GB2481373A - Treatment of hypercholesterolaemia by ubiquitination of PCSK9 - Google Patents

Treatment of hypercholesterolaemia by ubiquitination of PCSK9 Download PDF

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GB2481373A
GB2481373A GB1010280.4A GB201010280A GB2481373A GB 2481373 A GB2481373 A GB 2481373A GB 201010280 A GB201010280 A GB 201010280A GB 2481373 A GB2481373 A GB 2481373A
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Weiming Xu
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00Drugs for disorders of the cardiovascular system
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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    • G01N2333/95Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
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    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2440/00Post-translational modifications [PTMs] in chemical analysis of biological material
    • G01N2440/36Post-translational modifications [PTMs] in chemical analysis of biological material addition of addition of other proteins or peptides, e.g. SUMOylation, ubiquitination
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value
    • G01N2500/02Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00Detection or diagnosis of diseases
    • G01N2800/32Cardiovascular disorders

Abstract

Methods for treating or diagnosing or preventing hypercholesterolaemia by ubiquitination of proprotein convertase subtilisin/kexin type 9 (PCSK9) are disclosed, along with compositions for use in such methods. PCSK9 plays a critical role in cholesterol metabolism by controlling the level of low-density lipoprotein receptor (LDLR). The methods may involve the use of the E3 ligase cellular inhibitor of apoptosis protein 1 (c-IAP1) or STIP1 homology and U-box containing protein 1 (stub1) to ubiquitinate PCSK9. The invention also relates to methods and for identifying compounds which enhance ubiquitination of PCSK9.

Description

New treatment of hypercholesterolaemia by ubiquitination of PCSK9
Field of The Invention
The invention relates to methods and composition for identifying molecules and therapeutic agents which ubiquitinate PCSK9 for controlling the level of low-density lipoprotein (LDL) in circulation. The therapeutic agents identified are particularly useful for treatment of hypercholesterolaemia and other cardiovascular diseases.
Background to The invention
Cardiovascular Disease (CVD) is among the main causes of premature death in the world. The most important risk factor for CVD is the plasma level of cholesterol. Recent genetic studies have shown, one of the key genes, Proprotein convertase subtilisinlkexin type 9(PCSK9), plays a critical role in cholesterol metabolism by controlling the level of low-density lipoprotein receptor. PCSK9 is the third locus of autosomal dominant hypercholesterolemia(ADH) in addition to LDLR (low-density lipoprotein receptor) locus and APOB (apolipoprotein B) locus(1). A wide spectrum of mutations of PCSK9 gene have been found to be directly associated with hypercholesterolemia or hypercholesterolemia. Due to its direct binding to LDLR and degradation LDLR, PCSK9 now regards as novel target for the treatment of hypercholesterolemia. The PCSK9-knock-out mice exhibits higher level of LDLR in liver and reduced serum cholesterol while over express PCSK9 reduced LDLR and increased serum cholesterol. Tnterestingly, those individuals with loss of function mutations of PCSK9 have low levels of LDL and protected from cardiovascular diseases. Therefore, PCSK9 is a validated and new target for treatment of heart disease. So far, only LDLR and its closest family members VLDLR and ApoER2 have been found to bind with PCSK9. These studies were based through the normal pathway association studies.
Summary of the Invention
In order to find new binding partners for PCSK9, we have invented a novel screening method that is to combine a shotgun proteomic method to analyse the protein complex pulled down by immunoprecipitation against FLAG-tagged PCSK9 protein and differential analysis of natural occurring mutations of the PCSK9 gene. Among 22 potential novel binding proteins identified (Table 1), we found that the cellular inhibitor of apoptosis protein 1 (c-IAP11O) and the TNF receptor-associated factor 2 (TRAF211) complex are regulated differently in different dominant PCSK9 mutations that occur naturally.
Further immunoprecipitation analysis showed that c-lAP 1 is a direct binding partner for PCSK9. One of the "gain-of-function" mutants, PCSK9-S127R, which has impaired autocatalytic activity, is defective in binding to c-IAP1. The other dominant mutation, PCSK9-D374Y, which is 10-fold more potent in degrading the LDLR protein than wild-type PCSK9, can be significantly ubiquitinated by c-IAP1 in vitro. The ubiquitinated PCSK9-D374Y is unable to degrade LDLR, which is its main cause of hypercholesterolaemia in patients. These results indicate that there is a novel cholesterol uptake regulation pathway linking PCSK9/LDLR to the E3 ubiquitin ligase c-IAP1 in a TNF-a response pathway. This highlights the possibility of developing new treatments for human cardiovascular diseases through ubiquitin ligase-mediated ubiquitination of target proteins in cholesterol metabolism.
According to the present invention there is thus provided a method for identifying a substance which enhance the ubiquitination of PCSK9 protein and modulating the PCSK9-mediated LDLR degradation, which method comprises determining whether a test substance is an agent to treat cardiovascular diseases.
The invention also provides: * use the E3 ligase c-lAP 1 for ubiquitination of PCSK9 for use in a method of treatment of the human body by therapy; * Use the E3 ligase c-lAP 1 in the manufacture a medicament for use in treatment of hypercholesterolemia.
* use the E3 ligase stub 1 for ubiquitination of PCSK9 for use in a method of treatment of the human body by therapy * use the E3 ligase stub 1 in the manufacture a medicament for use in treatment of hypercholesterolemia.
* use other the E3 ligase for ubiquitination of PCSK9 for use in a method of treatment of the human body by therapy * use other E3 ligase in the manufacture a medicament for use in treatment of hypercholesterolemia.
* a substance identified by a method of the invention for identifying a substance which enhances ubiquitination of PCSK9.
* a substance of the invention for use in a method of treatment of human body by therapy.
* Products containing a substance of the invention and a therapeutic agent of treating hypercholesterolemia.
* a method of treating a host suffering from hypercholesterolemia, which method comprises the step of administrating to the host effective amount of a substance of the invention and therapeutic agent.
