent with the reported dissociation constants of interaction of lUNG domains with E2s typically greater than 100 jiM 07kan etal. 2005; Das et al. 2009). Thus, the dynamics of E2:E3 and E2:Ll interactions play a role in controlling ubiquitination of the target protein (van ID Wijk and Timmers 2010). Too tight an E2:E3 complex would block the E2:El interaction and vice versa.

The finding that IDOL is an iron-binding protein raises die question as to whether the iron is regulating the activity of the protein. It is provocative to note that iron has been implicated in heart disease (Suffivan 1996) and that studies of iron depletion show a lowering of LDL-cholesterol (Facchini and Saylor 2002).

Based on the information provided by the crystal structure of the IJBE2D-IDOL complex, the inventors found that disruption of the interaction interface between IDOI. and UBL2D not only inhibited the autoubiquitination of 11)01. in an in vitro assay, hut also prevented functional ubic1uitination and degradation of the LDIR mediated by IDOL in cells. It has been generally assumed that the binding between an P2 and an E3 is the primary determinant of a functional E2-E3 pair. However, it has been shown that although c-Cbl and UhcFI7 form a complex, Ubci-15B, rather than UhcII7 appears to he the functional L2 for the c-Cbl-mediated uhiquitination (Huang et al. 2009). It has also been reported that the BRCA 1 /I3ARI) 1 E3 heterodimer can interact with UhcH5C and UhcIl7 with similar affinity, hut only UbcHSC is active in uhiquitination assays Qfr7ovic et al. 2003). Results from these studies suggest that the physical binding between E2-E3 pairs is not sufficient to infer Xi biological function, lughligliting the importance of complementary structural and functional assays of F2-F3 interactions.

-56 -

Summary

The inventors have previously identified the E3 uhiquitin ligase 11)01. as a sterol-dependent regulator of the LDL receptor GA)LR). The molecular pathway underlying IDOL action, however, remains to be determined. The inventors have now identified compeffing biochemical and structural characterization of an E2-E3 ubiquitin ligase complex for TJ)TR degradation. They identified the IJBE2D family (UBE2DI-4) as F2 partners for IDOL that support both auto-ubiquitination and IDOL-dependent ubiquitination of the LDLR in a cell-free system. NMR chemical shift mapping and a 2.1A crystal structure of the IDOL-RING domain-IJBL2D1 complex revealed key interactions between the dimeric IDOL protein and the E2 enzyme. Analysis of the IDOL-CBE2DI interface also defined the stereochemical basis for the selectivity of 11)01. for UBE2Ds over other Fl ligases. Structure-based mutations that inhibit IDOL dimerization or TDOL-UBE2D interaction block IDOL-dependent LDLR uhic1uitination and degradation. Furthermore, expression of a dominant-negative LBE21) enzyme inhibits the ability of IDOL to degrade the LDLR in cells. These results identify the IDOT-WW2I) complex as an important determinant of LI)TR activity and provide insight into molecular mechanisms underlying the regulation of cholesterol uptake.

Example 8 -The T1)0I. FERM domain binds directly to its targets To improve the understanding of the mechanisni whereby IDOl. triggers degradation ofLDLR, apoER2 and VLDLR, the inventors performed a series of structure-function analyses. IDOL contains two distinct domains:a C-terminal rea]ly interesting new gene (RING) domain, defining it as a member of the RING E3 llgase family; and an N-terminal FERM (Hand 4.1, ezrin-radixin-moesin) domain, a putative protein-protein interaction motif. The IDOL FERM domain is comprised of a classic tn-domain structure common to FERM proteins comprising three independently folded domains Fl-3 with the ES domain having a structure similar to that of a FIB phospho/tyrosine binding) domain. This P'FB-like domain is typically o involved in the interaction with cytoplasmic tails of plasma membrane proteins, commonly via an NPxY motif (with additional N-terminal sequences enhancing the affinity and specificity. Despite the name, PTB domains can have specificity for either -3/ -phosphorylated or non-phosphorylated tyrosine. Sequence alignments of IDOL with other FFA{M domain-containing proteins, including Talin, Radixin and Moesin, revealed that, although the FERI\1 domain would normally be expected to be around 290 residues, the sequence homology extends to residue 344 of IDOL, with an apparent insertion within tile 13 domain (residues 21 5-272, designated subdomain F3b) based on the alignment with Talin (see Figures l9A-C). Further examination and secondary structure prediction suggested that there might be a duplication of the C-terminal portion of the 13 PTB domain; i.e. the F3b and [Ic subdomains share significant homology.

Functional analysis indicated that each of the FERJ\1 subdontain regions is required for IDOL-mediated degradation of the TflLR, as deletion of any of them abrogated the ability of FLAG-tagged IDOL to promote TJ)LR degradation in an llFJK293T cell co-transfection assay (Figure l2A. These data indicate that, in addition to the RTNG P3 ligase domain, the FERM domain plays an essential role in the IDOL mechanism of action. The inventors hypothesi7ed that the FERM domain in T1)OL might function as a mechanism for the specific recognition of targets for IDOL-dependent degradation. To directly test whether a direct interaction occurs between the T1)OT FERM domain and the cytoplasmic C-terminal tail of its target proteins, the inventors used a fluorescence polarization FP) assay to monitor binding of the IDOl. FERM domain to a fluorescently-labeled synthetic peptide, based on the VLDL receptor (residues 820-842). VLDLR was chosen over LDLR as the model peptide since LDTJ( contains a cysteine residue in the tail and the peptide is labeled means of conjugation to a terminal cysteine residue. The inventors observed clear binding to \LLDLR peptide hut not to control non-specific peptide in this assay and the data fit a single-site binding model (see Figure 1 21-3). The dissociation constant for the interaction between the IDOL FERM domain (1-273) and the VLDLR peptide was 15.8 XivE +/-0.5. Although this is a relatively weak interaction (and was not detected in membrane-free cell-based assays or conventional pull-down assays; XI data not shown), it is nevertheless a relatively tight interaction compared with other FERM domain interactions and therefore significant (Anthis et al. 2009) -58 -Structural homology models of the IDOL FERI'vl domain were generated using PHYRE (Kelley LA and Sternherg. 2009). The inventors generated two models with different F3 sub-domain assignments: 1-344 with the deletion of residues 21 5-272 and 1-276 lacking residucs 277-344. These two regions are indicated F3a:F3c and F3a:13b, respectively, in Figure l3A. The inventors used these alternative models to generate predictions of residues that might he important for the recognition of the LDLR cvtoplasmic tail, based on the known mode of interaction between the talin FLRM domain and the beta-integrin cytoplasmic tail (see Figure l3B).

