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Review Article *Corresponding author Kathryn L Ball, The Institute of Genetics and Molecular Medicine, Edinburgh Cancer Research Centre, The Functions of CHIP in Age University of Edinburgh, Western General Hospital, Crewe Road South, EH4 2XR, UK, Tel: +44 0-131-651-8500; E-mail: Related Disease Submitted: 03 August 2016 Ball KL*, Ning J, Nita E, and Dias C Accepted: 29 September 2016 Institute of Genetics and Molecular Medicine, University of Edinburgh, UK Published: 01 October 2016 Copyright Abstract © 2016 Ball et al. CHIP is a key component of the protein homeostasis or ‘Proteostasis’ network OPEN ACCESS that maintains protein structure and function as a way to ensure the integrity of the proteome in individual cells and the health of the whole organism. Proteostasis Keywords influences the biogenesis, folding, trafficking and degradation of proteins. Originally • CHIP identified as a Hsc70 associated protein and a co-chaperone CHIP has E3-ubiquitin • E3-ligase ligase activity and also displays an intrinsic chaperoning ability. It has become clear • Chaperone that CHIP is a multi-functional protein with roles in cellular processes that go beyond • Structure function its co-chaperone activity. Not surprisingly, by unravelling the functions of CHIP, we are • Neurodegeneration beginning to appreciate that loss of CHIP’s integrity can lead to the development of • Cancer several serious pathological conditions. Here we will describe the key features of CHIPs structure and functions with an emphasis on the non-canonical activities of CHIP before concentrating on the role it plays in protecting against the age associated pathologies of neurodegeneration and cancer.

ABBREVIATIONS encompass the elaborate systems involved in maintaining protein structure and function as a way to ensure the integrity of the AD: Alzheimer’s Disease; CHIP: C-Terminus Of Hsc70 proteome in individual cells and the health of the whole organism. Interacting Protein; Hsc70: Heat Shock Cognate Protein 70; Hsp: Proteostasis involves a highly complex network of processes that Heat Shock Protein; RING: Really Interesting New Gene; Lys: Lysine; TPR: Tetratricopeptide Repeat; Cyp40: Cyclophilin 40; proteins. The dual function E3-ubiquitin ligase and molecular Hop: Hsp70/Hsp90 Organizing Protein; PP5: Protein Phosphatase influence the biogenesis, folding, trafficking and degradation of 5; HX-MS: Hydrogen-Deuterium Exchange Mass Spectrometry: chaperone CHIP (C-terminus of Hsc70 interacting protein) was Ppiase: Peptidylprolyl Isomerase; Bag-1: BCL2 Associated Athanogene 1; IRF-1: Interferon Regulatory Factor-1; CFTR: degradation. In addition to being a co-chaperone that targets originally identified as a link between protein folding and protein Cystic Fibrosis Transmembrane Conductance Regulator: AMP: partially-folded or unfolded client proteins for proteasomal Adenosine Monophosphate: AMPK: Adenosine Monophosphate also function independently of its Hsp-partners to control the degradation dependent on its E3-ligase activity, CHIP can Enos: Endothelial Nitric Oxide Synthase; TLR: Toll-Like Receptor; structure and function of proteins through its intrinsic chaperone Kinase;IFN β: Interferon HSF1: Heat β; PKC Shock ζ: Protein Factor KinaseProtein Cζ: 1; Sirt6: α-Syn: Sirtuin-6; α-Synuclein: LKB- and/or E3-ligase activities. Besides its functions in ubiquitin- 1: Liver Kinase B1; HIF1A: Hypoxia-Inducible Factor 1-Alpha; CMA: Chaperone-Mediated Autophagy; CASA: Chaperone Assisted Selective Autophagy; MEFS: Mouse Embryonic Fibroblasts; UPS: dependent proteasome-mediated degradation, CHIP is implicated Ubiquitin Proteasome System; APP: Amyloid Precursor Protein; beginningin chaperone to appreciate dependent that autophagy loss of CHIP’s and integrity protein can trafficking. lead to Epidermal Growth theNot development surprisingly, of by several unravelling serious the pathological functions ofconditions. CHIP, we Here are Factor Receptor; we will introduce the general functions of CHIP with an emphasis Aβ:nuclear Amyloid Factor β; Kappa-Light-Chain-EnhancerPD: Parkinson’s Disease; EGFR: Of Activated B Cells; Erbb2: Erb-B2 Receptor Tyrosine Kinase 2; NF-κB ER: Estrogen Receptor; GR: Glucocorticoid Receptor; PRMT5: age related process of neurodegeneration and tumorigenesis. Protein Arginine Methyltransferase 5; E2F1:E2F Transcription on its non-canonical activities, followed by the role of CHIP in the Factor 1; TG2: Transglutaminase 2; Bcl-2: B-Cell Lymphoma 2; Structure of chip TRAF2: TNF Receptor-Associated Factor 2; EMT: Epitheliaap1l- CHIP contains a conserved C-terminal U-box domain and Mesenchymal Transition; MMP9 :Matrix Metallopeptidase 9;

Factor Beta; AP1: Activator Protein 1 MMP2 Matrix Metallopeptidase 2; TGF-β: Transforming Growth N-terminal TPR domain, which are separated by a coiled-coil INTRODUCTION region [1]. Structurally, the U-box is like a RING-finger except that it lacks metal chelating residues, playing a similar role to the RING- Protein homeostasis or ‘Proteostasis’ is the term used to finger domain in targeting the substrate and mediating ubiquitin- conjugation to it [2]. RING-finger type E3 ligases were originally Cite this article: Ball KL, Ning J, Nita E, Dias C (2016) The Functions of CHIP in Age Related Disease. JSM Enzymol Protein Sci 1(1): 1006. Ball et al. (2016) Email:

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Central Bringing Excellence in Open Access considered to be ‘passive’ i.e. they do not contain a catalytic site C-terminal peptide the TPR-domain becomes more stable and but instead function as scaffolds by placing the substrate and crystallography data on RING domains in complex with E2 and organized. By using a mutation at Lys30 of the TPR of CHIP, ubiquitinubiquitin-charged partners E2 suggests into close a more proximity active [2]. role However, in determining recent the entire TPR domain can be ‘locked’ (Figure 1). Also, changes Hsp70/90to the flexibility peptide of bindingthe TPR or domain with theaffect replacement U-box conformation of Lys30 with[20]. anThe Ala; E3-ligase this indicates activity thatof CHIP an intra-moleculardecreased significantly allostery upon can dependentE2~Ub conformation on the E2-partner [3,4]. CHIP [7]. Whilstcan ubiquitinate Lys48-linked substrates substrates to be transmitted from the TPR domain to the U-box domain (Figure canform be either degraded Lys48-linked by the chains proteasome [5] or andLys63-linked Lys63-linked chains chain [6], 1). Similar results were reported for Cyp40 bound to the Hsp peptides ‘XXXMEEVD’ with evidence presented that the peptide structure (PDB ID: 2C2V) for ‘nearly’ full-length murine CHIP modification results in a variety of outcomes [8,9]. The crystal on CHIP allosteric regulation suggests that cellular experiments usingcan inhibit a K30A the mutant PPIase of activityCHIP must of Cyp40be interpreted [21]. Finally, with care the [20]. data can(aa25-304) catalyze was the solvedformation in 2005, of Lys63-linked and showed ubiquitin the C-terminus chains. Inof thisCHIP structure in complex a hydrophobic with Ubc13-Uev1a, ridge in Ubc13 an E2 binds heterodimer into a U-box that Chip functions hydrophobic groove [10]. A subsequent structure (PDB ID: 2OXQ) Canonical activity of chip as a co-chaperone: CHIP was for the CHIP U-box from Danio rerio in complex with UbcH5a has discovered by the Patterson group [1] as a binding protein also been solved and shows a similar architecture to the murine for Hsc70; it was enriched in muscle tissue and could act as a negative regulator of Hsp70/Hsp40 chaperone function in vitro structureThe TPR-domain [11,12]. of CHIP (residues 26-131 in human) is located at the N-terminus. A TPR-domain is a 34 amino acid glucocorticoid[1]. Subsequently, receptor CHIP leading was shown to its degradationto interact with through Hsp90, the motif which forms a pair of anti-parallel helices that has been as well as Hsc/Hsp70, and could induce ubiquitination of the protein triage decisions impacting on the balance between folding tandem arrays of 3-16 motifs [14] and the distribution of these andubiquitin-proteasome degradation of chaperone pathway client [16]. proteins. Thus, The CHIP role modulated of CHIP in arraysidentified can in be a broad found range separated of proteins from each [13]. other It is assembled or arranged in have U-Box dependent E3-ubiquitin ligase activity [22]. This led as a mediator of protein-protein interactions that facilitates the protein triage was further defined when it was demonstrated to together [15]. Generally speaking, the TPR motif can be described units generate six helices that consist of three pairs of anti- to the definition of CHIP as a co-chaperone, which together with assembly of protein complexes [15]. In CHIP, three TPR domain from a protein folding- to a protein degradation-machine [22- parallel helices. The hydrophobic surface of the TPR domain is BAG-1 [6,23], could shift the activity of the Hsc/Hsp70 chaperones responsible for the interaction with the carboxy-terminus of Hsc/ an E3-ligase that ubiquitinated unfolded or mis-folded proteins targeting24]. Thus, them the early for ubiquitin days of CHIPdependent research proteasome focused on degradation its role as of murine CHIP in complex with a Hsp90 C-terminal decapeptide only in the presence of its chaperone partners [23-25]. This wasp70 orpublished. Hsp90 [1,16]. It showed In 2005, that the the crystal TPR domain structure of (PDBCHIP ID:is similar 2C2L) canonical CHIP pathway has been covered in detail in a number of recent reviews [26-28]. to TPR domains in other proteins, e.g. HOP/Cyp40/PP5 etc. In Non-canonical functions of chip: clamp.this structure, The electrostatic the Hsp90 peptide interaction was bound is contributed in the TPR primarily domain the functions of CHIP may be more complex than simply a byhydrophobic an aspartic pocket, acid from which Hsp90 forms and alysine distinct residues two-carboxylate in the TPR constitutive ubiquitinating co-factor for The the core first molecular hint that domain of CHIP [10]. A recent structure of CHIP and Hsp70 lid-tail

