WO2017178838A1 - Binding modulation - Google Patents

Abstract

The present disclosure provides assay methods for identifying agents which modulate binding between DHHC-PAT molecules and their protein binding partners, in particular those binding partners that bind the N- or C-terminal DHHC-PAT tail portion. Further the disclosure provides methods and uses which exploit certain DHHC-PAT/protein binding modulators with the aim of modulating downstream S-acylation (including palmitoylation) events. The disclosure also relates to uses of such modulator molecules and methods and uses for treating diseases and/or conditions associated with protein palmitoylation.

Description

BINDING MODULATION

FIELD OF THE INVENTION

The present disclosure provides assay methods for identifying agents which modulate the binding of substrate proteins to DHHC-PAT molecules, uses of such modulates and methods and uses for treating diseases and/or conditions associated with protein palmitoylation.

BACKGROUND OF THE INVENTION

The post-translational modification protein S-acylation plays a critical role in regulating a wide- range of biological processes including cell growth, cardiac contractility, synaptic plasticity, endocytosis, vesicle trafficking, membrane transport and biased-receptor signalling. As a consequence, DHHC-protein acyl transferases (DHHC-PATs), the enzymes that catalyse the addition of fatty acid groups to specific cysteine residues in target proteins, are important pharmaceutical targets.

At present, there are no therapeutic drugs used in the clinic that act by changing the palmitoylation status of specific target proteins. Existing efforts to discover inhibitors of DHHC- PATs have focussed exclusively on the enzyme's active site. Even if isoform-specific active site inhibitors can be identified they will not be useful in the clinic, as they will block palmitoylation of the entire substrate ensemble of the DHHC-PAT(s) where they bind not just the protein of interest.

SUMMARY OF THE INVENTION

The present disclosure is based on the finding that protein palmitoylation occurs as a consequence of the binding of substrate proteins to either the N- or C-terminal tail regions of DHHC-protein acyl transferases (DHHC-PATs). Specifically binding occurs between the DHHC- PAT C-terminal tail binding region of a protein and the DHHC-PAT C-terminal tail itself.

Disclosed herein are methods which can be used to alter or modulate the palmitoylation status of some specific proteins. These methods involve modulating (for example preventing or inhibiting) the recruitment of those specific proteins to the C-terminal N- or C-terminal tail regions of a DHHC-PAT molecule.

Thus, disclosed herein is a method (for example an in vitro method) of modulating the palmitoylation status of a protein, said method comprising modulating the recruitment of the protein to the N- or C-terminal tail region of a DHHC-PAT molecule.

Within the context of this disclosure, proteins which bind to or associate with the N- or C-terminal

l tail region of DHHC-PAT molecules, the palmitoylation status of which can be modulated via any of the methods disclosed herein, may be referred to as "DHHC-PAT binding partners". Thus the methods of altering protein palmitoylation status may focus on modulating the interaction between DHHC-PAT molecules and their (proteinaceous) DHHC-PAT binding partners.

An advantage associated with the methods (and subsequent or related uses, compounds, and/or compositions) is that general, or prior art, inhibitors of the DHHC-PAT active site (e.g. DHHC isoform specific active site inhibitors) have little clinical utility as they tend to block the total palmitoylation capability of the molecule (in other words they block palmitoylation of the entire substrate ensemble of the DHHC-PAT).

Thus, the palmitoylation status of a protein may be modulated by using a modulation molecule that either enhances or prevents recruitment of protein substrates to the N- or C-terminal tail regions of a DHHC-PAT molecule. A useful modulation molecule may bind to a specific region of either the N- or C-terminal tail of a DHHC-PAT molecule (or to some part (ideally the C-tail DHHC-PAT binding part) of the protein that is palmitoylated by a DHHC molecule), enhancing or blocking recruitment of a protein binding partner (i.e. a protein to be palmitoylated). Alternatively, a useful modulation molecule may bind to or have affinity and/or specificity for, a region or portion of the DHHC-PAT binding partner that is involved in or associated with binding to the N- or C-terminal tail region of a DHHC-PAT molecule, either enhancing or preventing interaction between the substrate protein and the DHHC-PAT

A modulation molecule may take any form and may comprise, consist essentially of or consist of a protein, a peptide, a carbohydrate, a nucleic acid (synthetic or natural PNA, DNA and/or RNA), an oligonucleotide (including siRNA, antisense oligonucleotides and the like), small molecules, aptamers and antibodies (including monoclonal antibodies, polyclonal antibodies and/or antigen binding fragments thereof).

Thus, the disclosure relates to a method of modulating the palmitoylation status of a protein, said method comprising modulating the recruitment of the protein (that is to be palmitoylated) to the N- or C-terminal tail region of a DHHC-PAT molecule, wherein recruitment of the protein to the N- or C-terminal tail region of a DHHC-PAT molecule is modulated by:

(i) a molecule that binds to the N- or C-terminal tail portion of a DHHC-PAT molecule; and/or

(ii) a molecule that binds to the DHHC-PAT C-terminal tail binding part of a protein that is palmitoylated by the DHHC-PAT; and/or (iii) a molecule that binds to or has affinity and/or specificity for, a region or portion of the DHHC-PAT C-terminal tail which is involved in or associated with binding to a protein to be palmitoylated; and/or

(iv) a molecule that binds to or has affinity and/or specificity for, a region or portion of the DHHC-PAT binding partner that is involved in or associated with binding to the C-terminal tail region of a DHHC-PAT molecule.

Modulation molecules of the type described herein may have clinical and/or therapeutic utility. For example, a modulation molecule may be used in the treatment or prevention of a disease or condition which is associated with or linked to protein palmitoylation via DHHC-PAT molecules. One of skill will appreciate that a molecule which binds to the C-terminal tail portion of a DHHC- PAT molecule may be used to block the binding of a protein to the C-terminal tail portion of the DHHC-PAT molecule. Conversely a molecule which binds to the C-terminal tail binding portion of a protein may also neutralise any binding event between the protein and the C-terminal tail portion of the DHHC protein.

By way of example, and without wishing to be bound or limited by theory, it is suggested that G- coupled proteins, ion channels, transporters, ion transport regulators, receptors and protein kinases are all subject to S-acylation (including palmitoylation) via DHHC-PAT molecules. Thus, diseases and/or conditions which may be treated and/or prevented by any of the modulation molecules described herein may include diseases and/or conditions associated with or linked to S-acylation and/or palmitoylation events in any of these specific proteins and/or systems.

For example diseases and/or conditions which can be treated and/or prevented with any of the modulation molecules described herein may include those that are associated with or caused or contributed to by, aberrant (increased or decreased) protein S-acylation events (including palmitoylation). Further (and again without wishing to be bound by theory) since S-acylation (including palmitoylation) is associated with the regulation of a wide variety of biological processes (including, for example, cell growth, cardiac contractility, synaptic plasticity, endocytosis, vesicle trafficking, membrane transport and biased receptor signalling), the diseases and/or conditions that may be treated or prevented using the modulation molecules described herein may relate to any one or more of these biological processes.

Thus this disclosure provides methods of treating or preventing diseases and/or conditions:

(i) associated with or linked to protein palmitoylation via DHHC-PAT molecules; and/or

(ii) associated with or caused or contributed to by, aberrant protein S-acylation and/or palmitoylation events; said method comprising administering to a subject in need thereof, one or more of the modulation molecules described herein.

Further, the disclosure relates to the use of any one or more of the modulation molecules described herein for use in treating or preventing diseases and/or conditions:

(i) associated with or linked to protein palmitoylation via DHHC-PAT molecules; and/or

(ii) associated with or caused or contributed to by, aberrant protein S-acylation and/or palmitoylation events.

For the avoidance of doubt, a modulation molecule which may be used in a method of treating a subject in need thereof may be a modulation molecule which binds to the (DHHC-PAT binding portion) of a protein which is palmitoylated by a DHHC-PAT or which binds to the protein binding region of the C-terminal tail region of the DHHC-PAT molecule.

A "subject in need thereof" is any subject suffering from, suspected of suffering from or susceptible or predisposed to any of the diseases and/or conditions outlined in this disclosure. Specific diseases treatable or preventable using any of the molecules described herein may include, for example, cancer, diabetes, heart failure, metabolic syndrome, neuropathic pain, Parkinson's disease, neurodegeneration, renal tubular acidosis, neuromyelitis optica, stroke, Huntington Disease, ischaemic damage, epilepsy and amyotrophic lateral sclerosis.