To test which of the two models was correct, the inventors introduced designed mutations into the surfaces that could potentially be involved in the interaction with the LDLR. Since there are no antibodies currently available that are capable of efficiently detecting native IDOL protein, and since epitope tags have the potential to affect protein function and interaction, they performed their initial analyses using native T1)OL constructs. Construct expression was monitored in these studies by RNA expression (not shown. Mutation of the key mino adds denoted in Figure l3\ demonstrated that Y265A and T269R. both of which reside in the IDOL F3b subdomain, appear to be especially important for IDOL-induced degradation of tile LDTR (see Figure 13B, left panel). Indeed, the lack of ability of Y265A and T269R to degrade the IA)LR was comparable to that of the RING domain mutant IDOL, C387A) \vhich lacks uhiquitination activity (Zelcer et al. 2009). When lower levels of IDOL were used in the degradation assy, it became apparent that Q232 was also involved in 11)01. action, as the Q232A mutant also led to a substantial reduction in activity on the LDLR (see Figure l3B, right panel). Mutations of M285 and Y323, which lie in the F3c subdomain, had a more modest effect on LDLR degtadation and an R327 mutant was comparable to WI in activity. These data suggest that the FERM 3b subdomain of IDOL is particularly important for interaction with other proteins and that the model of IDOl. FI-Rvl domain structure shown on the left in Figure 13A is the correct one. Xi

Given that the LDLR internalizes L1)I particles and reduces plasma 11)1] cholesterol levels when expressed at the cell surface, the inventors assessed the effects of IDOL -59 -FLRM domain mutants on the degradation of surface LDLR protein levels. Using a hiotin-laheling approach they found that the T269R and Y265A mutants were defective in their ability to clear LDLR protein from the plasma membrane, consistent with their observations with total cellular LDLR (see Figure 14A). To test the functional consequence of these mutations, they assayed the uptake of tluorescently labeled LDL particles by transfected 293T cells. As expected, the inhibitory activity of IDC)l T269R on LDT. uptake was dramatically reduced compared to WT IDOL (see Figure l4B). Q232A IDOL exhibited a partial defect, consistent with its effects on L1)LR protein.

In order to further validate the effects of the IDOL mutants on endogenous LDLR degradation, the inventors stably expressed the WT IDOL, RING mutants or F3h domain mutants in IDOL null (IDOL-/-mouse embryonic fihrohlasts MLFs).

Figure 14C demonstrates that in the presence of lipoprotein-deficient serum (LPI)S), stable expression of WT IDOL or IDOL A273E (a mutant with an intact ability to degrade LDLR) was associated with lower LDLR expression than control I1)OL-/-MEFs. In contrast, cells stably expressing RING mutant, Q232A, Y265A or T269R IDOL all exhibited greater LDLR abundance. Furthermore, they observed reduced uptake of LDL particles in 1DOL-/-MEFs expressing \VT and A273E IDOL compared to those expressing RING mutant, Q232A, Y265A or T269R (see Figure I 413).

Example 9 -The FERM F3h subdomain of IDOL is required for IA)LR ubic1uitination hut does not affect intrinsic E3 licrase activity Given that 11)01. is an E3 ligase, the lack of degradation and subsequent increased uptake of LDL particles associated with mutants in the IDOL F3h subdomain would he consistent with a reduced ability to ubiquitinate the LDLR. To test this hypothesis, the inventors performed immunoprecipitation assays. The data in Figure 14E confirmed enhanced uhiquitination of the LDLR in the presence of \VT and A273E Il)OL. I lowever, mutants Y265A and T269R showed markedly reduced ability to ubiquitinate JI)JR. Note, background levels of uhiquitinated Ll)I.R in the -60 -absence of transfected IDOL constructs (lane 1) reflect endogenous IDOL activity in 293 cells.

In order to address potential differences in the expression or stability of the various mutants employed above, the inventors repeated their analysis using tagged IDOL constructs. None of the fib mutants substantially altered the abundance of FLAG-IDOL protein, hut Y265A and T269R again sho\ved markedly reduced ability to degrade the LDLR (Figure 14F and data not sho\vn. Furthermore, mutations in the II)OT. protein that affect intrinsic E3 ligase activity (such as the RING mutant) would be expected to lead to stabilized IDOL protein due to loss of auto ubiquitination (Zelcer et al. 2009). The observation that all of the FERi1 domain mutants in Figure 14F showed comparable stability to WT IDOT. indicated that the intrinsic E3 ligase activity of these mutants was intact. Thus, these mutants appear to decouple LDLR recognition from intrinsic E3 ligase activity, and strongly suggest that the FFIth1 domain is involved in L1)I.R recognition (see below).