* A method of identifying the function unit, such as the region containing the baculoviral lAP repeat 3 (BIR3) domain of c-IAP1 could be used to develop pharmaceutical compounds, either activate or inhibit the Ubiquitin ligase 3 activity on ubiquitination of PCSK9.
Brief description of the Drawings
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.
All publications and patents referred to herein are incorporated by reference.
Table 1 List of the potential PCSK9 binding proteins identified through affinity purification and shotgun LC-MSIMS analysis. Each protein was identified by at least two matched spectra (95% confidence minimum) in all three experiments with no spectrum identified in the control samples (empty vector).
Fig. 1. Immunoprecipitation (IP)/western blot analysis of novel PCSK9 binding proteins in PCSK9-FLAG pull-down assay. Cellular extracts from a T-Rex 293 cell line stably overexpressing FLAG-tagged wild-type PCSK9 or a negative control cell line, T-Rex-293 cells transfected with the empty vector pcDNA3.1(control) were subjected to anti-FLAG IP and blotted to nitrocellulose and probed with the indicated antibodies. The western blots shown are representative of three separate experiments.
Fig. 2. c-TAP 1 interacts with PCSK9. (a) Co-TP of PCSK9-wt and c-TAP 1. Myc-tagged c-TAP 1 or c-TAP 1 -BTR3 was co-transfected with either empty vector (pCMV6-entry vector) or FLAG-tagged wild-type PCSK9 into T-Rex-293 cells. Cell lysates were immunoprecipitated with an anti-myc tag antibody followed by immunoblotting with either anti-PCSK9 or anti-c-lAP 1 antibodies. (b): Co-IP of PCSK9-wt, TRAF2 and LDLR. Myc-tagged TRAF2 or LDLR was co-transfected with FLAG-tagged wild-type PCSK9 into T-Rex-293 cells. Cell lysates were immunoprecipitated with an anti-myc tag antibody followed by immunoblotting with either anti-PCSK9 or anti-TRAF or LDLR antibodies. The western blots shown are representative of three separate experiments.
Fig. 3. Silencing of c-IAP1 by pGB c4AP1 siRNA mixture. (a) A wild-type PCSK9 overexpressed T-Rex-293 stable cell line was transfected with pGB c-IIAP1 siRNA. A stable c-TAP1 siRNA clone was established with nearly 100% knockdown of endogenous c-lAP 1 protein (lane 1) in comparison with empty pGB control vector (lane 2). Western blot was probed with anti-c-IAP1 or anti-PCSK9 antibodies. Scanning densitometry analysis of three western blots is shown below. Data are presented as the percentage conversion to mature PCSK9 (p63), calculated as the p63 value divided by the sum of p63 + p75 (Pro-PCSK9), divided by the tubulin, multiplied by 100. ** indicates a significant difference (P=0.009) from c-lAP siRNA treated cells from control siRNA cells.(b) Western blot analysis of the LDLR level in pGB-c-IAP1 siRNA knockout cells. Myc-tagged LDLR-CMV6 or empty vector was transfected into c-lAP 1-siRNA-knockout cells or control siRNA cells. After 48 h, the cell lysates were analysed by western blot with anti-myc antibody to detect LDLR protein. LDLR amounts were quantified and normalised to the amount of -tubulin. The ratio of LDLR/tubulin in control siRNA cells was assigned a value of 1.00. * indicates a significant difference (P =0.04) between treated cells and control siRNA cells.
Fig. 4. Identification of PCSK9 as a substrate of c-IAP1 ubiquitin ligase in vivo and in vitro.
a. Western blot analysis of the LDLR level in transient transfection of wild-type FLAG-tagged PCSK9-pcDNA3 or PCSK9-D374YpcDNA3 with or without cTAP-1pCMV6 in HepG2 cells.
LDLR was quantified and normalised to the amount of a-tubulin. The ratio of LDLR/tubulin in the cells transfected with empty vector control was assigned a value of 1.00. P-values are from comparisons between transfection with empty vector and the PCSK9 expression vectors with or without c-lAP 1 expression vector. Values are means + s.d. from three separate experiments. * denotes P<0.05; ** denotes P<0.01.
b. Proteasomal and lysosomal inhibitors prevent D374Y-PCSK9-mediated LDLR degradation.
HepG2 cells were transiently transfected with a plasmid expressing PCSK9-D374YpcDNA3 or empty vector (pCDNA3.1). 24 h after transfection, cells were grown in serum-free DMEM medium for an additional 16 h and then treated with 10 iM of proteasomal inhibitor MG 132 or the lysosomal inhibitor Ammonium Chloride (NH4CI) for 4 h in serum-free DMEM medium. Western blotting of total cell lysates using anti-LDLR antibody. Anti-tubulin antibody was used as a control. LDLR was quantified and normalised to the amount of a-tubulin. The ratio of LDLR/tubulin in the cells transfected with empty vector control was assigned a value of 1.00. The western blots shown are representative of three separate experiments.
c. Hep G2 cells were co-transfected with wild-type PCSK9 and D-374Y pCSK9 with or without c-lAP 1 and the Haemagglutinin (HA-ubiquitin) expression plasmid. After 24 h, lysates were subjected to IP with anti-ubiquitin antibody and probed with anti-PCSK9 antibody. The western blots shown are representative of three separate experiments.
d. FLAG-tagged PCSK9-D374Y was subjected to a ubiquitination assay in the presence of recombinant c-lAP 1. The poly-ubiquitination of D374 was detected by immunoblotting with anti-PCSK9 antibody.
e. Effect of ubiquitination of mutant D374Y on LDLR expression in whole cell extracts of Hep G2 cells. Lane 1, Untreated; Lane2, D374Y (1jtg); Lane 3, c-TAP1-ubiquinated D374Y (1 jig); Lane 4, ubiquitination buffer only. LDLR was quantified and normalised to the amount of a-tubulin. The ratio of LDLRltubulin in the untreated cells was assigned a value of 1.00. P-values are from comparisons between untreated samples and recombinant D379Y treatment and ubiquitination treatment. Values are means + s.d. from three separate experiments. * * denotes P<O.O1.