As an example, a modulation molecule may be used to modulate the interaction between the DHHC5-PAT and its C-tail binding partners. One such binding partner is the sodium pump accessory protein phospholemman (PLM) which is recruited to the intracellular C-tail portion of the DHHC5-PAT molecule. Once recruited, it is acylated (or palmitoylated) and this inhibits the sodium pump. Sodium pump activators are sought after as they are of clinical benefit to those patients that have cardiac disease, heart failure and/or have (or are predisposed to) myocardial infarction.

Thus PLM is palmitoylated by DHHC5-PAT and that process involves a ca. 120 amino acid region of the DHHC5-PAT intracellular C-terminal tail (located immediately after its fourth transmembrane domain). By interference with binding between the PLM protein and this 120 amino acid region of the DHHC5-PAT C-terminal tail region, it is possible to modulate (for example reduce) PLM palmitoylation. A reduction in PLM palmitoylation increases sodium pump activity and this is of therapeutic benefit.

Thus a method of modulating PLM palmitoylation may comprise contacting PLM and DHHC5- PAT in the presence of a modulation molecule and under conditions which permit binding between the modulation molecule and the PLM and/or DHHC5-PAT, wherein binding of the modulation molecule to either the PLM and/or the DHHC5-PAT, modulates PLM palmitoylation. Useful modulation molecules may include those derived from or comprising fragments or portions of the DHHC-PAT C-terminal tail. For example, the modulation molecule may comprise the full length C-terminal tail region of any DHHC-PAT molecule or a fragment thereof. Useful fragments may comprise n-1 amino acids from the C-tail of the DHHC-PAT protein, wherein "n" is the total number of amino acids of the C-terminal tail region.

A useful modulation molecule may comprise a fragment of the intracellular C-tail region of a DHHC5-PAT protein, wherein the fragment comprises a region located immediately after the 4^th transmembrane domain region of the DHHC5-PAT protein.

Modulation molecules useful in the treatment and/or prevention of some of the diseases listed herein (including but not limited to cardiac events (including myocardial infarction or heart failure), diabetes, metabolic syndrome, neuropathic pain, Parkinson's disease, neurodegeneration, renal tubular acidosis and neuromyelitis optica) may include those which comprise a fragment derived or obtained from the C-terminal tail region spanning amino acids 213-677 of DHHC5-PAT. In this case a useful fragment may comprise n-1 consecutive amino acids from the C-terminal tail region spanning amino acids 213-677 (wherein n is the total number of amino acids present across residues 213-677). For example, a useful fragment may comprise, consist essentially of or consist of amino acids 213-267, 223-247, 233-257 and 243- 267 from the C-terminal tail region of the DHHC5-PAT. Modulation molecules which comprise these peptides may be useful in the modulation of the palmitoylation of phospholemman and a range of other proteins (see for example Figure 5 and Table 1 below).

A peptide comprising amino acids 223-247 may have the amino acid sequence (SEQ ID NO: 1):

GKFRGGVNPFTNGCCNNVSRVLCSS

A peptide comprising amino acids 233-257 may have the amino acid sequence (SEQ ID NO: 2):

TNGCCNNVSRVLCSSPAPRYLGRPK

A peptide comprising amino acids 243-267 may have the amino acid sequence (SEQ ID NO: 3):

VLCSSPAPRYLGRPKKEKTIVIRPP

Useful fragments may be obtained from other parts of the DHHC5-PAT C-terminal tail including from example regions 463-487, 523-557 and 653-677. There are various uses for modulation molecules comprising these peptides including, for example modulation of the palmitoylation of those proteins identified in Figure 5 (Synaptotagmin-7, Band 3 Anion transport protein, Aquaporin 4 and the like) and Table 1. Hereinafter these particular modulation molecules may be referred to as "DHHC5-PAT based modulation molecules".

As a further example, a modulation molecule may be used to modulate the interaction between the DHHC9-PAT and its various C-tail binding partners. One such binding partner is H-ras (a small G protein) which is recruited to the intracellular C-tail portion of the DHHC9-PAT molecule. Other DHHC9-PAT binding partners include Excitatory amino acid transporter 1 and Glutamate receptor 1. The use of molecules which modulate binding between these partners and the DHHC9-PAT protein can increase and/or decrease (block or enhance) partner recruitment to the DHHC9-PAT tail region and subsequent acylation (or palmitoylation) processes. Molecules which modulate binding between H-ras, Excitatory amino acid transporter 1 (EAT1) and Glutamate receptor 1 (GR1) may have clinical benefit to those patients that have or are susceptible/predisposed to cancer, stoke, Huntington Disease, Ischaemic damage, Epilepsy and Amyotrophic Lateral Sclerosis.

H-ras, EAT1 and GR1 are palmitoylated by DHHC9-PAT and that process involves amino acids within the region of the DHHC9-PAT intracellular C-terminal tail (again located immediately after its fourth transmembrane domain). By interference with binding between the H-ras, EAT1 and/or GR1 proteins and the relevant amino acid region of the DHHC9-PAT C-terminal tail region, it is possible to modulate (for example reduce or increase) H-ras, EAT1 and/or GR1 palmitoylation.

Thus a method of modulating H-ras, EAT1 and/or GR1 palmitoylation may comprise contacting H-ras, EAT1 and/or GR1 and DHHC9-PAT in the presence of a modulation molecule and under conditions which permit binding between the modulation molecule and the H-ras, EAT1 and/or GR1 proteins and/or the modulation molecule and DHHC9-PAT, wherein binding of the modulation molecule to H-ras, EAT1 and/or GR1 and/or DHHC9-PAT, modulates H-ras, EAT1 and/or GR1 palmitoylation.

A useful modulation molecule may comprise a fragment of the intracellular C-tail region of a DHHC9-PAT protein, wherein the fragment comprises a region located immediately after the 4th transmembrane domain region of the DHHC9-PAT protein.

Modulation molecules useful in the treatment and/or prevention of diseases and/or conditions may include those which comprise a fragment derived or obtained from the C-terminal tail region spanning amino acids 270-324 of DHHC9-PAT. In this case n = 55 amino acids and a useful fragment may comprise n-1 consecutive amino acids from the C-terminal tail region spanning amino acids 270-324. For example, a useful fragment may comprise, consist essentially of or consist of amino acids 270-314, 290-294, 270-294, 280-304, 290-314 or 300-324 from the C- terminal tail region of the DHHC9-PAT. Modulation molecules which comprise these peptides may be useful in the modulation of H-ras, EAT1 and/or GR1 palmitoylation and the treatment and/or prevention of cancer (in the case of H-ras palmitoylation modulation), Huntington Disease and Stroke (in the case of EAT1 palmitoylation modulation) and Ischaemic damage, epilepsy and amyotrophic lateral sclerosis (in the case of GR1 palmitoylation modulation) (see for example Figure 6 and Table 1 below).

A peptide comprising amino acids 270-294 may have the amino acid sequence (SEQ ID NO: 4):

VQNPYSHGNIVK CCEVLCGPLPPS

A peptide comprising amino acids 280-304 may have the amino acid sequence (SEQ ID NO: 5):

VK CCEVLCGPLPPSVLDRRGILPL

A peptide comprising amino acids 290-314 may have the amino acid sequence (SEQ ID NO: 6):

PLPPSVLDRRGILPLEESGSRPPST

Useful fragments may be obtained from other parts of the DHHC9-PAT C-terminal tail and any of the assay methods described herein may be used to identify and obtain such fragments.

Hereinafter these particular modulation molecules may be referred to as "DHHC9-PAT based modulation molecules".

Table 1

Table 1 shows that depending on the DHHC fragment used, one can achieve variable modulation effects. For example using a fragment from DHHC5-PAT (comprising amino acids 213-267) can enhance Glucose transporter 4 recruitment to the DHHC5-PAT tail (and hence enhance palmitoylation) whereas the same fragment might inhibit or block PLM recruitment.

The DHHC5-PAT based modulation molecules may find application in any of the methods of treatment or prevention outlined herein or may be for use in the treatment or prevention of any of the described diseases and/or conditions. In particular, the DHHC5-PAT based modulation molecules (especially those comprising peptides derived from the region spanning amino acids 213-267 of the C-terminal tail region of DHHC5) in the treatment (prophylaxis) and/or prevention of heart failure, myocardial infarction and other types or forms of cardiac disease. As stated, the DHHC5-PAT based modulation molecules may prevent PLM palmitoylation and thus increase sodium pump activity.