Example 10-A conserved SI/MxF sequence as an IDOL recognition motif The inventors have previously shown that the 50 amino acid cytoplasmic tail of the TflTJ{ is required for IDOL-induced degradation (Zelcer et al. 2009; Hong et al. 2010). Since only three proteins LDLR, VLI)LR and apoER2 appear to he targeted by IDOI. for degradation, the inventors hypothesized that these proteins must harbor a speciflc recognition sequence. To identify amino acids within the tail that make up the TDOT. degradation motif, they combined sequence analysis and structural modeling (see Figure 1 SA, B and C). They hypothesized that the IDOL FERM domain might bind directly to lipoprotein receptor tails and generated a homology model of the IDOL FERM domain (1 -276) with PIJYItE using the structure of the Protein 4.1R core domain (P1)13 ID: lgg3). The LDLR cytoplasmic tail was docked with reference to the structures of talin in complex with layillin, PIPKI-' and integrin-l1) (PDB IDs: 2k00, 2G35 and 369W respectively). The O resulting model suggests that F823 and 1821 in the LDLR cytoplasmic tail should be key residues mediating the interaction with IDOL F3h suhdomain (see Figures 1 SB and I SC). Interestingly, the model of IDOL reveals a pocket adjacent to residues -61 -Y265 and T269 that is not present in other RIB domains (see Figure 15C. The phenvlalanine, F823 at the -S position relative to the NPVV motif (where Y is position 0) on the LDLR tail is positioned opnmally to fit into this poclcet Another key determinant suggested from the complex model was the interaction with 1821 of the LDLR tail interacting with a non-polar surface on the surface of the FERAl domain (see Figure ISB).

Extensive site-directed mutagenesis of the LDLR tail led to the identification of 3 amino acids that conferred resistance to TOOL-mediated degradation when mutated (see Figure 1 51) and Figure 20). These data served to strongly validate the structural modeling. Point mutants in the phenylalanine at position 823 (F823A) and the isoleucine at position 821 (182FF) appeared to he completely resistant to degradation, whereas mutation of the serine at position 820 (S820D) was partially resistant (see Figure 1SC). It is noteworthy that basal LDLR protein levels observed following transfection of constructs containing the F823A mutation were consistentiy higher compared to WT TA)TR constructs (see Figure 151) and Figure 20A). This observation is consistent with enhanced LDTA{ stability due to lack of degradation by endogenous IDOL. Mutation of other conserved residues in the LDLR tail, including the key tyrosine in the NPVY internalization motif (Y828C; see Figure 20A, did not inhibit L1)T.R degradation. A chimeric protein in which LDTJ{ was fused to GFP just after V823 retained its ability to he degraded by IDOl, but a fusion after 1821 (lacking F823) was resistant, indicating that the sequences upstream of Y823 are sufficient for IDOL targeting (see Figure 20B). fransfection of these mutated LDLR constructs into 293T cells revealed that resistance to IDOL-dependent degradation also translated to resistance to IDOL-dependent inhibition of LDL uptake (see Figure 1511).

To establish a link between the 1 Dl R Sl/i'vlxF motif and IDOl -dependent ubiquitination, the inventors analy2ed the ability of WT and mutant LDLR proteins to he uhiquitinated in 293T cells. Mutations at positions F823, 1821 and 8820 each led to reduced uhiquitination by 1001. (see Figure 1SF), consistent with the results of the degradation assays.

-62 -To verify the importance of the SI/MxF motif for endogenous TJDLR degradation, the inventors stably expressed \VT or mutant lipoprotein receptors in LDLR-/-MEFs. Treatment with the LXR agonist GW3965, known to induce IDOl expression (Zelcer et al. 2009), effectively reduced the expression of \Vf I Dl R and inhibited LDL uptake, but had little effect on any of the LDLR mutants (see Figure 20C and data not shown).

Example II -A conserved IDOL reconnition site in ApoER2 and VLDLR io Surprisingly, both F823 and S820 are conserved across all three IDOL tat-gets (see Figure 1 5/k). In place of the isoleucine, the VLDLR and ApoER2 have a conservative substitution of the neutral and non-polar methionine. The inventors investigated the importance of the amino acids corresponding to F823 and S820, as well as the methionine aligning with 1821, for degradation of VLDLR and ApoER2.

For both receptors, mutation of the phenylalanine and the methionine rendered the receptor resistant to degradation, further suggesting that these amino acids are part of the IDOL recognition motif (see Figure ISG and 1511). In contrast, mutation of the equivalent serine of S820 in the LI)LR had a minor effect on \LI)LR degradation (5829D) and a small effect on ApoER2 degradation (S858D). Again, these results are good agreement with the structural modeling pointing to the particular importance of the -5 position (relative to the NPxY motif) for TOOT. recognition of LDLR, VLDLR and apoER2. Together, the results of Figures 13-15 define structural determinants for IDOL target recognition that explain the stringent speciticity of this unusual E3 ligase.

Example 12-Key residues in the IDOL FLRM domain and LpR are functionally conserved Given that integral physiological processes tend to be conserved through evolution, the inventors utilized protein sequence alignment to examine whether the key IDOL XI FERIV[ residues were conserved across species. Figure 16A demonstrates that the most important residues for LDLR recognition in the IDOL F3h subdomain are conserved in vertebrates as well as in the insect IDOL homolog, DNR1. b -63 -determine whether the function of these residues was also conserved, the inventors examined the effect of mutation in these residues in drosophila 1)NRI. Consistent with previous observations, DNR1 was capable of degrading human LDLR when expressed in 2931 cells (I long et al. 2010). Remarkably, however, DNRI point mutations in the tyrosine and threonine residues corresponding to human Y265 and T269 (drosophila Y405 and T409) were associated with reduced ability to degrade the LDLR compared to \VT DNR1 (see Figure 1 6B). The reduced inhibitory activity towards LUL uptake of DNR1 Y4OSA and T409R compared to WT DNRI further confirmed the functional importance of these residues for regulation of cholesterol uptake (see Figure I 6C).

Sequence alignment also revealed conservation of the S1/lvIxF motif in LDLRs across vertebrate species (see Figure 16D). Remarkably, this sequence was even present in the lipophorin receptor pR), the major lipoprotein carrying receptor in insects. As shown in Figure I 5E, TOOT. promoted the degradation of TpR in 293T cells, indicating that this receptor can indeed he recogni7ed by the FERM domain.

Furthermore, consistent with the mutational analysis of \LDLR and ApoER2, the phenylalanine residue corresponding to LDLR F823 (F992) was critical for IDOL-dependent degradation of LpR. Thus, key aspects of the I[)OT. mechanism of action on the LDLR appear to be highly conserved through evolution.