chaperones, came from Meacham et al., (2001) [28]. This study (HsHsc70541-646Δ626-638) revealed that residues in addition CHIPshowed over that expression CHIP has ledsubstrate to the ubiquitinationspecificity and anddid degradationnot catalyse to the Hsp70 C-terminal peptide ‘PTIEEVD’, namely the α-helical ofthe the degradation Hsp70 client of allprotein Hsp70-dependent CFTR it had no clients. effect Thus, on a whereasrange of lid sub-domain, interact with CHIP in order for Hsp70 to act as a other Hsp70 substrates (including the transferin receptor and CHIP substrate and post-translational modifications of the lid can regulateThe coiled-coilHsp70-CHIP region binding of affinity CHIP exists [17]. as a linker between the TPR- and U-box domains. It is essential for dimer formation ofluciferase CHIP in [29,30]).cells could Furthermore, increase the in folding contrast capacity to the ofability Hsp70 of CHIPin an which in turn is required for CHIP to be fully active as an E3 E3-ligaseto inhibit Hsp70independent mediated manner folding [30]. in vitro This [1,16], lead to over-expression the suggestion that the association of CHIP with Hsp70 did not always convert an asymmetric dimer with different conformations in the coiled- ligase [18]. It is of note that, in the murine structure, CHIP forms this class of chaperone from a folding to a degradative mode [30]. In agreement with this we have shown that CHIP over expression coil region for each protomer. In one subunit, a hairpin is formed or depletion differentially affects the levels of the interferon by two straight helices. In the other subunit, one of the helices regulated transcription factor IRF-1 dependent on the cellular overlaidis broken the into TPR two domain separated in the α-helices. dimer will When block the access structures to one for of CHIP-Hsp90 peptide (2C2L) and CHIP- Ubc13-Uev1a (2C2V), are CHIP is a negative regulator of IRF-1 steady state levels whereas inconditions. cycling cells Thus, CHIP in enhances cells that IRF-1 are recovering protein levels from [31]. heat stress the U-box binding sited for the E2, thus only one U-box can bind We now know that CHIP has several non-canonical functions with E2 [10] and is presumable sufficient for catalysis. Recently, using HX-MS, it was shown that the apo-CHIP which either do not require chaperone partners, which can be TPR domain is very flexible [19]. When bound to a Hsp70/90 JSM Enzymol Protein Sci 1(1): 1006 (2016) 2/10 Ball et al. (2016) Email:

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a

b

Figure 1 Inter-domain allosteric regulation of CHIP. The allostery signal transmits from the TPR domain of CHIP to the U-box domain. The TPR domain of CHIP is very

cartoon showing that locking of the TPR domain makes the U-box domain more stable. (b) A structure showing the conformational inhibition signal transfers from the flexible. By binding to Hsp70, or by introducing a mutation in the TPR domain, the TPR domain becomes ‘locked’ and allosterically reduces the flexibility of U-box. (a) A

N-terminal TPR domain to C-terminal U-box. Red means flexible. Blue indicates stable. negatively regulated by CHIP association with Hsp’s or where CHIP and Hsp70 cooperate to regulate processes other than domain is reported to prevented p53 from irreversible the degradation of unfolded or mutant proteins through the thermalubiquitination inactivation [20]. In [33]. addition, In this binding study ofCHIP CHIP was to anfound N-terminal to bind proteasome. Some examples of this are given below: preferentially to the mutant conformation of p53 and to restore the DNA binding activity of p53 following heat inactivation. Chip chaperone activity: Although CHIP was originally The authors proposed that CHIP might therefore be a direct chaperone for chromatin bound p53 helping it to maintain a apparent that CHIP also had its own intrinsic chaperone activity. identified as a co-chaperone for Hsc/Hsp70, it later became transcriptionally active conformation under stress conditions [33]. Perhaps the best evidence for the physiological role of CHIP the refolding of proteins in cells following thermal denaturation as a chaperone comes from studies on the AMP activated kinase [30].Thus, Underover expression these conditions of CHIP inhibitionin fibroblasts, of Hsp70 was able abolished to increase the effects of CHIP on protein folding suggesting that CHIP might be functioning to enhance Hsp70-dependent chaperone activity. (AMPK). In the case of the AMPK, CHIP can regulate the catalytic α-subunit by promoting LKB-1-mediated phosphorylation, which in turn leads to an increase in its specific activity. CHIP binding Furthermore, enhancement of Hsp70 ‘folding’ function was suggesting that CHIP can chaperone native as well as non-native studies went on to show that CHIP could bind to and refold to AMPK causes a conformational change in the α-subunit independent of CHIP E3-ligase activity. However, subsequent proteins to affect changes in their activity [34]. Hsp70. We can therefore conclude that CHIP has an intrinsic Chip as an activator of hsf1-mediated transcription: In chaperoneheat-denatured activity luciferase that is elevatedin vitro and during in cells, heat stressindependent [32] and of response to heat stress a portion of total CHIP protein moves from the cytoplasm into the nucleus where it can be found in complex in a substrate folding pathway [30]. that it may also function to activate, or cooperation with, Hsp70 CHIP can physically interact with native p53 leading to its with active HSF1, a transcription factor that regulates heat shock protein expression [35,36], at heat shock elements [37]. Thus, JSM Enzymol Protein Sci 1(1): 1006 (2016) 3/10 Ball et al. (2016) Email:

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Central Bringing Excellence in Open Access whereas over expression of CHIP leads to trimerization and Regulation of protein activity by chip: One of the areas to of HSF1 and these changes occur independently of any change more general role in protein trafficking [46]. activation of HSF1, knock-down of CHIP decreases the activity adaptive immunity. CHIP plays a role in antigen aggregation mechanism by CHIP promotes the activity of HSF1 remains andreceive presentation recent attention, during in terms maturation of CHIP of activity, antigen is presentinginnate and unclearin the half-life CHIP and of HSF1the transcription are reported factor to form [37,38]. a direct Although interaction the cells as part of the co-chaperone network [47]. Perhaps more under conditions of heat stress [39]. Fascinatingly CHIP appears to cause the re-localisation of HSF1 [37]. The interaction between leadingsurprisingly, to the CHIP activation can coordinate of downstream the assembly signal of components active Toll-Like and there is evidence that CHIP might recruit Hsp70 to the HSF1 Receptor signalling complexes, specifically for TLR4 and TLR9, transcriptionCHIP and HSF1 complex can occur under independently some conditions of Hsp70, [37]. and Inindeed this authors of this study suggest that as well as acting as scaffolding the production of antiviral cytokines including IFNβ [48]. The CHIP function leading to inhibition of HSF1 [40]. situation however, Hsp70 would oppose rather that support Docking dependent substrate ubiquitination by chip: protein for active TLR complex assembly, CHIP may be involved in As described above the canonical pathway for CHIP is that it howeverthe recruitment evidence of Src is and presented PKCζ to that TLR4/9 the and K63-ubiquitin their subsequent ligase ubiquitinates unfolded or mutant substrates in collaboration activation. How CHIP activates these signalling kinases is unclear, with Hsp70 and targets them for degradation. In this pathway CHIP playing a more direct role in regulating protein activity is CHIP does not interact directly with the substrate but substrate SirT6.activity SirT6 of CHIP is a maylysine be deacetylase/ADP required [10,48]. ribosylase Another involved example in of DNA repair and metabolism. In this case CHIP maintains SirT6 evidence that CHIP can ubiquitinate substrates independently of activity by preventing its degradation. This requires CHIP E3- recognition is mediated by Hsp70. However, there is also ligase activity and the authors speculate that CHIP-dependent binding of CHIP to its substrate. We have shown that CHIP binds Hsp70 and that in this pathway modification requires the direct K48-linked polyubiquitnation and degradation of SirT6 [49]. ubiquitination of the IRF-1 DNA-binding domain [41]. In this ubiquitination of SirT6 at K170, prevents other E3-ligases from directly to IRF-1 [31] to carry out docking-mediates site specific Autophagy and chip: In addition to the well-studied link between CHIP and proteasome mediated degradation there is scenario conditions which block access to the E3-binding site, growing evidence that CHIP can also take part in autophagy. As the link between autophagy and age related diseases are well unfoldingsuch as when during IRF-1 mild is in heat its DNAstress bound binding conformation of CHIP is inhibited [41], inhibit and substrate ubiquitination. Interestingly, when IRF-1 is subject to

inestablished CHIP protein [50], levels autophagy in neuronal regulation cells could induces be import autophagosome for the role whetherit becomes Hsp70 a poor wouldsubstrate restore for CHIP ubiquitination E3-ligase activity under (Landre, these formationof CHIP in degradationneuroprotection of the (see classic below). autophagy Thus, whilst marker a decrease protein conditionsMativa, Ning remains and to Ball, be seen. unpublished Docking-dependent observation). ubiquitination However, p62 is decreased [51] leading the authors to speculate on the Hsp70 as binding leads to inhibition of U-box activity. Thus of native IRF-1 and, a second substrate p53, are inhibited by a role for CHIP in mitophagy and the maintenance of energy metabolismrole of CHIP in theautophagic CNS [52]. flux. The Studies functions in CHIP of CHIP null spread mice indicate beyond TRP-domain of CHIP can act as allosteric regulators that affect CHIPbinding substrate-docking of full-length Hsp70, and or U-box Hsp70 activities mimetic [20]. peptides, This to study the as a component of the ubiquitin-dependent chaperone-assisted is supported by the observation that CHIP-dependent mono- canonical autophagy, thus it plays a role in antigen presentation ubiquitination of Smad1/5 [42] is inhibited by Hsp70 and by data takes part in a second form of chaperone-dependent autophagy selective autophagy (CASA) process [47,53]. Further, CHIP using α−synuclein where suppression of mono-ubiquitination by example of CHIPs role in CMA is the hypoxia-inducible factor and is a transcriptional factor it is interesting to speculate that the re- named Chaperone-Mediated Autophagy (CMA) [54,55]. An localisationBAG-5 is Hsp70-mediated of CHIP to the [43].nucleus As IRF-1,under likestress HSF1 conditions (see above), may have wider implications for transcriptional regulation than the transcription factor HIF1A. In the case of HIF1A, CHIP is required control of Hsp expression [37]. for its ubiquitination by K63 linked ubiquitin, which is an early Role for chip in regulation of protein trafficking: step in CMA-dependent degradation. However, the requirement CHIP in age related disease: Perhaps one of the most As referred to above over expression of CHIP can lead to a for CHIP activity in CMA is likely to be substrate specific [56,57]. striking phenotypes of surviving CHIP null mice is the effect on redistribution of HSF1 suggesting that CHIP may play roles longevity [58]. The CHIP null-mice have a 60% decrease in life- span compared to wild-type animals and this is accompanied by in protein trafficking. For example, CHIP has been shown to increased anatomical ageing. These mice have signs of increased apoptoticinteract with activity. and ubiquitinate The ubiquitination Daxx, a death of Daxx domain does associated not signal protein and transcriptional suppressor, to counter its pro- DNA-damaging agents [49]. Ageing and cellular senescence degradation but temporarily sequesters it into an insoluble oxidative stress in the brain [58] and, are hypersensitive to fraction from which it can later be released when the cells are in and DNA- are enhanced by various stress conditions, including oxidative for ubiquitination and proteasome-dependent degradation but stress, defects in proteasome-mediated degradation the recovery phase [44,45]. Similarly, CHIP does not target eNOS activated during cellular senescence (i.e. ageing) in normal cells partitions soluble eNOS into an insoluble and inactive cellular damage [59]. Likewise, CHIP expression is dynamic and is

to facilitate targeting of proteins to the proteasome, including the compartment [46]. However, unlike Daxx, eNOS regulation by degradation of p53, a key modulator of senescence. However, CHIP occurs in unstressed cells, suggesting that CHIP may play a JSM Enzymol Protein Sci 1(1): 1006 (2016) 4/10 Ball et al. (2016) Email:

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Central Bringing Excellence in Open Access when CHIP is silenced (using siRNA) in human embryonic lung of the molecular chaperons and ubiquitin ligases in the fate of hyperphosphorylated tau and the toxic form of the amyloid precursor protein (APP). fibroblasts (HFL-1), hallmarks of premature senescence are seen: CHIP appears to have an important role in APP metabolism. roleirreversible of CHIP cell in growth preventing arrest, oxidative induction stress; of p16, CHIPand accumulation knockdown APP undergoes ubiquitination on its C-terminal cytoplasmic resultsof β-gal inpositive pronounced cells [60]. accumulation This could be of explained oxidized in proteins part by andthe region [65]. Stable CHIP-APP interactions are detected in the addition of antioxidants only slightly delayed senescence different cell cultures and post-mortem human brain. Evidence of this is the increase in the levels of normal APP after CHIP over [59,60]. Oxidative stress is likely to result from an impaired expression and proteasome inhibition. Pull-down experiments have shown an increase in the levels of ubiquitinated APP ubiquitin-proteasome system (UPS), which in turn promotes-/-) interacting with CHIP. APP undergoes proteolytic cleavage by oxidativeshow increases stress, longevity triggering of a polyubiquitin- vicious cycle. Mouseand proteasomal- embryonic - and - secretases to form C99 and C83 amino acid C-terminal fibroblastssubstrates [60](MEFs) as derived well as from in-creases CHIP knock-out in oxidized mice protein (CHIP and β α effect due to CHIP-mediated degradation of oxidized proteins APP fragments (CTFs), respectively. In cells- amyloid over expressing peptide CHIP(A ) bylipids the [58]. proteasome Conversely, preventing over expressing imbalances CHIP inhas protein cytoprotective quality CTFsproduct [66] of accumulate and are more stable. In addition, CHIP removal might also influence the accumulation of the β β one hand CHIPγ- secretase interacts action with HSPson C99, to stabilize accelerating normal Aβ APP and betweencontrol and proteostasis reduced longevity and the epigenetic [60]. Further, regulation CHIP bindsin aging to, [49]. and onand the protecting other it againstassists inaccompanying the ubiquitination toxicity. of Ina subpopulationconclusion, on Inregulates, addition SirT6to the andeffects this of interactionCHIP on longevity defines and an cellular intersection aging of APP molecules that are destined for proteasome degradation there is also growing evidence that CHIP can protect form aging [66]. related diseases such as neurodegeneration and cancer. An important characteristic of AD is the formation NFTs Neurodegeneration that are composed of the microtubule-associated protein tau. General neuroprotective function(s) of chip linked to During the development of disease pathology tau becomes oxidative stress: There is evidence that the neuroprotective and aggregates [67]. Tau lesions in post-mortem human tissue hyperphosphorylated, detaches from the axonal microtubules Ineffect turn of the CHIP antioxidant can be linked, effects at of least CHIP in appear part, to to its be ability linked to ubiquitinates tau in vitro and in vivo [68]. CHIP decreases the protect neurons and CNS cells from oxidative stress [52,58,60]. amountare immune-positive of total tau in for a CHIP TPR-domain which interacts dependent and manner specifically [69] Oxidoredctase 1 (NQO1); an enzyme that is induced in response suggesting a role for CHIP chaperone activity or Hsp binding. toits ability electrophilic to ubiquitinate and/or oxidative a flavoenzyme, stress NAD [61] (P) and H: interacts Quinone with the 20S proteasome to prevent the degradation of some while the level of tau mRNA is unchanged suggesting that the proteins [62]. Whilst the level of CHIP is down-regulated substantialCHIP deficient increase mice have in phospho-tau reduced Hsp70 and mRNA total-tau and is protein due to level role with ageing NQO1 shows a corresponding increase [61]. CHIP endonuclease responsible for mediating caspase-independent phosphorylatedof CHIP in tau protein tau species degradation in the [70]. presynaptic Thus, in the compartment. absence of DNAalso interacts fragmentation with endonuclease which leads to G cell (EndoG), death a following mitochondrial acute CHIP there is an increased accumulation of ubiquitin-negative, susceptibility to cell death following treatment with the oxidant In addition, these mice exhibit increased caspase-3, cleaved oxidative stress [63,64]. Cells depleted of CHIP have an increased caspase-3 and caspase-3-cleaved tau. However, neither H2O2 (~1.7 fold higher) and increased levels of EndoG both before ubiquitinationhyperphospholylation has an importantnor caspase-3 role cleavedin the formation tau are sufficient of the tau to with Hsp70 and is ubiquitinated by CHIP in a Hsp-dependent aggregatesinduce aggregation [70]. of tau in the absence of CHIP, suggesting that and after treatment. Under normal conditions, EndoG interacts manner. Treatment with the oxidizing agent H2O2 abolishes the and EndoG translocates to the nucleus for DNA fragmentation. In cell cultures, CHIP increases the insoluble tau fraction interaction with Hsp70, and in turn with the TPR domain of CHIP, Observed age-dependent decreases in CHIP levels most likely taucomposed is an Hsp90 of high substrate molecular and it weightfacilitates aggregated its dephosphorylation tau species, explain the corresponding increase in EndoG and enhanced andwhist refolding expression preventing of Hsp70 degradation.reduces it [68]. Thus Moreover, inhibition phospho- of the susceptibility to oxidative stress. Hsp90 refolding pathway and over expression of CHIP result Chip in alzheimer’s disease: AD is one of the most common in the degradation of phosphorylated tau [71]. In AD patient language disturbances and executive dysfunction as well asforms a decline of late-onset in cognitive dementia, function. characterized Pathological by memory hallmarks loss, of expressionbrains, there of isfull an length inverse tau relationship or a tau isoform between results CHIP in and changes PHF- intau mitochondrial (paired helical distribution filaments) a phenomenon accumulation that [72]. can Finally, be rescued over plaquesthe disease composed are intracellular of extracellular neurofibrillary deposits of amyloid- tangles (NFT) (A ). Chip in parkinson’s disease: PD is a progressive Accumulationcomposed of filamentousof these abnormal hyperphosphorylated protein aggregates tau is andassociated senile by CHIP [69,73]. β β neurodegenerative disease caused by selective degeneration of with neuronal loss. There is growing evidence for the importance dopaminergic neurons. The pathological hallmark of the disorder