f. FLAG-tagged wild-type -PCSK9, S127-PCSK9 and PCSK9-D374Y were subjected to ubiquitination assays in the presence of recombinant c-lAP 1. The ubiquitinated PCSK9 was detected by immunoblotting with anti-PCSK9 antibody.
g. Effect of ubiquitination of wild-type PCSK9 on LDLR expression in whole cell extracts of Hep G2 cells. Lane 1, Untreated; Lane2, D374Y (1 fig); Lane 3, wild-type PCSK9 (4 fig); Lane4, c-lAP 1-ubiquinated wt-PCSK9 (4 jig). LDLR was quantified and normalised to the amount of a-tubulin. The ratio of LDLR/tubulin in the untreated cells was assigned a value of 1.00. P-values are from comparisons between untreated samples and recombinant wild-type PCSK9 treatment and ubiquitination treatment. Values are means + s.d. from three separate experiments. * denotes P<0.05; ** denotes P<0.01.
h. Flow cytometric analysis of BODIPY-labelled LDL uptake in Hep G2 cells. Cells were incubated in the presence or absence of D374Y protein with or without ubiquitination. Graph is representative of three separate experiments with similar results.
Supplementary Fig. Si. SPR analysis of interactions of PCSK9 and c-lAP 1. Representative overlays for various concentrations of purified PCSK9-wt to immobilised c4AP1. Coloured lines represent data; black lines indicate a theoretical good fit to a simple 1:1 kinetic model with a Kd of 44.3±5 nM (n=3).
Fig. S2. Co-localisation of PCSK9 and c-lAP 1 in the cytoplasm. Cells stably overexpressing PCSK9 were immunostained with mouse anti-FLAG M2 monoclonal antibody (for detection of PCSK9) using secondary anti-mouse antibody labelled with Alexa488(Green); Rabbit anti c-lAP was used to detect the c-IPA1 protein, with an anti-rabbit-cy3 labelled antibody. Cells were subjected to confocal microscopy examination. Green fluorescence indicates PCSK9; red indicates c-lAP 1. In the merged images, yellow staining indicates co-localisation. Bar, 20 jim.
Fig.53. IP/westem blot analysis of interaction between the FLAG-tagged PCSK9 and stub 1 protein in PCSK9-FLAG pull-down assay. Cellular extracts from the T-Rex 293 stable cell line overexpressing FLAG-tagged wild-type PCSK9 was subjected to anti-FLAG IP, blotted to nitrocellulose and probed with the stub 1 antibody and PCSK9 antibody. The western blots shown are representative of three separate experiments.
Fig. S4. Proposed model of c-TAP1/TRAF2 regulation of the PCSK9-mediated LDLR degradation. Upon activation by tumour necrosis factor a, TNFR complex recruit TRAF-2 to its cytoplasmic tail, leading to recruitment of c-lAP 1. c-lAP 1 can bind to PCSK9 and promote its maturation. The second role of c-lAP 1 is its E3-ubiquitin ligase activity. When extra-cellular PCSK9/LDLR complexes re-enter the cell, c-lAP 1 binds to PCSK9, leading to its proteasomal degradation, releasing LDLR to recycle back to the membrane. There may be a switch for LDLR/PCSK9 to be shuttled either to the proteasome or to the lysosome. Due to extremely tight binding to LDLR, PCSK9-D374Y/LDLR will lead to more destruction of LDLR in the proteasome, whereas wild-type PCSK9 may depend mostly on the lysosomal pathway. Due to its inability to bind to c-IAP1, PCSK9-S127R may bind to other E3 ligases (such as Stub 1 ligase) to form a complex with HSP7O, DNAJ family, ERP72, or APOB, leading to the destruction of LDLR..
Detailed Description of the Invention
Proprotein convertase subtilisinlkexin type 9 (PCSK9), in addition to LDLR (low-density lipoprotein receptor) and APOB (apolipoprotein B), is one of three loci implicated in autosomal dominant hypercholesterolaemia (ADH)'. A number of PCSK9 gain-of-function mutations and loss-of-function mutations have been identified from families afflicted with ADH with hypercholesterolaemia or hypocholesterolaemia, respectively'4. In humans, the main function of PCSK9 appears to be the post-transcriptional regulation of the number of cell-surface LDL receptors57. To date, only LDLR and its closest family members VLDLR binding studies. We found that only the region containing the baculoviral lAP repeat 3 (BIR3) domain of c-lAP 1 could be used to pull down wild-type PCSK9 (Fig. 2a, lane 3), indicating that c-TAP 1-BIR3 is the binding site for PCSK9. The binding of PCSK9 to c-lAP 1 was further confirmed using surface plasmon resonance (SPR) experiments, in which the binding affinities of the wild-type PCSK9 protein for c-TAP 1 were determined at pH 7.4 with kinetic constants for dissociation at a Kd of 44.3 + 5 nM (n=3, T100 evaluation software, supplementary Fig. Si). We also used confocal microscopy to conduct immunostaining studies to confirm the colocalisation of PCSK9 and c-TAPi in the cytoplasm of 293 cells with stable overexpression of PCSK9 (Supplementary Fig. S2).
We next determined whether TRAF2 binds to the wild-type PCSK9. We co-transfected wild- type FLAG-tagged PCSK9 and myc-tagged TRAF2-pCMV6 expression vectors into T-Rex- 293 cells. After 24 h, the cell lysates were immunoprecipitated with anti-myc antibody. We were unable to detect the PCSK9 protein in the IP product (Fig. 2b, lane 2), indicating that TRAF2 was not a direct binding partner for PCSK9. c-lAP 1 was the only physical binding partner of PCSK9 in the c-lAP 1/TRAF2 complex. As a positive control, we co-transfected wild-type FLAG-tagged PCSK9 and myc-tagged LDLR-pCMV6 into T-Rex-293 cells. After 24 h, the cell lysates were immunoprecipitated with anti-myc antibody. We were able to detect the PCSK9 protein in the IP product (Fig. 2b, lane 3), confirming LDLR binding to PCSK9 in our system.