Example 13-TI)OI. uhiquitinates itself on the FERM 3c subdomain As is common for E3 ligases, IDOL controls its own by stability through autouhiquitination, presumahl of one or more lysine residues. By introducing a series of point mutations at lysine residues throughout the TOOL protein, the inventors identified specific sites in the F3c subdomain that appear to influence the abundance of TOOL protein (see Figure 17A). Interestingly, K293R and K309R had the greatest influence on IDOL abundance (see Figure 1713) and are also the most highly conserved of the lysines in the F3c suhdomain (see Figure i7A. Subsequent io compound mutations, each including K293R and K3OQR, were all associated with increased TOOL abundance compared to WT, as well as increased degradation of the L1)LR (see Figure 17(T). The abundance of each of the 4X lysine mutants was similar -64 -to that of RING mutant IDOL (which does not undergo autouhiquitination), suggesting that mutation of these lysines is sufficient to inhibit 11)01.

autodegradation. Studies in the absence or presence of the proteasomal inhibitor, MG-132 revealed little difference in the protein levels of each of the 4X mutants, further confirming that they are no longer undergoing proteasomal degradation (see Figure 21). Analysis of 293T cells treated with MGI 32 contirmed that mutation of lysine residues in the 173c subdomain strongly reduced IDOL autoubiquitination as predicted (see Figure PD).

Interestingly, drosophila I)NRI also appears to undergo RING domain-dependent autodegradation in both drosophila S2 cells and mammalian 293 cells (see Figure 17E and data not shown). Alignment of the 1I)NRI and IDOL F3c domains revealed two conserved lysine residues (see Figure l7A. Mutation of the highly consened lysine corresponding to IDOL 1<293 (K433R) increased DNRI protein stability and increased LDLR degradation, consistent with reduced capacity fbr autoubiquitinaton and degradation (see Figure 17E). Thus, the mechanism for IDOL protein turnover also appears to be largely conserved from insects to humans.

Example 14 -Membrane context is important for IDOL-dependent LI)LR deuradation Since the 1 DLR is a transmembrane protein, if IDOL interacts directly \vith the LDLR, it must do so in the context of the plasma membrane or membrane vesicles such as endosomes. Interestingly, IDOL was unable to promote the degradation of a fusion protein consisting of the LDLR cytoplasmie domain fused to GFP (see Figure 22). This observation suggested that a simultaneous IDOL-membrane interaction nught be required for efficient LDLR recognition and uhiquitination. Consistent with this hypothesis, the inventors were unable to demonstrate a stable interaction between the soluble LDLR cytoplasmic tail and IDOL in immunoprecipitation assays (data not shown). To investigate the ability of 11)01. to interact with membrane-o hound LDLR, they established a membrane interaction assay. They isolated membrane fractions froni 293T cells transfected with LDLR and \VT or FLRM domain mutant IDOL constructs. They then analyzed the ability of IDOL to -65 -associate with these membranes by immunoblotting. Figure ISA demonstrates that the abundance of \VT and mutant (Q232A, Y265R) IDOL proteins in total 293T cell lysates from transfected cells was similar. However, in isolated membrane fractions, the inventors readily detected the presence of \Vf IDOL protein in cells transfected with LDLR, but not those transfected with vector alone, suggesting that IDOL associates with membrane fractions in an IDLR-dependent manner. Furthermore, the Q232A and Y263R IDOL mutants, which were defective in LDLR degradation, showed reduced ability to associate with the membrane fraction (see Figure ISA).

Note, even in the presence of much higher levels of LDLR in the membrane (due to lack of IDOT. degradation), very little mutant IDOT. was recovered in the membrane fraction.

The cell-based assays suggested that IDOL requires membrane-inserted LI)LR tail for a fight and measurable interaction. [o explore this further, the inventors employed in vitro assays that are able to detect weak hut relevant protein-lipid and protein-peptide interactions. Their finding that IDOL localizes to membranes in an LDLR-dependent manner raised the question as to whether the FERM domain interacts directly with membranes. Structural modeling suggested that, similar to other FERM proteins such as Talin and Radixin, the IDOL FERM domain has a high proportion of positively charged residues, predominantly on one face of the protein (see Figure lSB). Ibis face is predicted to he proximal to the plasma membrane. To determine whether there was a direct interaction between the 11)01.

FERM domain and the membrane, the inventors perfbrmed vesicle cosedimentadon assays in which a solution containing protein and vesicles was separated by centrifugation into a pellet consisting of vesicles plus bound protein and a supernatant containing unbound material (see Figure 18C).

In the absence of vesicles ot-in the presence of neutral phosphatidylcholine vesicles (or even vesicles containing 20% negatively charged phosphatidylserine), the TII)OT.

o FLRvi remained in the supernatant fraction. However, increasing the negatively charged content of the vesicles to 100% phosphatidvlserine caused 80°/h of\\T 11)01. to precipitate with the vesicles. Phosphatidylserine was used due to its -66 -negatively charged head groups that had previously been shown to he a good model when the mode of binding is due to the general interaction with negatively charged lipid head groups (Goult et ad. 2010). Interestingly, the interaction of IDOL with these vesicles was considerably weaker than that of the Talin FERM domain (see Figure 1 SC). This is suggestive of a more transient IDOL-LDLR-memhrane interaction, and is consistent with the requirement for the L1)TR tail in the cell-based membrane association assays.

To confirm that membrane-ficing residues in the IDOL FERM domain were important for TA)TR degradation, the inventors performed cosedimentation assays and LDLR degradation assays with mutant IDOL proteins. A FLAG-IDOL expression construct encoding R73E/1K75E (residues in subdomain Fl) showed a partial reduction in LDLR degradation activity and a R193E/K199L/R259E mutant (subdomain F3) construct showed a prominent deficit (see Figure 1SD). A K137E/TK146L (subdomain F2) mutant effectively degraded the TA)LR, indicating that the primary membrane contacts are mediated by the Fl and F3 subdomains.

Cosedimentation assays further verified the importance of the F3 domain residues l93/Kl99/R259) for association with phospholipid vesicles (see Figure 18C).