As stated, useful modulation molecules may be obtained or derived from the region of the DHHC-PAT binding partner protein that binds to the C-terminal tail of the DHHC-PAT molecule. For example, a fragment of the PLM protein may be used to prevent binding between PLM and the DHHC5-PAT C-terminal tail. For example a protein or peptide comprising, consisting essentially of or consisting of amino acids 37-72 of the PLM protein may be used. Any fragment of this protein or peptide (a fragment being a peptide comprising n-1 amino acids from the PLM protein where n=36 (i.e. the amino acids from residues 37-72)) may be useful provided it has the ability to bind the DHHC5-PAT C-terminal tail and prevent PLM palmitoylation.

Similarly, fragments of for all other DHHC-PAT binding partner proteins may be used to bring about modulation of palmitoylation events.

A modulation molecule according to this disclosure is a molecule which can be used to modulate (for example prevent) the binding of a protein to the C-terminal tail region of a DHHC-PAT molecule and the subsequent protein palmitoylation event. As such, the disclosure further provides an assay method for determining whether or not a test agent is a modulation molecule, said method comprising:

contacting a test agent with a peptide comprising a protein or peptide sequence from a DHHC-PAT C-terminal tail; and

identifying those test agents that interact or associate with or bind to the DHHC-PAT peptide, wherein those test agents that interact or associate with or bind to the DHHC-PAT C- terminal tail protein/peptide are potential modulation molecules. The test agent may comprise a peptide library may comprise peptides derived from a DHHC-PAT C-terminal tail and/or peptides derived from a protein known to bind to or interact with a DHHC- PAT molecule. For example a method for identifying molecules which might modulate protein palmitoylation via a DHHC5-PAT protein may use a library based on peptides derived from the DHHC5-PAT C-terminal tail and/or peptides derived from proteins which are known to bind the DHHC5-PAT C-terminal tail; for example peptides derived from PLM.

A useful library may comprise a series of 20-30 mer peptides (for example 25 mer peptides) all derived from a DHHC-PAT protein or a protein binding partner therefor. The peptides of the library may overlap in sequence. For example they may overlap by anywhere between about 5 and 20 amino acids, for example 10 or 15 amino acids. The peptide library may comprise (optionally overlapping) peptides that cover or span the entire DHHC-PAT C-terminal tail region (in the case of DHHC5-PAT, this would be from residues 213 to 715).

The peptide library may then be contacted and/or incubated with, the protein or peptide sequence from a DHHC-PAT C-terminal tail.

In some cases the protein or peptide sequence from a DHHC-PAT C-terminal tail may be provided in the form of a cell extract, which cell extract comprises the DHHC-PAT protein. For example if the method is intended to identify molecules that might bind to DHHC5-PAT, the protein or peptide sequence from a DHHC-PAT C-terminal tail may comprise a cardiac cell lysate.

The proteins or peptides of the library may be tagged or labelled. For example the proteins or peptides may be biotin tagged or labelled.

The library may be contacted/incubated together with the protein or peptide sequence from a DHHC-PAT C-terminal tail for a time (for example about 1 hour) and under conditions, suitable to permit binding between a peptides/proteins of the library and the protein or peptide sequence from a DHHC-PAT C-terminal tail.

Library peptides that have not bound to the protein or peptide sequence from a DHHC-PAT C- terminal tail may be removed by washing.

After any washing steps, bound library peptides (i.e. those that have interacted with a protein or peptide sequence from a DHHC-PAT C-terminal tail) may be isolated or obtained by affinity chromatography. For example, depending on the nature of the tag or label applied to the peptides or proteins of the library, an agent capable of binding the tag or label may be used. For example biotin labelled peptides may be obtained or purified using a streptavidin based system - for example a streptavidin coated bead.

Isolated test agent/DHHC-PAT C-terminal tail complexes may be further analysed by, for example PAGE and/or mass spectrometry based techniques in order to determine the identity and features (for example sequence) of the test agent.

A further variant of a method for determining whether or not a test agent is a modulation molecule may involve contacting or incubating a DHHC-PAT C-terminal tail peptide with a protein known to bind the same, in the presence of a test agent. If the test agent is a modulation molecule it will modulate (perhaps prevent or increase) binding between the DHHC-PAT C- terminal tail peptide and the protein capable of binding the same. Such a method may, for example exploit any of the DHHC-PAT/protein binding pairs described herein. For example a method may exploit a peptide derived from the C-terminal tail portion of DHHC5-PAT and a peptide derived from PLM.

A further assay method provided by this disclosure is a method of reported element based method which can also be used to identify molecules that modulate binding between proteins and DHHC-PAT molecules - in particular binding between certain proteins and the C-terminal tail region of the DHHC-PAT molecule. The method may involve a reporter element which is split into two parts (for example report unit A and reporter unit B). Individually, neither unit is capable of acting as a functional reporter element, however when the two reporter units are brought together, a functional reporter element is generated that is capable of reporting some form of signal. This technology may be applied to the methods described herein where a DHHC-PAT peptide is conjugated, bound or joined to one reporter unit (unit A) and a protein known to bind the same is conjugated, bound or joined to the other reporter unit (unit B). A functional reporter element is generated when the two units are brought together and reconstituted through interaction between the DHHC-PAT peptide and the protein capable of binding the same.

The protein capable of binding a DHHC-PAT protein may comprise a fragment or portion of a protein which is known to be recruited to (or which binds) the C-terminal tail portion of a DHHC- PAT molecule. For example, where the DHHC-PAT peptide is a peptide derived from the C- terminal tail portion of the DHHC5-PAT molecule, the peptide derived from a protein capable of binding the same may be a protein (or peptide) derived from the PLM molecule (the PLM molecule being known to bind to the C-terminal tail portion of the DHHC5-PAT molecule). The (reporter unit A-tagged) DHHC-PAT protein/peptide and the (reporter unit B tagged) protein/peptide capable of binding the same may be contacted and/or incubated in the presence of a test agent. The incubation step may be conducted under conditions which might permit binding between the DHHC-PAT protein/peptide and the protein/peptide capable of binding the same. In use, if the assay system generates a signal from the reporter element then reporter units A and B must have been brought together or reconstituted via binding between the DHHC- PAT protein/peptide and the protein/peptide capable of binding the same. Under such circumstances one may conclude that the test agent is not a molecule which modulates binding between the DHHC-PAT protein/peptide and the protein/peptide capable of binding the same. On the other hand, if the assay reports a greater than expected signal, one might conclude that the test agent has somehow positively enhanced binding between the DHHC-PAT protein/peptide and the protein/peptide capable of binding the same.

The assay may not report a signal in which case on might conclude that the test agent has prevented binding between the DHHC-PAT protein/peptide and the protein/peptide capable of binding the same. Under such circumstances the test agent may be identified as a potential modulation molecule.

Thus the invention provides an assay method for identifying test agents which modulate binding between proteins and DHHC molecules, said method comprising:

providing a split reporter element;

providing a DHHC-PAT peptide or protein tagged, fused or conjugated to/with one part of the split reporter element;

providing a DHHC-binding partner tagged, fused or conjugated to/with the other part of the split reporter element;

contacting or incubating the DHHC-PAT peptide or protein with the DHHC-binding partner in the presence of a test agent and under conditions which permit binding between the DHHC-PAT peptide/protein and the DHHC-binding partner, wherein:

(i) detection of a signal from the reporter element indicates that the test agent has not prevented binding between the DHHC-PAT and the DHHC binding partner;

(ii) detection of an enhanced signal from the reporter element indicates that the test agent has increased binding between the DHHC-PAT and the DHHC binding partner; and

(iii) failure to detect a signal from the reporter element indicates that the test agent has prevented binding between the DHHC-PAT and the DHHC binding partner.

Modulation molecules identified by any of the methods described herein may find application as molecules potentially useful in the treatment of any of the diseases described herein. Thus the disclosure relates to methods of treating or preventing one or more of the diseases described herein, said methods comprising administering one or more modulation molecules identified by an assay method of this disclosure.