We next used a siRNA-c-IAP1 construct to knock down the endogenous c4AP 1 in a human T-Rex-293 stable cell line that overexpressed FLAG-tagged wild-type PCSK9. We observed significantly increased pro-PCSK9 bands (90% PCSK9 protein is still pro-PCSK9, only 10% is converted to the mature band) in comparison to the non-silencing RNA control (over 90% of PCSK9 is converted to the mature band), indicating that c-lAP i is directly involved in processing PCSK9 from a proprotein to the functionally mature protein (Fig. 3a). We have also detected high molecular weight aggregates of PCSK9 formed in c-lAP 1 siRNA treated samples. Because of a very low LDLR protein level in the PCSK9-overexpressed 293 cells, we transfected the myc-tagged LDLR-pCMV6 plasmids into a c-lAP 1 siRNA knocked-out cell line or a control cell line with empty vector as control. There was a more than 30% reduction of LDLR protein level in c-TAP 1 siRNA knockout 293 cells when compared to that of control cells, indicating that c-lAP 1 negatively regulated PCSK9-mediated LDLR degradation (Fig. 3b).
To further investigate the role of c-lAP 1 in PCSK9-mediated LDLR reduction, we transiently co-transfected wild-type FLAG-tagged PCSK9 or a gain of mutation' construct (PCSK9-D374Y-pcDNA3) with or without clAP-i into HepG2 cells. As shown in Fig. 4a, the LDLR level was decreased slightly (i 8% less than empty vector transfection) after wild-type PCSK9 transfection. The PCSK9-D374Y mutation was significantly more potent in reducing the LDLR protein level (over 80% less than empty vector transfection). The co-transfection with CMV6-cIAP 1 vector attenuated both PCSK9-wt-and PCSK9-D374Y-mediated LDLR decrease, indicating the c-lAP 1 protein inhibits PCSK9-mediated LDLR degradation (Fig. 4a).
We next tested whether D374Y-PCSK9-mediated LDLR degradation was dependent upon lysosomal function or ubiquitin-mediated proteasomal degradation, as c-lAP 1 is a well-known E3 ubiquitin ligase in the proteasome-mediated protein degradation pathway'4. We transiently transfected a gain-of-function mutation construct of PCSK9-D374Y-pcDNA3 into HepG2 cells. After 24 h, we treated the transfected cells with either 20 tmMofMG132 (a proteasome inhibitor) or 100 tM of NH4CI (a lysosomal inhibitor), or left the cells untreated. As shown in Fig. 4b, the LDLR level was significantly decreased after transfection with PCSK9-D374Y.
Treatment with MG 132 was able to increase the LDLR level back to 44% of the original level, whereas treatment with NH4C1 was able to increase the LDLR level back to normal. The result indicated both lysosomal and proteasome-mediated protein degradation pathways were involved in mutant PCSK9-D374Y-mediated LDLR degradation.
We then tested c-lAP 1 s ability to ubiquitinate the PCSK9-D374Y in vivo and in vitro. T- Rex-293 cells were co-transfected with wild-type FLAG-tagged PCSK9 or a gain-of-function' mutation construct of PCSK9-D374Y-pcDNA3 and pcDNA3.1 -(HA-ubiquitin) with or without c-TAP 1-pCMV6. After 24h, the whole cell lysates were immunoprecipitated with anti-ubiquitin antibody and probed with PCSK9 antibody. As shown in Figure 4c, only the HA-UB/cIAP-1 and D374 mutant combination resulted in an appearance of multiple high-molecular weight bands representing the polyubiquitinated PCSK9 (Fig. 4c, Lane 4). For the in vitro ubiquitin assay, the FLAG-tagged D374Y-PCSK9 was purified and subjected to ubiquitination in the presence or absence of recombinant c-lAP 1 (Fig. 4d). PCSK9 was found to be ubiquitinated by c-lAP 1, as shown by the appearance of multiple high-molecular weight bands on an SDS-PAGE gel representing the polyubiquitinated PCSK9 in the presence of recombinant c-lAP 1 (Fig. 4d, Lane 3). In control experiments, there were no detectable polyubiquitinated PCSK9 bands in the samples without c-IAP(Fig.4d, Lane 2) or without D374Y protein(Fig.4d, Lane 4).
To further elucidate the functional significance of D374Y-PCSK9 ubiquitination, we added 1 jig/mi of ubiquitinated D374Y-PCSK9 recombinant protein into DMEM (without 10% serum) to test its ability to reduce the levels of endogenous LDLRs in HepG2 cells. As shown in Fig. 4e, the LDLR level was significantly decreased in the PCSK9-D374Y-treated sample (lane 2, over 90% decrease in LDLR level compared to the untreated sample), but there was no LDLR level decrease in the samples treated with 1mg of ubiquitinated D374Y-treated (lane 3). The ubiquitin buffer itself has no significant effects on LDLR protein levels in HepG2 cells (lane 4).
We also carried out an in vitro ubiquitin assay on the purified wild-type PCSK9 protein. We noticed that wild-type PCSK9 could also be ubiquitinated at a much higher protein concentration (4 tg, Fig. 4f, Lane 3). The other dominant mutation, Si 27R, which was unable to bind clAP-i, was only very weakly ubiquitinated by c-lAP 1 at the 4 tg concentration (Fig. 4f, Lane 4). In the functional analysis, there was a modest effect on the LDLR level (20% decrease in LDLR) after administration of 4mg/mi wild-type PCSK9 in HepG2 cell culture (without serum) for 16 h (Fig. 4g. lane 2) in comparison to D374Y-PCSK9 administration (85% decrease of LDLR, Fig. 4g, lane 3). Ubiquitination of wild-type PCSK9 abrogates its ability to reduce LDLR levels in HepG2 cells (Fig. 4g. Lane 4).