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aggregatedis the presence -Syn. of intracytoplasmic Under certain fibrillary cellular conditions aggregates called-Syn Alternatively, epigenetics is a plausible explanationCHIP for promoter the low Lewy Bodies (LB) the main component of which is misfolded, CHIP mRNA levels. Gastric, colorectal and breast cancer patients α α methylationhave significant and hyper-DNA-methylation it was hypothesized thatin the other epigenetic degradationstarts to form and oligomers, protein which chaperone ultimately machinery leads to in aggregation regulated [84,88]. Although CHIP levels did not correlate with promoter into amyloid-like fibrils [74-76]. Thus, the role of proteasome discrepancy [89]. Thus the mechanism causing reduced CHIP changes, such as histone deacetylation, may account for this α-Syn levels and conformation is of major interest in PD research. differ between cancer type and from patient to patient. co-localizes with CHIP and Hsp70 in intracellular inclusions. expression in tumours is controversial, and the mechanism may OverIn expression cell culture of modelsCHIP results and in a human reduction brain in tissues,the number α-Syn, of Effects of chip on cell growth parameters: Typical of cells containing morphological changes transforming large inclusions into micro-aggregatesα dispersed-Syn inclusions throughout [77]. the Moreover, cytoplasm CHIP in a causes TPR- independenta tumour suppressor growth and protein, induces CHIP cell death. over expressionOn the other inhibits hand dependent manner [77]. It is reported that the degradation of reducedoncogenic levels signaling of the pathways, CHIP protein cell promote migration tumour and anchorage formation -Syn can be achieved both by the proteasome pathway in a TPR metastasis and this is thought to be mediated primarily by the dependent manner and by U-box domain-mediated lysosomal degradation of oncogenic proteins [85]. An example of this degradationα [74]. It is not surprising therefore that CHIP over- expressed in 50-70% of invasive breast cancers and can even be a molecular weight -Syn oligomers and rescues the resultant is CHIP’s effect on glucocorticoid receptor (GR) levels, GR is cytotoxicityexpression reduces[78]. the levels of intermediate, toxic and high luminal breast cancer. GR has anti-proliferative and anti-apoptotic α functionsbiological and and prognostic its levels biomarker decrease duringof lower cancer grade, progressionER-positive In vitro and in vivo [90]. In situ ubiquitination assays confirmed the ability and transfecting an E3 ligase-defective CHIP shows dominant- , CHIP ubiquitinates GR in a dose-dependent manner of CHIP to mono-, multi mono- and poly-ubiquitinate α−Syn [79]. the HC8 particle of the S5a proteasome subunit and such direct The E3 ligase activity of CHIP, and thus α-Syn ubiquitination,-Syn interactionnegative effects. most likelyImportantly, occurs inCHIP the contextco-immunoprecipitates of the assembled with 26S canexist bewithin inhibited the same by complex BAG5, which and their causes interaction enhanced is dependent dopamine neuron death in a model of PD [43,79]. BAG5, CHIP and α proteasome complexes. It may seem counterintuitive in the context of cancer that inon decreasedthe Hsp70 chaperone. Finally, α-Syn co-immuno precipitates tumours show lower levels of CHIP compared to surrounding with Hsp70 and Hsp90 [80], and inhibition of Hsp90 resulting-Syn is a Hsp90 substrateα-Syn oligomerizationwhich could be ubiquitinated and toxicity inand Drosophila, degraded yeast and cellular models of PD [81-83], suggesting that α healthy tissue. As discussed above, cells depleted of CHIP perhaps introduce a note of caution as much of the research on become prematurely senescent, possibly due to defects in redox CHIPby CHIP in relation in an Hsp70 to -Syn dependent has used manner. cell systems However, in which we shouldeither and protein homeostasis [59,60], which could prevent cancer mutant forms of CHIP or tagged forms of -Syn are over expressed. PRMT5growth causes [91]. However,epigenetic CHIP changes causes that degradationpromote tumourgenicity of strategic α andoncoproteins, is upregulated such as during protein the arginine development methyltransferase and progression 5 [85]. ubiquitination of endogenous -Syn in αhealthy individuals or in humanTherefore, pathologies it is still is not physiologically clear whether relevant. direct CHIP-dependent What is clear is that CHIP plays a central role inα maintaining healthy aging in the methyltransferaseof several cancers functions including as lymphoma, an oncoprotein leukemia by methylating and breast, histonegastric, colorectal4 at arginine and 3 ovarian and/or cancers histone [92-94]. H3 at arginine This type 8 to II brainCancer and CNS [52,58]. Accumulating evidence indicates that CHIP predominantly silence the expression of tumour suppressors, including p53 and RB [87,94]. Alternatively, PRMT5 can methylate non-histone substrates to activate signalling molecules such as E2F1, p53, functions as a tumour suppressor, preventing cell proliferation RelA/p65, epidermal growth factor receptor (EGFR), RAD9, and Theseand/or cellular promoting effects apoptosisare the result in of breast, CHIP-mediated gastric, regulation prostrate, rendingprogrammed cancer cell cells death with 4 [87,93]. a survival Methylation advantage. of E2F1 Methylation reduces pancreatic and colorectal cancers, glioma and hepatoma [84,85]. its ability to suppress cell growth and to promote apoptosis, of oncogenic proteins, including the estrogen receptor ERα, targetingof p65 activates it for proteasomal the NF-κβ degradation. and TNF signalling Such post-translational pathways [87]. ErbB2, androgen receptor, histone deacetylase 6, hypoxia- CHIP mediated the K48-linked polyubiquitination of PRMT5, mesenchymalinducible factor-1A transition (HIF-1a), [85-87] C-Myc, (Figure p65 and2). EGFR, which in turn influences NF-kβ signaling, the Akt pathway and the epithelial- Hsp90modification inhibitors was dependent decreased on PRMT5 both the protein TPR and expression U-box domains in a Different groups have tried to understand the mechanism dose-dependentof CHIP for PRMT5 recognition and ubiquitination, respectively. underlying the low CHIP gene expression detected across tumours. Two cancer gene databases (The Cancer Genome Atlas may be regulated manner,by the molecular and this chaperone was dependent system involving on CHIP expression. Again, this suggests that the degradation of PRMT5 analyses of copy-number variations detected loss of the gene under these conditions increased cell death and inhibited cell inand 17.5% COSMIC) of renalrevealed and no 45% “loss-of-function” of ovarian cancer mutations. patient However, samples. growth;CHIP, Hsp90 such effects and Hsp70. may be Interesting, mediated by over CHIP-dependent expression of down- CHIP