The various molecules described herein may be provided in the form of a composition comprising a modulation molecule of this disclosure and some form of excipient, carrier or diluent. For example the modulation molecules may be provided as pharmaceutical compositions comprising pharmaceutically acceptable excipients. The compositions may be prepared for topical, parenteral and/or oral administration. The modulation molecules may be provided in controlled release or delayed release form and/or encapsulated.

The disclosure further provides kit which provide components necessary to conduct any of the methods (or assay methods) described herein. For example the kits may comprise DHHC-PAT peptides, peptides derived from DHHC-PAT binding partners (for example PLM and/or any of the other partners identified herein). Some kits may comprise DHHC-PAT or DHHC-PAT binding partner peptides conjugated to one or other part of a split reporter element. The kits may provide instructions for use and receptacles in which incubations are performed.

DETAILED DESCRIPTION

The present disclosure will now be described in detail with reference to the following figures which show:

Figure 1 : Potential approaches to blocking the palmitoylation of a target protein with small molecules (A) active site inhibition: current efforts to identify inhibitors of protein palmitoylation have focussed on the enzyme active site. However, even if DHHC-PAT specific molecules can be identified they are unlikely to have any clinical utility, as they will block palmitoylation of all the enzyme's substrates not just the target protein. (B) preventing substrate recruitment: here, we propose a novel strategy for modulating protein palmitoylation of specific proteins through identifying molecules that prevent their recruitment to (a) partner DHHC-PAT isoform(s). This approach takes advantage of the natural diversity of the DHHC-PAT N- and C-termini, which will facilitate selective intervention.

Figure 2: Schematic diagram showing an unbiased medium— throughput approach for identifying DHHC5 binding partners. The complete ensemble of interacting proteins (both substrates and regulators) that bind to the disordered C-terminal region of DHHC5 (αα 213-715) in the heart can be determined using a peptide library in combination with tandem mass spectrometry.

Figure 3: SDS-PAGE analysis of all purifications from cardiac muscle using a biotinylated DHHC5 peptide library. Lysates were prepared by solubilising homogenised cardiac tissue with (A) C12E10, (B) CHAPS or (C) DDM/CHS. The lysates were individually incubated with all of the DHHC5 peptides before interacting proteins were purified using streptavidin beads. All pull- downs were analysed by SDS-PAGE. Those purification reactions containing protein (denoted by red box) were analysed by tandem mass spectrometry. Figure 4: Identifying potential DHHC5 substrates and regulators from the LC-MS/MS data.

All DHHC5 peptide pull-downs containing protein were analysed by tandem mass spectrometry. The data sets obtained were filtered to remove those proteins present in a 'no peptide' control, as well as those found in three or more purification fractions. Specific binding partners were then further filtered into potential DHHC5 substrates (proteins annotated as being palmitoylated in Uniprot and/or being found in either locally-determined palmitoyl proteomes or those deposited in the SwissPalm database) and potential DHHC5 Regulators (non— palmitoylated proteins).

Figure 5: (A) SDS-PAGE analysis of all purifications from mouse cerebellum using a biotinylated DHHC5 peptide library Homogenised cerebellum was solubilised with C12E10, and then individually incubated with all of the DHHC5 peptides before interacting proteins were purified using streptavidin beads. All pull-downs were analysed by SDS-PAGE. Those purification reactions containing protein (denoted by red box) were analysed by tandem mass spectrometry. (B) Schematic diagram showing where different DHHC5 substrates bind to the enzyme's C-tail. Figure 6: Locating the H-ras binding site within the DHHC9 C-tail A series of overlapping peptides from the DHHC9 C-tail were used to pull-down H-ras from solubilised mouse cerebellum. Subsequent Western Blot analysis revealed that only three DHHC9 peptides (αα 270-294, 280-304 and 290-314) could pull-down H-ras, showing that its' recruitment site is located in the region αα 270-314 of the C-tail and is centred on residues 290-294.

Figure 7: Validating potential substrates of individual DHHC-PATs. (A) Schematic diagram of acyl-RAC Free thiol groups are rendered chemically inert by reaction with MMTS. Palmitate groups can then be removed from the protein by treatment with neutral hydroxylamine. Those proteins from which palmitate groups have been removed can be pulled down using thiol-reactive sepharose beads, before analysis by SDS-PAGE and Western Blotting. (B) Validation of CD36 and Gia2 as DHHC5 substrates The relative palmitoylation of EGFP-tagged CD36 and Gia2 in HEK cells was compared with that in a Cas9-generated DHHC5 KO cell line as well as in KO cells transfected with either DHHC5 (positive control) or DHHC17 (negative control). As both proteins had reduced palmitoylation in the KO cell line compared to WT, and had increased palmitoylation in the KO cells on transfection with DHHC5 but not DHHC17 they were confirmed as DHHC5 substrates.

Figure 8: (A) Locating the PLM binding site within the DHHC5 C-tail A series of overlapping peptides from the DHHC5 C-tail were used to pull-down PLM from cellular extracts. Subsequent Western Blot analysis revealed that only three DHHC5 peptides (αα 223-247, 233-257 and 243- 267) could pull-down PLM, showing that the PLM recruitment site is located in the region αα 223- 267 of the C-tail and is centred on residues 243-247. (B) PLM interacts with DHHC5 via a disordered-disordered protein interaction The C-terminal region of PLM (αα 37-72) contains both palmitoylation sites (C40, C42), is predicted to be disordered (values > 0.5), and is located on the same side of the membrane as the DHHC5 C-tail (which is also predicted to be disordered14). Altogether, this means that PLM must be recruited to DHHC5 via a disordered-disordered protein interaction between the DHHC5 and PLM C-tails.

Figure 9: The kinetics of DHHC5-PLM binding as revealed by Bio-Layer Interferometry (A)

Specific binding sensograms of the PLM C-tail (αα 37-72) to a DHHC5 peptide (αα 233-257) at six different concentrations (3.1 , 6.3, 12.5, 25, 50 and 100 μΜ) were determined. The kinetics data shows that binding of PLM by DHHC5 is a slow process but once the protein-protein interaction has been formed the enzyme does not release the substrate (an observation consistent with the pulldown data (Figure 8A)). In vivo, substrate release must occur following palmitoylation of PLM by DHHC5, and is likely driven by the thermodynamic consequences of attaching a hydrophobic fatty acid to a polar protein surface. (B) Saturable specific binding between DHHC5 and PLM was observed with a Kd of 16.7 +/- 1.9 mM.

Figure 10: Interaction of an amphipathic a-helix (aas 740-757) from NCX1 with the C-tail of DHHC9 (αα 280-304) is required for NCX1 palmitoylation (A) Using a peptide library approach, the NCX1 binding site in the DHHC9 C-tail was localised to region αα 280-304. (B) Deletion of 21 amino acids on the C terminal side of the NCX1 palmitoylation site (αα 745-765) completely abolished NCX1 palmitoylation in HEK cells. (C) The region 740-757 of NCX1 is predicted form an amphipathic a-helix with a small hydrophilic face (coloured black) with the remainder of the helix hydrophobic in character and rich in aromatic amino acids (grey). (D) Deletion of the predicted amphipathic a-helix (Δ740-756) or breaking it by introducing 3 proline residues (M744P/H745P/F746P) both largely abolished palmitoylation but not cell surface delivery of full- length NCX1. Fusing αα 738-756 of NCX1 (including both palmitoylated cysteine and amphipathic a-helix) to the C terminus of YFP caused YFP to be (E) palmitoylated and (F) tethered to intracellular membranes in a manner indistinguishable from that observed for YFP- NCX1. In short, an amphipathic a-helix (αα 740-757) from NCX1 interacts with the C-tail of DHHC9 (αα 280-304), and that this interaction is required for NCX1 palmitoylation.

Figure 11 : PLM palmitoylation can be blocked using a peptide that disrupts recruitment of PLM to DHHC5. HEK293 cells expressing human PLM were incubated overnight with either 3 or 30 μΜ of a TAT-tagged DHHC5 disruptor peptide (αα 233-257). The next morning cells were harvested and the extent of PLM palmitoylation determined by acyl-RAC. For both concentrations of DHHC5 peptide tested, PLM had reduced palmitoylation compared to untreated cells. (*, p< 0.05).