These results strongly suggest that TI)OT. FERM interaction with cellular membranes is important for efficient targeting and therefore subsequent degradation of the LDLR. Taken together, the results suggest that the IDOL FERM domain mediates direct interactions with negatively charged membrane surfaces and with the cvtoplasmic peptides of its targets. High affinity interaction, sufficient to be detected in cell-based assays, clearly requires both membrane and the LDLR tail.

Discussion Examples 8 to 14 provide compelling data concerning the FERM-dependent E3 ligase recognition. Induction of the K3 uhiquitin ligase IDOL in response to sterol activation of LX1( provides a complementary pathway to SREBPs for feedback o inhibition of the LDLR pathway. I lowever, until the present invention, the mechanism by which IDOL specifically targets the LDLR had not been elucidated.

The inventors previously showed that increased IDOL levels in cells correlate with -67 -LDLR uhiquitination, but a central unresolved issue has been the question of whether 11)01. interacts directly with the T1)1J(, or whether the primary target for binding or ubiquitination is an intermediate protein. Surprisingly, the inventors have now demonstrated that IDOL binds directly to the cytoplasmic tails of lipoprotein receptors and cellular membranes through its FLItI'vi domain. Furthermore, they have defined the structural requirements for these interactions using complementary cell-based, computational and biochemical approaches. The inventors also establish that FLRM domain binding to both membrane and the LDLR tail is critical for the ability of T1)OL to trigger ubiquitination and degradation of the TA)TR receptor.

These studies provide mechanistic insight into sterol-dependent regulation of lipoprotein receptor expression.

In the uhiquitin system for protein degradation, the role of the E3 ligase is to confer target specificity. Regulation of E3 targeting can be achieved by changes in abundance or activity via transcriptional or post-translational mechanisms. Uhiquitin-mediated protein degradation often involves direct binding of the E3 and subsequent ubiquitination the target, but this is not always the case. For example, cbl-mediated degradation of the LGFR requires an intermediate factor. Since IDOL is the only L3 ligase that contains a FERM domain (Zelcer et al. 2010), the inventors postulated that this domain may be responsible for target recognition. Structural homology modeling and niutagenesis studies revealed that FERIV[ subdomains (denoted F3h and F3c harbor the critical residues for lipoprotein receptor recognition.

Interestingly, the lF3bc subdomains do not align with other FERM domain sequences. Thus, the structural basis for IDOL target recognition is unique amongst FERM domain proteins.

The inventors have described lysine residues within TJ)TR, \QDLR and apoER2 that serve as sites for uhiquitination. However, the recognition sequence for IDOL in its target proteins has remained elusive. E3 ligase recognition signals are commonly o short amino acid sec1uences, such as RXALGDCIXN in the case of the destruction box, the first uhiquitination signal to he identified (Glotzer etal. 1991). The LDLR has previously been shown to interact with proteins that have a PTI3-like domain -68 -such as sorting nexin-17 (SNXI7). This interaction requires residues within and downstream of the NPVY endocytosis signal in the LDLR cytoplasrnic tail (Burden et al. 2004). 17ERM-NPxY interactions are common among membrane proteins, such as that reported for the Talin FERM F3 domain and integrin (Garcia-Alvarez et al. 2003). However, the F3b subdomain of IDOL binds to a distinct recognition sequence (820s1/Mx[7823) immediately N-terminal to the NPxY motif in the cytoplasmic tail of the I D1 R. In fact, mutation of each of the 4 residues in the NPVY sec1uence of LDLR, or complete deletion of this motif, had no effect on IDOL-induced degradation, suggesting that the IDOL degradation pathway may be independent of clathrin-mediated endocytosis.

The structural modeling also strongly supported the importance of the -5 position (relative to the NPxY motif) to the specificity of 11)01. for LDLR, VLDLR and apoER2. The model predicts the existence of a pocket in the F3h subdomain adjacent to the critical amino acids required for target degradation (Y265 and T269) that accommodates the phenylalanine residue in-S position of the LDLR, \/LDTR and apoER2 cytoplasmic tails. Interestingly, the sequence motif FxNPxY only occurs in 13 other proteins of which only one, seizure6-like protein, is located in a cytoplasmic tail of a membrane protein. Seizure6-like lacks the key ubiquitinated lysine, however, and is therefore unlikely to he an TDOT. target. Disabled-I (DAB1) also binds the LDLR cytoplasmic tail and the structure of DAB I in complex with the apoER2 cytoplasmic tail has been solved Pl)B ID: 1NTY). In this structure the phenylalanine equivalent to F823 in the LDTR also seems to play an important role in the interaction.

Fluorescence polari2ation-based interaction assays confirmed that the IDOL FFRI binds to a single site in lipoprotein receptor tails. Moreover, the structural requirements for this interaction are in agreement with the amino acid se9uence requirements for Ti)0T -dependent LDLR degradation. Xi

Despite the fact that the LDLR internalization factor ARIT readily associates with the soluble L1)LR cytoplasntic tail in biochemical assays, the inventors' attempts to co- -69 -immunoprecipitate IDOL with the soluble LDLR tail were unsuccessful. This led them to hypothesize that the cell membrane may he a key component of 1DOL-lipoprotein receptor interactions. Indeed, they found that IDOL interacts with negatively charged phospholipid membranes, although this association is weaker than that reported for talin @nthis et al. 2009). [hey further defined positively charged residues on the predicted membrane-facing surface of the 11)01. FERM domain important for this interaction. The IDOL-membrane interaction may serve several different purposes. Since the affinity between IDOL and the LDLR tail is relatively weak, simultaneous membrane interaction likely provides stability to the complex. In addition, by helping 11)01. to locali2e with its targets in the cells, membrane association of IDOL imparts a spatial constraint on IDOL-dependent protein degradation. Finally, it is likely that the FERM-membrane interaction positions IDOL in die correct orientation to bind lipoprotein receptor tails.