Finally, we measured the uptake of LDL in ubiquitinated PCSK9-D374Y and non-ubiquitinated PCSK9-D374Y treated HepG2 cells (Fig. 4h). Paralleling the levels of LDLR on western blot, we found that the mean level of LDL uptake in the 1 tg/ml D374Y treatment was reduced on average by 50% (Fig. 4g, green peak, mean intensity 8.7±1, n=3) when compared with untreated samples (brown peak, mean intensity 17.2±i, n=3, P=O.03). In contrast, ijg/ml of ubiquitinated D374Y was unable to reduce LDL uptake (blue peak, mean intensity i6.5±1.2, n=3) compared with untreated sample (mean intensity 17.2±1,n3, P=O.22). In the control experiment, the ubiquitin reaction without clAP-i had no significant effect on the mutant D374Y's ability to reduce LDL uptake (red peak, mean intensity 7.8±0.2, n=3) in comparison to the D374Y treatment (green peak, mean intensity 8.7±1, n=3, P0.45, Fig. 4h).
In summary, we report here that E3 ubiquitin ligase c-lAP 1, also known as Baculoviral lAP repeat-containing protein 2 (BIRC2), plays an important role in regulating PCSK9-mediated LDLR degradation. By binding to wild-type PCSK9, it promotes its maturation from its proprotein form. One of the gain-of-function' mutations, PCSK9-S127R, has a defect in binding to c-IAP1, and so has impaired autocatalytic activity. The precise mechanism for how the S127R mutant proprotein promotes LDLR degradation and causes hypercholesterolaemia is still unknown. However, in c-lAP 1 siRNA knockout cells, in addition to the significant defect in PCSK9 maturation, there is significant LDLR depletion, indicating c4AP1 negatively regulates PCSK9-mediated LDLR degradation. By not binding to c-lAP 1, Si 27R could work more effectively to degrade LDLR. The other possibility is that Si 27R could bind to other E3 ligases to regulate the LDLR/PCSK9 pathway differently. In fact, from our preliminary results, the other E3 ligase we detected in our shotgun proteomic analysis, stub 1 (STlPihomology and U-box containing protein 1)' has been found to bind much more strongly to S127R than to wild-type PCSK9 (Supplementary Fig S3). Stub 1 is a ubiquitin ligase/co-chaperone that participates in protein quality control by targeting a broad range of chaperone protein substrates, including Hsp7O, Hsc7O and Hsp9O. The proteins in the DNAJ (Hsp4O) subfamily we detected in the shotgun proteomic analysis are also known to form complexes with Hsp7O. A close relative of LDLR, LDLR related protein lb (LRP1b) has been shown to bind to DNAJA1'6 Furthermore, PDTA4, also known as ERP72, has been shown to be in a chaperone complex with the DNAJ family and APOB'7. Therefore, S127R potentially bind to these complexes through the Hsp7O /APOB'8 pathway to exert its effect on LDLR degradation (Supplementary Fig. S4).
Interestingly, the other dominant mutation, PCSK9-D374Y, binds to c-lAP 1 and can be cleaved normally and secreted from cells. We showed that by binding to PCSK9-D374Y, c- TAP1 ubiquitinates it very effectively. Function analysis showed that ubiquitinated PCSK9-D374Y has lost its ability to degrade LDLR in culture cells in vitro. Given previous observations showing that there is a good correlation between the effect of PCK9 mutations on LDLR in cultured cells in vitro and their effect on the plasma cholesterol level of heterozygous carriers of the mutations'9, we have envisaged a novel approach, using c-IAP1 or another E3 ligase to inactive some of the most severe hypercholesterolaemic mutations, such as PCSK9-D374Y'2.
The E3 ligase is a large isoenzyme family, defined by one of several motifs. These include a HECT, RING or U-box (a modified RThG motif without the full complement of Zn2+-binding ligands) motif. The c-TAP 1, traf2 and stub 1 E3 liagses we identified in our assay comprising RNG or U-box box. E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrate's ubiquitination. Some can apparently act alone, others are found much larger multi-protein complexes, such as the Stub 1/Hsp7O /APOB'8 pathway to exert its effect on LDLR degradation.
Furthermore, E3s represent a class of "drugable" targets for pharmaceutical intervention.
Especially the C-lAP 1 protein has containing one or several FIR (baculoviral lAP repeat) domains that are required for regulation by a mitochondrial protein Smac/DIABLO. Smac physically interacts with multiple TAPs and relieves their inhibitory effect on caspases 3, 7 and 9. Smac binds to the BIR3 domain of c-TAP 1 via the N-terminal four residues (AVPI) that recognize a surface groove on BTR3. These four amino acids are conserved in three Drosophila proteins, Reaper, Grim, and Hid that induce apoptosis by eliminating Drosophila TAP binding to caspases. Targeting TAP's ubiquitin ligases could therefore be a feasible approach for pharmapeutical intervention. This invention of discovaery of E3 ligases regulating PCSK9-mediated LDLR degradation will open completely new avenues for exploring new treatments for cardiovascular and infectious diseases.
EXAMPLE
Cell culture HepG2 cells were obtained from European collection of cel culture(Wiltshire,UK). T-Rex 293 cells were obtained from (Invitrogen, Paisley, UK). Cell were grown in DMEM containing 25mIVl glucos and 10% fetal calf serum, as described27.
DNA constructs, trasfections and western blot analysis C-terminal flag-tagged Wild-type PCSK9, 51 27R-PCSK9 and F2 1 6L-PCSK9 were kindly provided by Jay D. Horton(University of Texas Southwestern Medical Center, Dallas, TX,USA). D374Y mutation was introduced by oligonucleiotide-direct mutagenesis with forward primer 5' -CATTGGTGCCTCCAGCTACTGCAGCACCTGC-3' and reverse primer -GCAGGTGCTGCAGTAGCTGGAGGCACCAATG-3' using QuickChange XL Mutagenesis kit(Stragenen,La Jolla,CA, USA). The intergrity of the construct was confirmed DNA sequencing. C-terminal myc-tagged LDLR, clAP 1, TRAF2 in CMV6-based mammalian expression vector were obtained from Origene, Inc. PCR was used to generate deletion constructs of myc-tagged c-lAP 1, containing only BIR1 or BIR2 or BIR3 or Ring domains.
The PCR products were cloned in frame to the pCMV6 entry clone(Origene, USA) with Sgfl and Mlul restriction sites. The successful creation of all constructs was confirmed by DNA sequencing. HA-tagged ubiquitin plasmid was purchased from Addgene(Cambridge, MA).