Figure 12: Regulation of PLM recruitment to and palmitoylation by DHHC5 via post- translational modification. (A) Palmitoylation DHHC20 palmitoylates the DHHC5 C tail and regulates PLM recruitment, (i) Palmitoylation of the DHHS5 (catalytic cys removed) C tail in HEK cells is enhanced by co-expression of DHHC20 only in the presence of DHHS5 C236 and C237. (ii) The presence of the DHHC5 C tail cysteines is required for DHHC20 to regulate PLM recruitment by DHHC5 in HEK cells. [UF: unfractionated lysate, P: palmitoylated proteins, **: p<0.01 , ***: p<0.005]. (B) GlcNAcylation (i) DHHC5 S241 fits an O-GlcNAc transferase consensus sequence motif, (ii) Affinity purification with recombinant, tagged catalytically inactive OGA indicates DHHC5 is GlcNAcylated in rat hearts (upper) and transfected HEK cells (lower), (iii) Enhancing DHHC5 GlcNAcylation at S241 increases PLM palmitoylation in HEK cells. [UF: unfractionated lysate, Palm: palmitoylated proteins, *: p<0.05, **: p<0.01]. (C) DHHC5 phosphorylation. The broad-spectrum Cdk inhibitor Purvanalol A increases PLM palmitoylation in HEK cells via enhanced phosphorylation of DHHC5 S247, which fits a Cdk consensus phosphorylation motif²⁷. (D) PLM phosphorylation Phosphorylation of the PLM C-tail by PKA is known to cause an increase in PLM palmitoylation²⁸. Here, we show that phosphorylation of the PLM C-tail with PKA causes an increase in the rate of association between PLM and DHHC5 compared to untreated protein.

Figure 13: A split— protein assay for identifying modulators of PLM recruitment to DHHC5

(A) A reporter protein can be made in two parts, and subsequently reconstituted through interaction between DHHC5 αα 223-247 and the PLM C-tail (αα 37-72) generating a signal. By incubating the fusion protein containing the PLM C-tail with a molecule for a defined period (e.g. 1 h) first before adding the DHHC5-fusion protein, it will be possible to identify molecules that bind to the PLM C-tail that either block (reduced signal) or enhance (increased signal) PLM recruitment to DHHC5. (B) Molecules that bind to the substrate protein and alter its recruitment to the DHHC enzyme can act in one of two ways. First, the molecule may physically block the interaction between substrate and the DHHC-PAT. Alternatively, the molecule may alter the conformation of the substrate protein by an allosteric-type mechanism either enhancing or reducing its recruitment to the DHHC-PAT.

Background information

S-acylation is the reversible covalent post-translational attachment of a fatty acid (chain length ranging between 16 and 20 Carbons (C)), typically palmitic acid (C = 16), to the thiol group of a specific cysteine residue in a particular substrate protein via an acyl-thioester linkage.² Acylation induces substantial changes in the secondary structure and, therefore, function of the intracellular regions of target proteins through their recruitment to the surface of a membrane bilayer via the acylated cysteine. Protein S-acylation is catalysed by a family of DHHC containing protein acyltransferases (DHHC-PATs), reversed by protein thioesterases, and occurs dynamically and reversibly throughout the secretory pathway in a manner analogous to protein phosphorylation.² The diversity of proteins now known to undergo S-acylation includes G- proteins,³ ion channels,⁴ transporters,⁵ ion transport regulators,⁶ receptors,⁷ and protein kinases.⁸ Not surprisingly, it is becoming increasingly clear that S-acylation plays a critical role in regulating a wide-range of biological processes including cell growth, cardiac contractility, synaptic plasticity, endocytosis, vesicle trafficking, membrane transport and biased-receptor signalling. The DHHC family of protein acyl-transferases (DHHC-PATs) DHHC-PATs are zinc-finger-containing enzymes characterised by a cysteine-rich region with a conserved Asp-His-His-Cys (DHHC) motif within the active site;⁹ there are 23 human isoforms. They typically have 4 transmembrane (TM) domains, with a conserved ca. 50 amino acid cytosolic core that is located between TM2 and 3 and contains the DHHC motif. In contrast, the intracellular amino and carboxyl termini are poorly conserved, and likely contribute to DHHC isoform substrate selectivity.¹ DHHC-PATs are expressed throughout the secretory pathway (including the cell surface).¹⁰

Substrate recognition by DHHC enzymes

Determining the molecular basis of substrate recognition by signalling enzymes is the key to understanding the specificity of the signalling pathways in which they participate. The study of kinases, for example, has been significantly aided by experimentally determined substrate primary sequence requirements for individual kinases. Hence, kinases are defined and classified using well-established consensus phosphorylation motifs, and prediction algorithms make the identification of candidate phosphorylation sites in any given target protein relatively facile. Although prediction algorithms exist to identify acylation sites in proteins based on primary sequence,^{11 ,12} they are frequently inaccurate. This is largely due to the different ways in which kinases and acyltransferases recognise their substrates. Unlike kinases, which only form transient complexes with their target proteins, several DHHC enzyme isoforms form stable complexes with their substrates facilitating acylation. For example, DHHC13 and DHHC17 contain N terminal ankyrin repeats that are essential for substrate binding and acylation.¹³ Furthermore, DHHC5 has recently been shown to recruit one of its substrates, the Na pump accessory protein phospholemman (PLM), through its disordered, intracellular C tail (αα 213- 715¹⁴). DHHC5 has also been shown to bind to post-synaptic density protein-95 (PSD-95) through a PDZ binding motif located at its C-terminus.¹⁵ With the exception of PLM^14,16, δ- catenin17 and the ubiquitously expressed protein flotillin 2¹⁸, however, few substrates for DHHC5 have been identified thus far. Furthermore, little is currently known about the closely related family member DHHC8, for which only a handful of substrates have been reported^5,19,20 among them is ankyrin-G (a key regulator of both membrane height in polarised epithelia and neuronal function).^{21 ,22}

Pharmaceutical targeting of protein palmitoylation for therapeutic purposes

Unlike kinases where numerous inhibitors have been created and tested for clinical efficacy in the treatment of disease, no therapeutically useful molecular modulators of protein palmitoylation have been developed thus far. This, in no small part, is due to a fundamental lack of knowledge regarding the molecular basis of both enzyme catalysis and substrate recruitment by DHHC- PATs. To date, efforts to discover inhibitors of DHHC-PATs have exclusively focussed on the enzyme's active site as the catalytic pocket is thought to be drugable. Even if isoform-specific active site inhibitors can be identified they will not be useful in the clinic, as they will block palmitoylation of the entire substrate ensemble of the DHHC-PAT(s) where they act not just the protein of interest (Figure 1A). In contrast, selectively blocking the recruitment of particular substrates to the tail regions of specific DHHC-PATs yields therapeutically useful molecules (Figure 1 B).

An unbiased medium-throughput approach for the identification of DHHC-PAT substrates

The N- and C-termini of DHHC enzyme isoforms are highly divergent, implying control of substrate specificity, subcellular localisation and regulation of activity all reside here.

Bioinformatic analysis of the DHHC5 primary sequence with the regional order neural network tool (RONN)²³ suggests that the enzyme has an Ordered' amino terminal half, that includes the transmembrane (TM) domains and catalytic site, with an extensively 'disordered' intracellular C- terminal region. Such disorder is characteristic of regions of proteins involved in protein-protein interactions, and strongly suggests that the C-terminal half of DHHC5 is involved in determining substrate specificity and/or DHHC5 localisation. In published experiments we have previously shown that the cardiac phosphoprotein PLM is a substrate of DHHC5.¹⁴ DHHC5 silencing in HEK cells abolishes PLM acylation. PLM co-immunoprecipitates with full length DHHC5, but not when the entire DHHC5 disordered intracellular C tail is removed. Therefore, disordered domain interactions underlie substrate recognition by (and hence substrate specificity of) DHHC5 and, given that the other 22 DHHC-PATs have disordered C- and in some cases N-termini also¹⁴, all other family members too.