The analysis also revealed a distinct regulatory function for tile EFIRM 173c subdomain: regulation of IDOL protein stability. Autouhiquitination is a strategy employed by many P3 ligases as a means of regulating turnover. [he inventors defined a series of lysine residues in the IDOL F3c subdomain domain critical for autouhiquitination and subsequent proteasomal degradation. Furthermore, mutation of these residues allowed them to establish that TDOI. autouhiquitination is functionally independent of I Dl R degradation. Indeed, an IDOI. protein lacking the F3c ubiquitinated lysines is resistant to proteasomal degradation, but shows enhanced ability to degrade the LDLR. Although certain other P3 ligases have been reported to undergo autoubiquitination on a single residue (e.g. cydin Dl), promiscuity is also commonly observed, as L2-h3 ligase complexes often favor lysine accessibility rather than sequence context once they have been recruited to their specific targets (l)anieisen et al. 201 1). The clustering of lysine ubiquitination targets in the lF3c subdomain suggests that this region may he particularly accessible to MNG-domain-catalyaed ubiquitin transfer (see the schematic model shown in Figure Xi 23).

-70 -The central role of the IDOL FERM domain in LDLR recognition is in line with recent studies showing that a non-synonymous single nucleotide polymorphism in this domain (N342S) is associated with total cholesterol levels in humans. The presence of a serine at this site results in attenuated uhiquitination of the LDLR and a concomitant increase in LDLR expression and uptake of LDL particles (Weissglss-Volkov, JCI). However, compared with mutation of key residues in the FTh subdomain (Y265 and T269) the N342S change exerts more modest functional effect.

These data suggest that residues in the F3c subdomain may make secondary contacts with the L1)LR that stabilize the complex or that the N342S polymorphism may lead to conformational changes that affect the F3b-TflI.R interaction. The fact that an IDOL protein lacking the F3c domain is inactive also supports the hypothesis that this region is important for the overall conformation of the FFR1\1 domain.

Although LXR nuclear receptors are not present in organisms lower than vertebrates, the 11)01. pathway for lipoprotein receptor degradation is conserved in insects. The inventors have shown here that the same molecular strategy for recognition of the LDLR receptor by 11)01. is employed by the drosophila I)NR1 ES ligase to bind the insect lipophorin receptor (LpR. Key residues predicted by the structural modeling to he involved in FERM domain-lipoprotein receptor interactions are conserved between IDOL and 1)NR I. Furthermore, the IDOl. S1/MxF recognition sequence is conserved in the mamniahan IDOI. targets (LDLR, apoF-tR2 and VLDLR) and the insect receptor LpR. Thus, the IDOL FERM-LDLR interaction is an evolutionarily-conserved mechanism for the post-translational control of membrane lipoprotein receptor actvity.

Summary

The E3 ubiquitin ligase TI)OL is an important regulator of cholesterol uptake, hut its mechanism of action, including the molecular basis for its stringent target specificity, is poorly understood. The inventors have also shown that IDOL employs a unique strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a Sl/MxF recognition sequence in the cvtoplasmic tails of lipoprotein receptors. This interaction is independent of IDOL's RING domain ES ligase -71 -activity and its capacity for autouhic1uitination. Surprisingly, the key interacting residues in TDOL and die L1)LR are functionally conserved in their insect homologues. The inventors have also demonstrated that target recognition by 11)01.

involves a tripartite interaction between the FERM domain, membrane phospholipids and the lipoprotein receptor tail. The data identify the IDOL-LDLR interacuon as an evolutionarily-conserved mechanism for the regulation of lipid uptake and suggest that this interaction can be exploited for the pharmacologic modulation of lipid metabolism using agents capable of preventing or inhibiting this interaction, i.e. the FERM-L1)LR interaction might he tractable target for the pharmacologic manipulation of lipid metabolism.

Example 15-Preparation of molecules which inhibit deadadon of L1)LR F lie inventors set out to develop agents or inhibitors, which are able to block the various protein interactions described herein in order to inhibit degradation of TJ)TR, VTA)LR or apoER2. In summary, these agents are as follows:- (a A molecule which inhibits binding of FERM domain of IDOL with the target receptor (i.e. the conserved motifs in TJ)TR, VTA)LR or apoER2); (b) A molecule which inhibits binding of FERM domain of IDOL with membrane phospholipids; (c A molecule which inhibits binding of RING' domain of IDOL with IJBE2I)1-4; (d) A molecule which inhibits binding of iron ions with the RING domain of IDOL; or (e) A molecule which inhibits the dimerisation of IDOL.

Peptide design, based on the sequence of a natural protein partner have been successfully used. For example, in the case of 1-3CL6, peptides based on the BCOR protein bind BCL6 and blocks SMRT from interacting at the same site and in doing so blocks BCL6-mediated transcriptional repression and kills lymphoma cells (Ghetu et al 2008). Similarly, the design of a synthetic, cell-permeable, stahilised peptide that targets the protein-protein interface in the NOTCI 1 transactivation complex has also been successfully used in leukaemic cells in culture.

-72 -In a similar way, a wildtype pepade corresponding to the LDLR tail sequence, SEQ ID No:2, would compete fbr binding to the TI)OT. FFRM and Irevent degradation of the LDLR receptor. Modifications/optimisation of the peptide sequence could be made to increase the affinity so that it is tighter than the wildtype which is a relatively weak and short lived interaction by design. A peptide from VTflLR or apoER2 or from other species could possibly have higher affinity, and this would be explored.

The inclusion of a uhiquitination motif, as in the LDLR tail, in the synthetic peptide would serve a secondary function; as well as binding the 17ERvi doniain it could be possible for it to be ubic1uitinated reducing the available pool of active E2 for uhiquitinating endogenous LDLR.

A peptide similar in nature to the conserved motif of the LDLR tail could clock into the unique surface on the IDOL lF3ah domain and prevent LI)TR interaction with IDOL and thus block LDLR ubiquitination. Structure-guided optimisation of the contacts would enhance the affinity. The inventors also consider the addition of sequence modules to enhance cell-permeability, and stability of transient structures.

Furthermore, a sequence with a TA)TA{-like motif and also a thiol-reactive moiety that could target the iron binding cysteines could have a dual mechanism. An antibody that targets the LDLR binding pocket on the FSah domain or the iron-binding cysteines could also he used to block LDLR ubiquitination.