All the transfection were done on T-Rex293 cells or HepG2 cells using superfect(Qiagen, UK) with 1-2ug DNA. The antibodies used were a rabbit antibody directed against amino acids 184-196 of human LDLR(Research Diagnostics Inc.) and a rabbit antibody against PCSK9(Cayman). Other antibdies, including c-IAP1, Traf2, PDTA4 and DNJA1 were from Abcam (Cambridge, UK).UGGT-1 antibody is from Santa Cruz Biotechnology, CA, USA).
Anti-Stub 1 antibody and anti-myc tag antibody are from Millipore, UK. All transfections were done with either T-Rex 293 cells and HepG2 cells using Superfect(Qiagen, UK). The methods of whole cell extract and western blots were carried out as described27. Western blot densitometry was carried out using the VisionworksLS software(UVP, Cambridge, UK). All data were analysed by GB-Stat V5.4.4 program(written by Dr. Philip Friedman, Howard University) using student t-Test(two-tailed).
Generation of stable cell lines, immunoprecitation and protein purification(Fig.2).
The PCSK9 expression plasmids were co-transefected with pTK-hygromycin (BD Clontech) into T-Rex 293 cells. 48h later, cells were subjected to selection with 5Otg/mI of hygromycin. The positive clones which over expressed flag-tgged PCSK9 were detected by western blot. Flag-tagged protein immunoprciptation was carried out by using Flag Tagged protein immunoprecipitation kit(FlagPT-1, Sigma) with final elution using 3xflag peptide(final concertation 15Ong/il 3xflag peptide). C-myc tagged protein was immunopreciptaed with Pierce Mammalian c-Myc Tag IP/Co-TP kit(Thermo scientific, UK).
PCSK9 proteins from stable expressed 293 cell lines, including wild-type, D374Y and 5127R PCSK9 were purified by using FLAG-M Purification Kit(Sigma, UK) following the manufacture's instruction. The final elution were concentrated with vivaspin 6 (Artoris Stedim biotech, UK) and dialysis against PBS using Slid-A-lyzer mini dialysis unit(Thermo scientific, UK). Protein concentration was measured using Bio-Rad Protein Assay kit, Cat:500-0006, Bio-Rad. UK). The protein purity was determined by SDS-PAGE and visualized by Coomassie Blue stain with over 90% purity.
Shotgun analysis of the FLAG-tagged PCSK9 and associated proteins complex
samples(Table 1).
A stable expressed flag-tagged PCSK9 cell line was grown in DEME medium with 10% FBS.
The flag-tagged PCSK9 protein was isolated using the flag-immunopereciptation kit(Sigma) following by elution with the Flag-peptide 3xflag peptide(final concertation l5Ongi'ul 3xflag peptide). The elution samples were then send to the Protein Analysis Facility, Center for Integrative Genomics, Faculty of Biology and Medecine, University of Lausanne, Switzerland where protein mixtures were separated by limited electrophoresis after which 3-5 molecular weight regions are cut and digested. Analysis is performed by LC-MS/MS on every fraction.
The resulting collections of spectra are pooled for every sample before database search.
The Lists of identified proteins for each sample with their scores are subjected to statistical validation and aligned for comparison with Scaffold program. A negative control cell line, T-Rex-293 cells transfected with the empty vector pcDNA3. 1 was used for background subtraction.
Surface Plasmon Resonance(Fig.S1) Surface Plasmon Resonance studies on the binding of c-lAP 1 to wild-type PCSK9 were carried out on a Biacore T100, essentially following the manufacturer's recommended conditions (Biacore, Uppsala, Sweden). Briefly, recombinant human c4AP1 protein(R&D, UK) was immobilized to CM5 chips (Biacore) with surface densities of 100 resonance units (RU) with amine-coupling kit (BlAcore) using immobilization wizard. Purified recombinant wild-type PCSK9(isolated from whole cell extract) diluted in the Hepes buffer( Hepes, pH7.4, 150mM NaCI and 0.1 mIVI CaCI2) were injected in a concentration range of 12.5-lOOnM at a flow rate of 30 jil/min. Regeneration buffer is 10mM glycine/HCI(pH2.5). Association and dissociation data from all concentrations were fit globally using Ti 00 evaluation software with 1:1 Langmuir binding model.
RNA interference(Fig.3).
c-IPA1 siRNA mix(pGB-cIAPJ siRNA, Biovision Research Products, CA, USA) or control siRNA (pGB-control) were transfected into a stable wild-type PCSK9 overexpress cell line using the superfect reagent(Qiagene, UK). After 481i, the cells were subjected G148 selection to obtain the stable cell lines with nearly 100% knock-out the c-lAP 1 protein by western blot analysis.
Degradation of the LDLR in the presence of MG132 or Ammonium Chloride(Fig.4b) HepG2 cells were transient transfected with plasmid expressing PCSK9-D374YpcDNA3 or empty vector(pCDNA3.1). 24h after transfection, the fresh DMEM culture medium(without serum) was added for 1 6h. Then cells were treated with 10 iM of proteasome inhibitors MG 132 or the lysosomal inhibitor Ammonium Chloride (NH4CI) for 4h in serum-free DMEM medium. Cells were washed twice in PBS and harvested for western blot analysis as previous described27.
Immunofluorescence Staining and Image Analysis(Fig. S2) PCSK9 overexpressed stable cell were grown to 50% confluence in coverslips in 6-well tissue culture plate, washed twice with PBS, and fixed with 4% paraformaldehyde in PBS for 10 mm. The cells were then washed with PBS and permeabilized in PBS containing 0,1% Triton X-100 and 5% normal horse serum for 30 mm. For detection of flag-tagged PCSK9, the cells were stained with a 1:1000 dilution of anti-FLAG M2 antibody (Sigma) and a 1:500 dilution of anti-mouse Alex 488-conjugated secondary antibody (Invitrogen). For detection of c-lAP 1, the cells were stained with anti-c-TAP 1 rabbit antibody (Abcam, Cambridge, UK) at a 1:500 dilution and a 1:500 dilution of anti-rabbit cy3 secondary antibody (Sigma). After washing three times in PBS, images were captured on a confocal microscope (Leica TCS SP, Germany).