To increase the discovery rate of DHHC enzyme binding partners, we have developed a novel unbiased medium-throughput approach for the identification of DHHC enzyme interacting proteins (encompassing both substrates and regulators) through the use of peptide libraries in combination with tandem mass spectrometry. We have successfully used a library of biotinylated 25mer peptides overlapping by 15 residues and covering the entire DHHC5 C tail (αα 213-715) to affinity purify the complete ensemble of proteins that bind to the enzyme's C-tail in rat hearts. Lysates were prepared by solubilising homogenised cardiac tissue with C12E10, CHAPS or a combination of dodecyl^D-maltoside (DDM) and cholesteryl hemisuccinate (CHS). Aliquots of each different lysate were individually incubated with the DHHC5 peptides for 1 h at 4°C before interacting proteins were purified using streptavidin beads (Figure 2). Each pull-down was analysed by SDS- PAGE (Figure 3). Those purification reactions containing protein were subsequently analysed by tandem mass spectrometry, enabling identification of the interacting proteins. The mass spectrometry data sets were filtered to remove all proteins also present in a 'no peptide' control. Furthermore, proteins present in three or more of the pull-down fractions were also removed from all of the datasets as they were deemed to be contaminants. The binding partners that remained were considered to be specific interactors that could be further filtered into potential DHHC5 substrates (proteins annotated as being palmitoylated in Uniprot and/or being found in either locally-determined palmitoyl proteomes or those deposited in the SwissPalm database, including several G protein a-subunits, glucose transporter type 4, sodium/calcium exchanger type 1 , CD36 and calnexin), and regulators (non-palmitoylated proteins, including ERK1 , ERK2, p38a, JNK2, MEK1 , AMPKa, β and γ subunits, ROCK1 , Cam kinase II δ and γ, integrin-linked kinase, protein phosphatases PP1 , PP2a and PP6 as well as O-GlcNAc transferase) (Figure 4). The DHHC5 peptide array has also been used with mouse cerebellum where further DHHC5 substrates have been identified, including regulator of G-protein signalling-7, potassium voltage-gated channel subfamily A member 2, protein EFR3 homolog A, synaptotagmin-7, band 3 anion transport protein and aquaporin-4 (Figure 5A). Combining the data from the rat cardiac and mouse cerebellum studies, it has been possible to generate a map of the DHHC5 C-tail showing the different regions where substrates bind (Figure 5B). To assess the usefulness of the peptide-library approach to identify substrates for other DHHC-PATs, a DHHC9 library was successfully used to identify the binding sites for several proteins known to undergo palmitoylation including glutamate receptor 1 , myelin-associated glycoprotein, glutamate decarboxylase 2 and excitatory amino acid transporter 1 (region αα 280-304; results not shown) as well as H-ras (a known DHHC9 substrate) (Figure 6).

Substrate validation

Protein palmitoylation is typically investigated using a technique called acyl-RAC (S-acylation resin assisted capture) (Figure 7A). With this method, a sample is first treated with MMTS (S- methyl-methanethiosulphonate), which reacts with any free thiol groups present rendering them chemically inert. Next, neutral hydroxylamine is added to the sample, which causes palmitate groups to be stripped from proteins through hydrolysis of the thioester linkages by which they are attached. The free thiols generated by this process can then be used to capture those proteins that were originally palmitoylated using thiol-reactive sepharose resin. Proteins captured by the beads can then be separated by SDS-PAGE under reducing conditions and analysed by Western Blotting. Successful enrichment of the protein of interest in the pull-down fraction compared to the starting material is indicative of the protein being acylated.

Putative DHHC5 substrates identified using the peptide library were validated by acyl-RAC, comparing the relative palmitoylation of a EGFP-tagged version of the protein of interest in HEK cells with that in a Cas9-generated DHHC5 KO cell line as well as in KO cells transfected with either DHHC5 (positive control) or DHHC17 (negative control). Those proteins that had reduced palmitoylation in the KO cell line compared to WT, and had increased palmitoylation in the KO cells on transfection with DHHC5 but not DHHC17 were confirmed as DHHC5 substrates (Figure 7B). Identifying the sites of interaction between DHHC— PATs and their substrates

Once an enzyme-substrate relationship has been established, it is then necessary to identify the sites of interaction on both the DHHC-PAT and the substrate protein. For the DHHC enzyme, the peptide(s) used to pull-down the substrate protein must contain the amino acid sequence responsible for the protein-protein interaction. For example, the DHHC5 peptides (αα 223-247, αα 233-257 and αα 243-267) all pull-down PLM (Figure 8A), showing that the PLM recruitment site within DHHC5 is located within the region αα 223-267 and is centred on residues 243-247. PLM consists of a single transmembrane domain with relatively short extracellular N- and intracellular C-termini. The C-terminal region of PLM (αα 37-72) contains both palmitoylation sites (C40, C42), is predicted to be disordered (Figure 8B), and is located on the same side of the membrane as the DHHC5 C-tail. Altogether, this means that PLM must be recruited to DHHC5 via a disordered-disordered protein interaction between the DHHC5 and PLM C-tails. This interaction can be visualised by direct measurement using biolayer interferometry (BLI), an optical analytical technique that allows binding between a ligand immobilised on the surface of a biosensor and an analyte in solution (e.g. small molecule, peptide) to be measured in real-time. Recombinant GST-PLM C-tail (αα 37-72) was immobilised upon a biosensor coated in anti-GST Ab. Control sensors were prepared by coating them with GST. A six point (3.1 , 6.3, 12.5, 25, 50 and 100 &M) concentration series was prepared for a single DHHC5 peptide (αα 233-257) previously shown to contain the PLM binding site (Figure 9A). Binding sensograms were recorded for both GST-PLM C-tail and the GST control in each of the DHHC5 peptide solutions. Specific binding between DHHC5 and the PLM C-tail was determined by subtracting the binding data for the GST control away from that for GST-PLM C-tail for each peptide concentration. The resulting binding sensograms showed saturable specific binding between DHHC5 and PLM with a Kd of 16.7 +/- 1.9 &M (Figure 9B). The kinetics data shows that binding of PLM by DHHC5 is a slow process but once the protein-protein interaction has been formed the enzyme does not release the substrate (an observation consistent with the pulldown data (Figure 8A)). In vivo, substrate release must occur following palmitoylation of PLM by DHHC5, and is likely driven by the thermodynamic consequences of attaching a hydrophobic fatty acid to a polar protein surface.

The electrogenic sodium/calcium exchanger type 1 (NCX1) mediates bidirectional calcium transport under the control of the transmembrane sodium gradient. NCX1 inactivation occurs in the absence of PIP2, and is facilitated by palmitoylation of a single cysteine at position 739 within its large intracellular loop. Previously, we have shown that NCX1 is palmitoylated by DHHC9 in the Golgi²⁴. The region of the DHHC9 C-tail that interacts with NCX1 was identified using a peptide library with NCX1 successfully pulled down using a biotinylated peptide corresponding to αα 280-304 of DHHC9 (Figure 10A). Sequentially mutating the residues immediately before and after C739 to Ala had negligible effect on NCX1 palmitoylation (results not shown). In contrast deletion of 21 amino acids on the C terminal side of the NCX1 palmitoylation site (αα 745-765) completely abolished NCX1 palmitoylation in HEK cells (Figure 10B). Residues 745-765 of NCX1 include a region initially annotated as a transmembrane helix, whose cytosolic location was later established by cysteine accessibility assays²⁵. The secondary structure prediction algorithm Jpred²⁶ suggests with high confidence that αα740-757 of NCX1 form an a-helix. A helical wheel projection indicates that this helix is expected to have a small hydrophilic face (residues D741 , H745, T748 and K752 highlighted in black), with the remainder of the helix hydrophobic in character and rich in aromatic amino acids, particularly on one face (F746, F750, F757) (Figure 10C). Deletion of the predicted amphipathic a-helix (Δ740-756) or breaking it by introducing 3 proline residues (M744P/H745P/F746P) both largely abolished palmitoylation but not cell surface delivery of full-length NCX1 (Figure 10D). In an additional 'gain of function' experiment, the αα 738-756 of NCX1 (including both palmitoylated cysteine and amphipathic a-helix) were fused to the C terminus of YFP. This caused YFP to be palmitoylated (Figure 10E) and tethered to intracellular membranes in a manner indistinguishable from that observed for YFP-NCX1 (Figure 10F). In short, through the combined use of a peptide library and site-directed mutagenesis it has been possible to show that an amphipathic a-helix (aas 740-757) from NCX1 interacts with the C-tail of DHHC9 (αα 280-304), and that this interaction is required for NCX1 palmitoylation.

These approaches (direct measurement by BLI or surface plasmon resonance (SPR) as well as site-directed mutagenesis in combination with acyl-RAC) can be readily used with other DHHC enzyme-substrate pairs to define the regions in both proteins that interact with each other. This information is an essential pre-requisite of efforts to identify molecules that modulate substrate recruitment to DHHC-PATs for therapeutic gain.