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CHIP * * *

Less ubiquitination of Reduced expression of Less ubiquitination of oncoproteins (directly) tumour suppressors (indirectly) signaling molecules

Ub-SRC-3 Ub-GR Ub-PRMT5 Ub-TG2 Ub-TRAF (?) SRC-3 accumulation 1 1 Transcription VHL Activates: causes increased Smad2 levels VHL VHL TG2-mediated VHL Canonical NF-κΒ RelA Proliferation activated crosslinking VHL VHL Smad2 VHL Epigenetic changes Polymers of VHL Non-canonical NF-κΒ RelB Methylation VHL Twist (unfunctional) B-Catenin Transcription VHL-mediated 2 repressed Vimentin Apoptosis ubiquitination HIF-1α RelA Epithelial-mesenchymal transistion or HIF-1α HIF-1α EPITHELIA (metastasis & invasion) MESENCHYMAL Hypoxia-inducible genes RelB p53 E2F1 1 1 Cell growth Chemotherapy Invasiveness Angiogenesis Integrin-B1 Proliferation resistance MMP-2 MMP-9 ECM 2 Activates non-canonical NF-κΒ

Apoptosis 3 Decreases PTEN causing AKT pathway 2 2 Angiogenesis Activates NF-κΒ to be constitutively active p-AKT (CHIP may act by increasing the levels of anti-apoptotic Bcl-2) Proliferation

Figure 2

Changes in CHIP activity reduce the ubiquitination of oncoproteins, tumour suppressors and signalling molecules. The net cellular effects include overall mesenchymal transition (thus promoting metastasis and invasiveness). suppression of apoptosis and enhanced cell proliferation, cell growth (including anchorage-independent growth), angiogenesis, chemotherapy resistance, epithelial- Interactions between signalling pathways are meant to illustrate protein-protein interactions downstream of CHIP. They do not necessarily mean that all processes occur

*E3 ligase activity that is TPR domain-dependent. in a specific cancer. Abbreviations: CHIP (C-Terminus of Hsc70 Interacting Protein); Ub (Ubiquitinated); SRC-3 (Steroid Receptor Co-Activator 3); GR (Glucocorticoid Receptor); PRMT5 (Protein Arginine Methyltransferase 5); TG2 (Transglutaminase 2); VHL (Von Hippel-Lindau); HIF-1a (Hypoxia-Inducible Factor 1a); P (Phosphorylated); NF-Kb (Nuclear Factor Kappa Beta) And MMP (Matrix Metalloproteinase’s)

regulation of PRMT5 expression [87].