Slaving identified the critical interaction surfaces on the proteins, the inventors expect that therapeutic antibodies can he developed that target these specific surfaces, block the interaction of TDOL with the relevant partners and hence prevent degradation of the TA)T receptor. Examples of such approaches in other systems can he found in Nature Reviews Immunology 10, 285, May 2010.

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Claims (1)

1. <claim-text>-76 -Claims I. An agent capable of: (a) inhibiting binding or interaction between a sub-domain of the FERM domain of TDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ ID No:i, or a functional fragment or variant thereof, and: (i) a Low-Density Lipoprotein receptor DLR, cii) a Very Low Density Lipoprotein Receptor (VLDLR and/or (iii) a Low density Jipoprotein receptor-related protein 8 (apoER2); (b) inhibiting binding or interaction between IDOL and a K/RNWXXIKNXXST/MXF motif present in the L1)LR, VLDLR and/or apoER2; (c inhibiting interaction or binding between IDOL and a member of the uhiquitin-conjugating en2ynl e (UIW2D) family; (ci) inhibiting or preventing binding of iron ions with IDOL; or (e) inhibiting or preventing the dimerisation of IDOL, for use in inhibiting LDLR, \IDTR and/or apoER2 degradation and/or promoting lipoprotein uptake.</claim-text> <claim-text>2. An agent capable of: (a) inhibiting binding or interaction between a sub-domain of the IFERM domain of IDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ Ti) No:l, or a functional fragment or variant thereof, and: (i) a Low-Density Lipoprotein receptor (LDLR, (ii) a Very I nw Density Lipoprotein Receptor (Vi DLR) and/or (iii) a Low density Jipoprotein receptor-related protein 8 (apoER2); inhibiting binding or interaction between IDOL and a K/RNWXX1KNXXST/MXr motif present in the L1)LR, VLDLR and/or apoER2; (c inhibiting interaction or binding between IDOL and a member of the ubiquitin-conjugating enzyme (UBE2D) family; d) inhibiting or preventing binding of iron ions with T1)OT; or (e inhibiting or preventing tile dimerisadon of IDOL, for use in die treatment, prevention or amelioration of hvperchoiesteroiaernia or cardiovascular disease.</claim-text> <claim-text>3. An agent according to either claim 1 or claim 2, wherein the agent is used for the treatment, amelioration or prevention of a cardiovascular disease selected from a group consisting of disorders of the heart and vascular system, such as congestive heart failure; myocardial infarction; ischemic diseases of the heart; ischemic cardiomyopathy; myocardial disease; all kinds of atriai and ventricular arrhythmias; hypertensive vascular diseases; peripheral vascular diseases; atherosclerotic coronary artery disease; heart failure; hvpertrophic cardiomyopathy; restrictive cardiomyopathy; congestive heart failure; cardiogenic shock; and hypertension.</claim-text> <claim-text>4. An agent according to any preceding claim, wherein the agent is capable of inhibiting binding or interaction between the receptor and the P3 sub-domain of the FERM domain of IDOL, wherein the P3 sub-domain is defined by amino add residues 183-344 of SEQ II) No:i, or a functional fragment or variant thereof 5. An agent according to any preceding claim, wherein the agent is capable of inhibiting binding or interaction between the receptor and an F3a, F3b or F3c sub-domain of the FERM domain of IDOL, wherein sub-domain F3a is defined by amino acid residues 183-214 of SKQ ID No:1, or a functional fragment or variant thereof, sub-domain lF3b is defined by amino add residues 21 5-272 of SEQ TI) No:1, or a functional fragment or variant thereoC and sub-domain E3c is defined by amino acid residues 272-344 of SEQ JE) No:l, or a functional fragment or variant thereof -78 - 6. An agent according to any preceding claim, wherein the agent is capable of inhibiting binding or interaction between an F3h sub-domain of the FFA{M domain of IDOL and the receptor, the F3b sub-domain being represented by amino acid residues 215-272 of SEQ ID No:l, or a functional fragment or variant thereof. )7. An agent according to claim 6, wherein the amino acid residues in the P3h sub-donnin of IDOl, which are targeted by the agent to prevent binding or interaction with the receptor, are selected from a group of residues consisting of residues: 232; 265; and 269 of SEQ II) No:l.8. An agent according to any preceding claim, wherein the agent is capable of inhibiting binding or interaction between an 173c sub-domain of the FERNI domain of IDOL and the receptor, the F3c sub-domain being represented by amino acid residues 273-344 of SEQ ID No:l.9. An agent according to claim 8, wherein the amino acid residues in the lF3c sub-domain of T1)OT, which are targeted by the agent to prevent binding or interaction with the receptor, are selected from a group of residues consisting of residues: 285; 323; 327 and 342 of SEQ Ii) No:1.10. An agent according to any preceding claini, wherein the agent is capable of inhibiting binding or interaction between a sub-domain of the FERI\4 domain of IDOL and amino acid residues conserved between (i) the LDLR, @i) the VTA)TM, and @ii) the apoER2 receptors.Ii. An agent according to any preceding claim, wherein the agent is capable of inhibiting binding or interaction between IDOl and: (i) a 820SI/?DCF823 motif; (ii) a 810WKNW813 motif; Qii) a 816KN81' motif; and/or (iv) a 821DNPVY828 motif, each motif being present in the LDLR, VLDLR and/or apoEl{2, as represented in SEQ ID No:2.