Selectively blocking the palmitoylation of specific DHHC-PAT substrate proteins

By using a peptide library to identify substrates for individual DHHC-PATs, the region(s) of the enzyme required for interacting with the target protein is (are) already known. This sequence information can be readily exploited to design disruptor peptides that block the recruitment of specific substrates to selected DHHC enzyme isoforms, preventing their palmitoylation. For example, a TAT-tagged DHHC5 peptide (αα 233-257) blocks the palmitolyation of PLM (Figure 1 1). Here, HEK293 cells expressing human PLM were grown in a 12-well plate. Once the cells had reached 80% confluency, they were incubated overnight with 3 or 30 μΜ of the TAT-tagged DHHC5 disruptor peptide. The next morning cells were harvested for acyl-RAC. The disruptor peptide approach is useful for generating proof-of-concept data, and is a significant first step towards the identification of therapeutically useful small molecules that selectively block the recruitment of specific substrates to particular DHHC-PATs preventing palmitoylation of the target protein.

Regulation of DHHC-PAT substrate recruitment by post-translational modification Like all biological processes, protein palmitoylation is regulated but the mechanism(s) by which this occurs is poorly understood. Using DHHC5-PLM as a model system, we have been able to show that recruitment to and palmitoylation of PLM by DHHC5 can be regulated by palmitoylation, phosphorylation and glycnacylation of amino acids in the DHHC5 C-tail close to the PLM binding site. DHHC20 palmitoylates the DHHC5 C tail at a di-cysteine motif C236/237 close to the PLM binding site causing enhanced recruitment and palmitoylation of PLM (Figure 12A). DHHC5 GlcNAcylation at S241 by O-GlcNAc transferase enhances PLM binding and palmitoylation (Figure 12B). Phosphorylation of a nearby consensus Cdk phosphorylation site in the DHHC5 C tail (S247)²⁷ inhibits recruitment of PLM to DHHC5 (Figure 12C). Furthermore, it has been previously shown that phosphorylation of the PLM C-tail by PKA caused an increase in PLM palmitoylation²⁸. Here, we show that phosphorylation of the PLM C-tail with PKA causes an increase in the rate of association between PLM and DHHC5 compared to untreated protein (Figure 12D).

In short, the covalent modification of both DHHC-PATs and their substrates in close proximity to the regions in both proteins that contact one another can alter (enhance or reduce) the extent of interaction between them, modulating the palmitoylation status of the substrate protein.

Split-protein assays for the identification of molecular modulators of DHHC-PAT substrate recruitment

Split-protein reporter systems are routinely used to identify proteins (or regions of proteins) that interact with one another²⁹. By fusing the regions of a DHHC-PAT and a target substrate protein known to interact with each other to opposite halves of a split-protein system, bespoke assays can be readily created that can be used for high-throughput screening to identify modulators of DHHC-PAT substrate recruitment and as a consequence substrate palmitoylation.

For example, a split-protein assay for identifying modulators of PLM recruitment to DHHC5 can be designed as follows (Figure 13A). A reporter protein split into two parts can be reconstituted through interaction between DHHC5 αα 223-247 and the PLM C-tail (αα 37-72) (Figure 8A, 9A) generating a signal. By incubating the fusion protein containing the PLM C-tail with a molecule for a defined period (e.g. 1 h) first before adding the DHHC5-fusion protein, it will be possible to identify molecules that bind to the PLM C-tail that either block (reduced signal) or enhance (increased signal) PLM recruitment to DHHC5. Molecules that bind to the substrate protein and alter its recruitment to the DHHC enzyme can act in one of two ways (Figure 13B). First, the molecule may physically block the interaction between substrate and the DHHC-PAT in an analogous way to that observed for the disruptor peptides. Alternatively, the molecule may alter the conformation of the substrate protein in an allosteric-type mechanism either enhancing or reducing its recruitment to the DHHC-PAT in an analogous way to that observed for regulation of PLM recruitment to DHHC5 by post-translational modification. Molecules that alter DHHC-PAT substrate recruitment through binding to the substrate rather than the enzyme will alter the palmitoylation status of the target protein alone. The split-protein assay strategy outlined here will work for any DHHC enzyme-substrate pair where the substrate is recruited to the enzyme through a physical interaction with either the enzyme's N- or C-tail regions, and will be useful for identifying molecules for the treatment of disease (Table 1).

References

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Claims

Claims

1. A method of modulating the palmitoylation status of a protein, said method comprising modulating the recruitment of the protein to the N- or C-terminal tail region of a DHHC-PAT molecule.

2. The method of claim 1 , wherein protein palmitoylation is blocked or inhibited by blocking or inhibiting recruitment of the protein to the N- or C-terminal tail region of a DHHC- PAT molecule and protein palmitoylation is enhanced by enhancing or increasing recruitment of the protein to the N- or C-terminal tail region of a DHHC-PAT molecule.

3. A method of modulating the palmitoylation status of a protein palmitoylated by interaction with a DHHC-PAT molecule, said method comprising modulating the recruitment of the protein to the C-terminal tail region of a DHHC-PAT molecule, wherein recruitment of the protein to the C-terminal tail region of a DHHC-PAT molecule is modulated by:

(i) a molecule that binds to the N- or C-terminal tail portion of a DHHC-PAT molecule; and/or

(ii) a molecule that binds to the DHHC-PAT N- or C-terminal tail binding part of the protein; and/or

(iii) a molecule that binds to or has affinity and/or specificity for, a region or portion of the DHHC-PAT C-terminal tail involved in or associated with binding to the protein; and/or

(iv) a molecule that binds to or has affinity and/or specificity for, a region or portion of the protein involved in or associated with binding to the N- or C-terminal tail region of a DHHC-PAT molecule.

4. A method of modulating the palmitoylation status of a DHHC5-PAT binding partner, comprising modulating DHHC5-PAT partner binding to the C-terminal tail portion of DHHC5- PAT.

5. The method of claim 4, wherein the DHHC5-PAT binding partner is selected from the group consisting of:

(i) Glucose transporter 4;

(ii) Phospholemman;

(iii) CD36;

(iv) KCNA2;

(v) Protein Deglycase DJ-1 (Park 7);

(vi) Synaptotagmin-7;

(vii) Band 3 Anion transport Protein; and

(viii) Aquaporin 4.

5. The method of claim 4, wherein the method uses a fragment or portion of the DHHC5-PAT C-terminal tail to modulate DHHC5-PAT partner binding to the C-terminal tail portion of DHHC5-PAT.

6. The method of claim 5, wherein the fragment or portion of the DHHC5-PAT C- terminal tail comprises an amino acid sequence derived from:

(i) amino acids 213-677 of the DHHC5-PAT C-terminal tail;

(ii) amino acids 213-267 of the DHHC5-PAT C-terminal tail;

(iii) amino acids 223-247 of the DHHC5-PAT C-terminal tail;

(iv) amino acids 233-257 of the DHHC5-PAT C-terminal tail; (v) amino acids 243-267 of the DHHC5-PAT C-terminal tail;

(vi) amino acids 523-557 of the DHHC5-PAT C-terminal tail;

(v) amino acids 463-487 of the DHHC5-PAT C-terminal tail; and/or

(vi) amino acids 653-677 of the DHHC5-PAT C-terminal tail.

7. The method of claim 6, wherein

(i) amino acids 223-247 has the amino acid sequence:

GKFRGGV PFTNGCCNNVSRVLCSS

(ii) amino acids 233-257 has the amino acid sequence:

TNGCCNNVSRVLCSSPAPRYLGRPK

(iii) amino acids 243-267 has the amino acid sequence:

VLCSSPAPRYLGRPKKEKTIVIRPP

8. The method of claim 4, wherein the method uses a fragment or portion of the DHHC9-PAT C-terminal tail to modulate DHHC9-PAT partner binding to the C-terminal tail portion of DHHC9-PAT.

9. The method of claim 8, wherein the fragment or portion of the DHHC9-PAT C- terminal tail comprises an amino acid sequence derived from:

(i) amino acids 270-324 of the DHHC9-PAT C-terminal tail;

(ii) amino acids 270-314 of the DHHC9-PAT C-terminal tail;

(iii) amino acids 290-294 of the DHHC9-PAT C-terminal tail;

(iv) amino acids 270-294 of the DHHC9-PAT C-terminal tail; (v) amino acids 280-304 of the DHHC9-PAT C-terminal tail;

(v) amino acids 290-314 of the DHHC9-PAT C-terminal tail; or

(vi) amino acids 300-324 of the DHHC9-PAT C-terminal tail.