CHIP expression is negatively correlated with a reduction in its phosphorylated, active form (p-AKT at either transglutaminase 2 (TG2) in renal cell carcinoma. The catalytic expressionSer473 or Thr308),of the anti-apoptotic leading to reduced gene Bcl-2 cellular proliferation. Additionally, CHIP over-expression markedly decreased the its K48-linked ubiquitination and degradation. CHIP increases , causing increased core of TG2 interacts with the TPR domain of CHIP, followed by the steady-state levels and stability of TG2. Pharmacological apoptosis [95]. Therefore, in both gastric and breast cancer cells, inhibition of Hsp70 inhibits CHIP-mediated TG2 degradation and where CHIP expression is low, Bcl-2 and Akt are upregulated, leading to anchorage-independent cell growth [87,88]. In breast cancer, SRC-3-mediated upregulation of Smad2 (a signal conversely, over-expression of Hsp70 enhances its degradation The latter is related to aggressive phenotypes in tumours by regulated by CHIP in the canonical pathway where it cooperates transducer in the TFG-β signalling pathway) was also observed. with[85]. the Therefore, core molecular TG2 andchaperones. PRMT5 The degradation increased expression seem to beof promote EMT and thus allowing benign tumours to metastasize. Suchdirectly invasive upregulating and metastatic twist, character β-catenin was and abolished vimentin, when which SRC- decreased in breast cancer and glioma possibly due to hyper- TG2 may be specific to renal cancer, since expression is greatly hindered during tumour development possibly due to alterations 3 was silenced [87,88]. The expression of RelA/p65 and RelB methylation [85]. In renal cancer, TG2 degradation might be respectively) were also upregulated. Driving this dysregulation was(involved the decrease in the canonical in CHIP-mediated and non-canonical suppression NF-κβ ofpathways, TRAF2 [85].in the balance between CHIP and TG2 expressions, conditions that favour cell growth, migration, metastasis and angiogenesis Effects of chip on cell signalling in cancer: Alterations expression (possibly by ubiquitination and degradation), a ofpositive the gelatinases regulator (matrixof both metalloproteinase)branches of the NF-κβsignalling. MMP-2 and MMP- NF- affecting multiple signalling pathways both directly and indirectly. κβ and AP-1 are transcription factors that bind to the promoter Whento CHIP over-expression expression lead CHIP to imbalancesin a human in gastric protein cancer homeostasis, cell line are closely associated with cancer invasion: MMP-2 promotes cleavage9 and of theof the integrin-β-1 extracellular gene, matrix regulating while their MMP-9 activity. modulates These the expression level of Akt was unchanged, although there was JSM Enzymol Protein Sci 1(1): 1006 (2016) 7/10 Ball et al. (2016) Email:

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Central Bringing Excellence in Open Access permeability of the vascular endothelium [95]. High expression of in kinase activation and cancer. Front Oncol. 2012; 2: 5. 10. al. Chaperoned ubiquitylation--crystal structures of the CHIP U box E3 integrin-β-1 has been detected during invasion, angiogenesis and ubiquitinZhang M, ligaseWindheim and a M, CHIP-Ubc13-Uev1a Roe SM, Peggie M, complex. Cohen P, Mol Prodromou Cell. 2005; C, 20: et metastasis of breast cancer and glioblastoma. In line with this, 525-538. CHIP over expression inhibited the activity of the NF-κβ pathway leading to diminished migration and invasion of gastric cancer 11. cellsand caused [95]. decreased levels of MMP-2, MMP-9 and integrin-β-1, ofXu HSP70 Z, Devlin interacting KI, Ford protein. MG, Biochemistry. Nix JC, Qin J, 2006; Misra 45: S. 4749-4759. Structure and DISCUSSION AND CONCLUSION interactions of the helical and U-box domains of CHIP, the C terminus 12. As we discover more about the dual functions of CHIP as an E3 the quality control ubiquitin ligase CHIP and ubiquitin conjugating ubiquitin-ligase and chaperone working as part of the molecular enzymes.Xu Z, Kohli BMC E, Devlin Struct KI, Biol. Bold 2008; M, Nix8: 26. JC, Misra S. Interactions between 13. chaperone network, or more independently, as a regulator of position in proteostasis. As the loss or impairment of CHIP 2012;Zeytuni 20: N, 397-405. Zarivach R. Structural and functional discussion of the function(s)native protein has function, the potential it is clearto cause that pronounced CHIP occupies homeostatic a central tetra-trico-peptide repeat, a protein interaction module. Structure. 14. ässle M. The tetratricopeptide repeat: a structural motif mediating protein-protein interactions. Bioessays. 1999; 21: 932-939. Blatch GL, L Yetdefects despite and growing disease pathologies,knowledge on it the has biology been put of forwardCHIP we asstill a 15. potential therapeutic target in a range of conditions [86,96,97]. know relatively little about how its structure is related to its Andrade MA, Perez-Iratxeta C, Ponting CP. Protein repeats: structures, 16. functions, and evolution. J Struct Biol. 2001; 134: 117-131.ö chaperone and E3-ligase activities of CHIP coordinated or The co-chaperone CHIP regulates protein triage decisions mediated Connell P, Ballinger CA, Jiang J, Wu Y, Thompson LJ, H hfeld J, et al. differentiallyfunctions and regulated activities. to ensure For example, that the howendogenous are the protein intrinsic is by heat-shock proteins. Nat Cell Biol. 2001; 3: 93-96. able to respond in multiple proteostatic pathways. Although the 17. use of mutated exogenous proteins expressed in cells can take us some way down the path to understanding the activities and ubiquitinationZhang H, Amick of J, Chakravarti chaperoned R, client Santarriaga proteins. S, Schlanger Structure. S, 2015; McGlone 23: 472-482.C, et al. A bipartite interaction between Hsp70 and CHIP regulates of mutations and interacting proteins on CHIPs activity though 18. regulation of CHIP, it has its limitations as illustrated by the effects Dimerization of the human E3 ligase CHIP via a coiled-coil domain is Nikolay R, Wiederkehr T, Rist W, Kramer G, Mayer MP, Bukau B. changes in structural flexibility linked to allosteric regulation E3-ligase or chaperone activity of endogenous CHIP would allow 19. essential for its activity. 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