-79 - 12. An agent according to any preceding claim, wherein the agent is capable of inhibrung interaction or binding between tile RiNG domain of 11)01 and a member of the ubicuitin-conjugating enzyme (UBE2D) family, which member is UIW2I) 1, L1BE2D2, UBE2D3 or UBE2D4. )13. An agent according to any preceding claim, wherein the agent is capable of inhibiting interaction of binding between the member of the ubiquitin-conjugating enzyme (UBE2D) family and one or more amino acid residues of the RING domain of TDOT. selected from the group of residues consisting of: C1u383; \al389; Leu4lS and Pro419 of SEQ TI) No:1.14. An agent according to any preceding claim, wherein the agent is capable of inhibiting interaction of binding between IDOL and one or more amino acid residues of the member of the uhiquitin-conjugating enzyme (UBE2D) family selected front the group of residues consisting of: LysS; Argl5; Pro61; Phe62 and Pro95 of SEQ TI) No:3.15. An agent according to any preceding claim, wherein the agent is capable of inhibiting interaction of binding one or more amino acid residues of the RING domain of 11)01. selected front the group of residues consisting of: G1u383; \tal3S9; Leu415 and Pro419 of SEQ ID No:l, and one or niore amino acid residues of the member of the ubiquitin-conjugating enzyme (UI3E2D) family selected from the group of residues consisting of: LysS; ArgiS; Pro6l; Phe62 and Pro95 of SEQ 11) No:3.16. An agent according to any preceding claim, wherein the agent is capable of inhibiting or preventing binding of iron ions with the RTN G donrain of II)OT.17. An agent according to any preceding claim, wherein the agent is capable of inhibiting or preventing binding of iron ions with amino acid residue C360, C363 and/or C383 of SEQ ID No:!.-80 - 18. An agent according to any preceding claim, wherein the agent is capable of inhibiting or preventing binding of membrane phospholipids with 11)01, preferably the FERM domain thereof 19. An agent according to claim 18, wherein amino add residues in the FER1v[ domain of TDOL, which are targeted by the agent to prevent binding or interaction with membrane phospholipids, are selected from the group of residues including 73; 75; 193; 199; 259; 137; and 146 of SEQ ID No:i.20. An agent according to any preceding claim, wherein the agent comprises a competitive polypeptide, or a derivative or analogue thereof, or a peptide-like molecule or a small molecule.21. An agent according to any one of claims 1-20, wherein the agent is an antibody or a fragment thereof.22. An anti-hypercholesterolaemia or anti-cardiovascular disease composition comprising a therapeutically effective amount of an agent capable of: (a) inhibiting binding or interaction between a sub-domain of the FERM domain of IDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ ID No:l, or a functional fragment or variant thereof, and: (i) a Low-Density Lipoprotein receptor (LDLR, (ii) a Very Low Density Lipoprotein Receptor (VLDLR and / or (iii) a Low density lipoprotein receptor-related protein 8 (apoER2); b) inhibiting binding or interaction between IDOl -and a K/RNWXXKNXXSI/T'vDCF motif present in the LDLR, \T1)LR and/or apoER2; (c) inhibiting interaction or binding between I[)0I. and a member of the ubiquitin-conjugating enzyme (UBL2D) family; -81 - (d) inhibiting or preventing binding of iron ions with IDOL; or (e) inhibiting or preventing the dimerisation of IDOL, and optionally a pharmaceutically acceptable vehicle.23. A process for making the composiflon according to claim 22, wherein the process comprises contacting a therapeutically effective amount of an agent capable of: (a) inhibiting binding or interaction between a sub-domain of the FERM domain of IDOL, the sub-domain being represented bvamino acid /0 residues 183-344 of SEQ ID No: 1, or a functional fragment or variant thereof, and: (i) a Low-Density lipoprotein receptor (LDTJ{), (ii) a Very Low Density Tipoprotein Receptor (VLDTA{) and/or (iii) a Low density lipoprotein receptor-related protein 8 (apoER2); (b) inhibiting binding or interaction between IDOL and a K/RNWXXKNXXSI/MXF motif present in the LDLR, VLDLR and/or apoER2; (c inhibiting interaction or binding between IDOL and a member of the uhiquitin-conjugating en7yme (UBE2D) family; (d) inhibiting or preventing binding of iron ions with IDOL; or (e inhibiting or preventing the dimerisation of IDOL, with a pharmaceutically acceptable vehicle.24. A composition according to claim 22 or a method according to claim 23, wherein the agent as detined in any one of claims 1-21.25. A method of inhibiting LDLR, VLDLR and/or apoER2 degradation and/or Jo promoting lipoprotein uptake in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable of: -82 - (a) inhibiting binding or interaction between a sub-domain of the FER\I domain of iDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ ID No:1, or a functional fragment or variant thereof, and: (i) a Low-Density Lipoprotein receptor (LDLR), (ii) a Very Low Density Lipoprotein Receptor (VTA)LR) and/or (iii) a Low density lipoprotein receptor-related protein S (apoER2); b) inhibiting binding or interaction between Ti)OL and a K/RNWXXKNXxS1/MxF motif present in the LDLR, VLDLR and/or apoER2; or (c inhibiting interaction or binding between TDOL and a member of the ubiquitin-conjugating enayme (UBE2D) faniilv; d) inhibiting or preventing binding of iron ions with Ti)0T4 or (e inhibiting or preventing the dimerisation of IDOL, to inhibit LDLR, VLDTR or apoER2 degradation and/or promote ilpoprotein uptake in the subject.26. A method of treating, preventing or ameliorating hypcrcholesterolaemia or cardiovascular disease in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of an agent capable of: (a) inhibiting binding or interaction between a sub-domain of the IFERM domain of IDOL, the sub-domain being represented by amino acid residues 183-344 of SEQ Ti) No:1, or a functional fragment or variant thereot, and: (i) a Low-Density Lipoprotein receptor (LDLR, (ii) a Very I ow Density Lipoprotein Receptor (Vi DLR) and / or (iii) a Low density lipoprotein receptor-related protein S (apoER2); -83 - (b) inhibiting binding or interaction between IDOL and a K/RNWXXIKNXXST/MXr motif present in the L1)LR, VLDLR and/or apoER2; or (c inhibiting interaction or binding betwccn IDOL and a member of the ubiquitin-conjugating enzyme (UBL2D) family; d) inhibiting or preventing binding of iron ions with T1)OT; or (e inhibiting or preventing tile dirnerisadon of IDOL, to treat, prevent or ameliorate hvperchoiesteroiaemia or cardiovascular disease in the subject.</claim-text>