10. The method of claim 9, wherein: (i) amino acids 270-294 has the amino acid sequence:

VQNPYSHGNIVK CCEVLCGPLPPS

(ii) amino acids 280-304 has the amino acid sequence:

VK CCEVLCGPLPPSVLDRRGILPL

(iii) amino acids 290-314 has the amino acid sequence: PLPPSVLDRRGILPLEESGSRPPST

1 1. The method of claim 4, wherein the method uses a fragment or portion of PLM to modulate PLM binding to the C-terminal tail portion of DHHC5-PAT.

12. The method of claim 1 1 , wherein the fragment or portion of PLM comprises amino acids 37-72 of the PLM protein.

13. A molecule that binds to the DHHC-PAT C-terminal tail binding part of a protein for use in the treatment or prevention of diseases and/or conditions associated with or linked to protein palmitoylation via DHHC-PAT molecules; and/or associated with or caused or contributed to by, aberrant protein S-acylation and/or palmitoylation events.

14. The molecule of claim 13, for use of claim 13, wherein the molecule that binds to the DHHC-PAT C-terminal tail binding part of a protein comprises:

(i) amino acids 213-677 of the DHHC5-PAT C-terminal tail;

(ii) amino acids 213-267 of the DHHC5-PAT C-terminal tail; (iii) amino acids 223-247 of the DHHC5-PAT C-terminal tail;

(iv) amino acids 233-257 of the DHHC5-PAT C-terminal tail;

(v) amino acids 243-267 of the DHHC5-PAT C-terminal tail;

(vi) amino acids 523-557 of the DHHC5-PAT C-terminal tail;

(v) amino acids 463-487 of the DHHC5-PAT C-terminal tail;

(vi) amino acids 653-677 of the DHHC5-PAT C-terminal tail;

(i) amino acids 270-324 of the DHHC9-PAT C-terminal tail;

(ii) amino acids 270-314 of the DHHC9-PAT C-terminal tail;

(iii) amino acids 290-294 of the DHHC9-PAT C-terminal tail;

(iv) amino acids 270-294 of the DHHC9-PAT C-terminal tail;

(v) amino acids 280-304 of the DHHC9-PAT C-terminal tail;

(v) amino acids 290-314 of the DHHC9-PAT C-terminal tail; or

(vi) amino acids 300-324 of the DHHC9-PAT C-terminal tail.

15. The molecule of claim 14, for use of claim 14, wherein:

(i) amino acids 223-247 of the DHHC5-PAT C-terminal tail has the amino acid sequence

GKFRGGVNP FTNGCCNNVSRVLCS S

(ii) amino acids 233-257 of the DHHC5-PAT C-terminal tail has the amino acid sequence :

TNGCCNNVSRVLCS SPAPRYLGRPK (iii) amino acids 243-267 of the DHHC5-PAT C-terminal tail has the amino acid sequence:

VLCSSPAPRYLGRPKKEKTIVIRPP

16. The molecule of claim 14, for use of claim 14, wherein: (i) amino acids 270-294 of the DHHC9-PAT C-terminal tail has the amino acid sequence:

VQNPYSHGNIVK CCEVLCGPLPPS

(ii) amino acids 280-304 of the DHHC9-PAT C-terminal tail has the amino acid sequence: VK CCEVLCGPLPPSVLDRRGILPL

(iii) amino acids 290-314 of the DHHC9-PAT C-terminal tail has the amino acid sequence:

PLPPSVLDRRGILPLEESGSRPPST

17. A molecule that binds to the N- or C-terminal tail portion of a DHHC-PAT molecule for use in the treatment or prevention of diseases and/or conditions associated with or linked to protein palmitoylation via DHHC-PAT molecules; and/or associated with or caused or contributed to by, aberrant protein S-acylation and/or palmitoylation events.

18. The molecule of claim 17, for use of claim 17, wherein the molecule that binds to the C-terminal tail portion of a DHHC-PAT molecule is obtained from a protein selected from the group consisting of:

(i) H-ras

Glucose transporter 4

(iii) Phospholemman (PLM) (iv) CD36

(v) KCNA2

(vi) Protein Deglycase DJ-1 (Park7)

(νϋ) Synaptotagmin-7

(viii) Band 3 Anion Transport Protein

(ix) Aquaporin 4

Excitatory amino acid transporter 1

(xi) Glutamate receptor 1

19. The molecule of claim 17 or 18, for use of claim 17 or 18, the molecule that binds to the C-terminal tail portion of a DHHC-PAT molecule is a fragment or portion of PLM.

20. The molecule of claim 19, for use of claim 19, wherein the fragment or portion of PLM comprises amino acids 37-72 of the PLM protein.

21. The molecules of any one of claims 17-20, for use of claims 17-20, wherein the disease or condition is one or more selected from the group consisting of cancer; diabetes; heart failure; metabolic syndrome; neuropathic pain; Parkinson's disease; neurodegeneration; renal tubular acidosis; neuromyelitis optica; stroke; Huntington Disease; ischaemic damage; epilepsy and amyotrophic lateral sclerosis.

22. A DHHC5-PAT C-terminal tail binding fragment or portion of PLM or a DHHC5-PAT C-terminal tail binding fragment or portion of PLM comprising amino acids 37-72 of the PLM protein, for use in treating heart failure or myocardial infarction.

23. A method or assay for determining whether or not a test agent modulates binding between a DHHC-PAT molecule and a DHHC-PAT binding partner, said method comprising contacting a test agent with a peptide comprising a protein or peptide sequence from a DHHC-PAT C-terminal tail; and identifying those test agents that interact or associate with or bind to the DHHC-PAT peptide, wherein those test agents that interact or associate with or bind to the DHHC-PAT C-terminal tail protein/peptide modulate binding between a DHHC-PAT molecule and a DHHC-PAT binding partner.

24. The method or assay of claim 23, wherein the test agent comprises a peptide library.

25. The method or assay of claim 24, wherein the peptide library comprise peptides derived from a DHHC-PAT C-terminal tail and/or peptides derived from a protein known to bind to or interact with a DHHC-PAT molecule.

26. The method or assay of any one of claims 23, 24 or 25, wherein the assay or method is an assay or method for identifying molecules which might modulate protein palmitoylation via a DHHC5-PAT protein and/or an assay or method which uses a peptide library comprising peptides derived from the DHHC5-PAT C-terminal tail and/or peptides derived from proteins which are known to bind the DHHC5-PAT C-terminal tail.

27. The assay or method of claim 26, wherein the assay or method uses a peptide library comprising peptides derived from PLM.

28. An assay or method for determining whether or not a test agent modulates binding between a DHHC-PAT molecule and a DHHC-PAT binding partner, said method comprising contacting or incubating a DHHC-PAT C-terminal tail peptide with a protein known to bind the same, in the presence of a test agent;

wherein if the test agent modulates binding between a DHHC-PAT molecule and a DHHC-PAT binding partner, binding between the DHHC-PAT molecule and a DHHC-PAT binding partner will be modulated.

29. The assay or method of claim 28, wherein the method uses a peptide derived from the C-terminal tail portion of DHHC5-PAT and a peptide derived from PLM.

30. An assay or method for identifying test agents which modulate binding between DHHC binding partners and DHHC molecules, said method comprising: providing a DHHC-PAT peptide/protein tagged, fused or conjugated to/with one part of a split reporter element; providing a DHHC-binding partner tagged, fused or conjugated to/with the other part of the split reporter element; contacting or incubating the DHHC-PAT peptide/protein with the DHHC-binding partner in the presence of a test agent and under conditions which permit binding between the DHHC-PAT peptide/protein and the DHHC-binding partner, wherein

(i) detection of a signal from the reporter element indicates that the test agent has not prevented binding between the DHHC-PAT and the DHHC binding partner; (ii) detection of an enhanced signal from the reporter element indicates that the test agent has increased binding between the DHHC-PAT and the DHHC binding partner; and

(iii) failure to detect a signal from the reporter element indicates that the test agent has prevented binding between the DHHC-PAT and the DHHC binding partner.