In vivo ubiqutination analysis(Fig.4c) pcDNA.lEmpty vector and plasmid containing HA-tagged ubiquitin gene(HA-ubiqutin, Addgene(Cambridge, MA) were co-transefected with PCSK9 expression vector with or without the pcDNA6 -c-lAP 1 plasmids into T-REX 293 cells. 24h later, the lysates were immunopreciptation with anti-ubiquitin anibody(Ubiquitin enrichment kit, Thermo Fisher) analyzed by Western blotting with anti-PCSK9 antibody.
In vitro Ubiquitanation Assay(Fig.4d and Fig.4f)) Purified Flag-tagged wild-type PCSK9(1-4ig), or D374Y-PCSK9(1 jig) or 5127R-PCSK9(1- 4ig) and recombinant c-IAP1(1ig, R&D system, UK) were incubated in a reaction buffer(5OmM Tris-HC1(pH7.5), 5mM MgC12, 2mM ATP, 0.6mM DTT) with recombinant rabbit E1(lOOng), UbcH5b(250ng) and ubiquitin(6ng) at 37°C for 2hr. The resulting mixtures were analyzed by immunoblotting using anti-PCSK9 antibody.
In vitro ubiquitination function assay on the purifed PCSK9 proteins(Fig.4e and Fig.4g) HepG2 cells were seeded in 6 well tissue plates at concentration of 2x 1 cells/ml. After 24h, the medium was replaced with DMEM medium without FBS and added the purified D374Y protein(1ig/ml) or wild-type PCSK9 (4tg/ml). In the control experiments, only c-lAP 1 ubiquitination buffer was added in tomedium without PCSK9 protein. After 16h, cells was washed twice in PBS and harvested for western blot analysis as previous described27.
LDL uptake assay by flow cytometry(Fig.4h) HepG2 cells were seeded in 6 well tissue plates at concentration 2x105 cells/mi. After 24h, the medium was replaced with DMEM medium without FBS and purified D374Y(1tg/ml PCSK9=13.4 nM) or 1-4tg/ml purified wild-type PCSK9. After 16h, the medium was replaced with fresh medium containing 1 Ojig/ml Bopipy FL LDL(Tnvitrogen, UK). The cells were incubated for 4 h at 37°C. The cells were washed with PBS and trypsinized and resuspended in FAC Flow solution. At lease 10,000 cells were analyzed on a FACSCalibur (BD Biosciences. UK) using Cellquest and FlowJo software.
References 1.Abifadel, M., ci' at. Mutations in PCSK9 cause autosomal dominant hypercholesterolemia.
Nat. Genet. 34:154-156(2003).
2. Maxwell, K.N. and Breslow, J.L. Proprotein convertase subtilisin kexin 9: the third locus implicated in autosomal dominant hypercholesterolemia. Curr. Op/n. Lip/dot. 16, 167-172(2005).
3. Cohen, J.C. et at. Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N. Engt. J. Med. 354, 1264-1272(2006).
4. Brown, M.S. and Goldstein, J.L. Lowering LDL -not only how low, but how long? Science 311, 172(2006).
5. Park, S.W. ci' at. Post-transcriptional regulation of low density lipoprotein receptor protein by proprotein convertase subtilisinlkexin type 9a in mouse liver. J. Biol. Chem. 279, 50630- 50638(2004).
6. Horton, J.D., Cohen, J.C. and Hobbs, H.H. Molecular biology of PCSK9: its role in LDL metabolism, Trends Biochem. Sd. 32, 71-77(2007).
7. Maxwell, K.N. and Breslow, J.L. Adenoviral-mediated expression of Pcsk9 in mice results in a low-density lipoprotein receptor knockout phenotype. Proc. Natt. Acad. Sci. USA.
101,7100-7105(2004).
8. Kwon, H.J., Lagace, T.A., McNuft, M.C., Horton, J.D., Deisenhofer, J. Molecular basis for LDL receptor recognition by PCSK9. Proc NattAcadSci USA. 105,1820-1825(2008).
9. Poirier, S. et at. The Proprotein Convertase PCSK9 Induces the Degradation of Low Density Lipoprotein Receptor (LDLR) and Its Closest Family Members VLDLR and ApoER28. J. Riot. Chem. 283,2363-2372(2008).
10. Li, X., Yang,L., Ashwell, J. D. TNF-R1I and c-lAP 1 mediate ubiquitination and degradation of TRAF2 Nature 416, 345-347(2002).
11. Wu, C.J., Conze,D.B., Li, X., Ying, S.,Hanover,J.A.and Ashwell,J.D.TNF-a induced c-lAP 1/TRAF2 complex translocation to a Ubc6-containing compartment and TRAF2 ubiquitination. EMBO 24, 1886-1898(2005).
12. Naoumova, R.P. ci' a!. Severe hypercholesterolemia in four British families with the D374Y mutation in the PCSK9 gene. Arterioscier Thromb Vasc Biol.25, 2654-60(2005).
13. Liu, H., Sadygov,R.G. and Yates, J, R. A Model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anat. Chem. 76, 4193-4201(2004).
14. Xu, L. et al., c-lAP 1 cooperates with myc by acting as a ubiquitin ligase for Madi Molecular Cell, 28, 9 14-922(2007).
15.Ballinger, C.A. et al., Identification of CHIP, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions.
Mo! Cell Blot. 19, 4535-45(1999).
16. Marschang, P. et al Normal development and fertility of knockout mice lacking the tumor suppressor gene LRP1b suggest functional compensation by LRP1. Mo! Cell Biol. 24, 3782- 3793(2004).
1 7.Linnik, K M. & Herscovitz, H "Multiple molecular chaperones interact with apolipoprotein B during its maturation. J. Biol.Chein. 273, 21368-21373(2008).
18.Fisher, E.A. et a!, The Degradation of Apolipoprotein B100 Is Mediated by the Ubiquitin- proteasome Pathway and Involves Heat Shock Protein 70 J. Biol. Chem. 272: 20427- 20434(1997).
19. Fasano, T., Sun, X.M., Patel, D.D., Soutar, A.K. Degradation of LDLR protein mediated by gain of function' PCSK9 mutants in normal and ARH cells. Atherosclerosis.
203, 166-171(2009).
20. Santoro,M,M., Samuel,T., Mitchell,T., Reed,J.C& Stainier,D.I.R. Birc2 (clapi) regulates endothelial cell integrity and blood vessel homeostasis. Nat Genet 39, 1397 -1402 (2007).
21. Gyrd-Hansen, M. et al., lAPs contain an evolutionarily conserved ubiquitin-binding domain that regulates NF-KB as well as cell survival and oncogenesis. Nat Cell Biol 10, 1309 - 1317 (2008) 22. Mbikay,M., Mayne,J., Seidah,N.G., Chrétien, M. Of PCSK9, cholesterol homeostasis and parasitic infections: Possible survival benefits of loss-of-function PCSK9 genetic polymorphisms. European Journa! of Cardiovascular Prevention & Rehabilitation, 69, 10 10- 1017(2007).
23. Maxwell, K.N, Fisher, E.A., Breslow, J.L. Overexpression of PCSK9 accelerates the degradation of the LDLR in a post-endoplasmic reticulum compartment. Proc Nail Acad Sd U SA 102,2069-2074 (2005).
24.Zelcer,N., Hong, C., Boyadjian, R., Tontonoz, P. LXR regulates cholesterol uptake through idol-dependent ubiquitination of the LDL receptor. Science 325, 100-104 (2009).
25.Vince J.E., et al. TWEAK-FN14 signaling induces lysosomal degradation of a cIAP1-TRAF2 complex to sensitize tumor cells to TNFct. JCellBiol. 182, 171-184(2008).
26.Abdur Rub A., et al., Cholesterol depletion associated with Leishinania major infection alters macrophage CD4O signalosome composition and effector function. Nature Immunology 10, 273 -280 (2009).
27.Xu, W., Liu, L., Charles, I.G. & Moncada S. Nitric oxide induces coupling of mitochondrial signalling with the endoplasmic reticulum stress response. Nat Cell Biol 6, 1129-1134 (2004)
Filename: Description.doc
Directory: C:\Documents and Settings\rmgzwgLWlBRDOM\Desktop\pcsk9 patent Template: C:\Documents and Settings\rmgzwgiWIBRDOM\Application Data\Microsoft\Templates\Normal.dot Title: New treatment of hypercholesterolaemia by ubiquitination of PCSK9 and modulating low density lipoprotein degradation Subject: Author: Weiming Xu Keywords: Comments: Creation Date: 6/19/2010 12:30:00 PM Change Number: 10 Last Saved On: 6/19/2010 1:26:00 PM Last Saved By: rmgzwgi Total Editing Time: 54 Minutes Last Printed On: 6/19/2010 1:36:00 PM As of Last Complete Printing Number of Pages: 12 Number of Words: 6,786 (approx.) Number of Characters: 38,685 (approx.)
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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000032787A1 (en) * 1998-12-01 2000-06-08 The University Of Leeds Therapeutic agents comprising an e3 ubiquitin ligase for use in degenerative disorders
US20030143579A1 (en) * 1995-08-08 2003-07-31 Tularik, Inc. Inhibitors of apoptosis
WO2008043753A2 (en) * 2006-10-09 2008-04-17 Santaris Pharma A/S Rna antagonist compounds for the modulation of pcsk9
WO2008063957A2 (en) * 2006-11-10 2008-05-29 The Board Of Trustees Of The University Of Illinois Cblb for treating endotoxin-mediated disorders
WO2008066776A2 (en) * 2006-11-27 2008-06-05 Isis Pharmaceuticals, Inc. Methods for treating hypercholesterolemia
WO2008125623A2 (en) * 2007-04-13 2008-10-23 Novartis Ag Molecules and methods for modulating proprotein convertase subtilisin/kexin type 9 (pcsk9)
WO2010029513A2 (en) * 2008-09-12 2010-03-18 Rinat Neuroscience Corporation Pcsk9 antagonists

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030143579A1 (en) * 1995-08-08 2003-07-31 Tularik, Inc. Inhibitors of apoptosis
WO2000032787A1 (en) * 1998-12-01 2000-06-08 The University Of Leeds Therapeutic agents comprising an e3 ubiquitin ligase for use in degenerative disorders
WO2008043753A2 (en) * 2006-10-09 2008-04-17 Santaris Pharma A/S Rna antagonist compounds for the modulation of pcsk9
WO2008063957A2 (en) * 2006-11-10 2008-05-29 The Board Of Trustees Of The University Of Illinois Cblb for treating endotoxin-mediated disorders
WO2008066776A2 (en) * 2006-11-27 2008-06-05 Isis Pharmaceuticals, Inc. Methods for treating hypercholesterolemia
WO2008125623A2 (en) * 2007-04-13 2008-10-23 Novartis Ag Molecules and methods for modulating proprotein convertase subtilisin/kexin type 9 (pcsk9)
WO2010029513A2 (en) * 2008-09-12 2010-03-18 Rinat Neuroscience Corporation Pcsk9 antagonists

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
Clinical chemistry, Vol. 55, epub Oct 2009, T Sawamura, "New Idol for cholesterol reduction?", pages 2082-2084. *
Future Cardiology, Vol. 4, No. 1, 2008, M Willis, et al., "Appetite for destruction: E3 ubiquitin-ligase protection in cardiac disease", pages 65-75. *
Nature Genetics, Vol. 39, 2007, M Santoro, et al., "Birc2 (cIap1) regulates endothelial cell integrity and blood vessel homeostasis", pages 1397-1402. *
Recent Pat DNA Gene Seq., Vol. 3, Nov. 2009, H Li, et al., "Recent patents on PCSK9: a new target for treating hypercholesterolemia", pages 201-212. *

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