Influenza A virus nucleoprotein exploits Hsp40 to inhibit PKR activation

Background

Double-stranded RNA dependent protein kinase (PKR) is a key regulator of the anti-viral innate immune response in mammalian cells. PKR activity is regulated by a 58 kilo Dalton cellular inhibitor (P58^IPK), which is present in inactive state as a complex with Hsp40 under normal conditions. In case of influenza A virus (IAV) infection, P58^IPK is known to dissociate from Hsp40 and inhibit PKR activation. However the influenza virus component responsible for PKR inhibition through P58^IPK activation was hitherto unknown.

Principal Findings

Human heat shock 40 protein (Hsp40) was identified as an interacting partner of Influenza A virus nucleoprotein (IAV NP) using a yeast two-hybrid screen. This interaction was confirmed by co-immunoprecipitation studies from mammalian cells transfected with IAV NP expressing plasmid. Further, the IAV NP-Hsp40 interaction was validated in mammalian cells infected with various seasonal and pandemic strains of influenza viruses. Cellular localization studies showed that NP and Hsp40 co-localize primarily in the nucleus. During IAV infection in mammalian cells, expression of NP coincided with the dissociation of P58^IPK from Hsp40 and decrease PKR phosphorylation. We observed that, plasmid based expression of NP in mammalian cells leads to decrease in PKR phosphorylation. Furthermore, inhibition of NP expression during influenza virus replication led to PKR activation and concomitant increase in eIF2α phosphorylation. Inhibition of NP expression also led to reduced IRF3 phosphorylation, enhanced IFN β production and concomitant reduction of virus replication. Taken together our data suggest that NP is the viral factor responsible for P58^IPK activation and subsequent inhibition of PKR-mediated host response during IAV infection.

Significance

Our findings demonstrate a novel role of IAV NP in inhibiting PKR-mediated anti-viral host response and help us understand P58^IPK mediated inhibition of PKR activity during IAV infection.     

Regulation of Interferon-Induced Protein Kinase PKR: Modulation of P58IPK Inhibitory Function by a Novel Protein, P52rIPK

The cellular response to environmental signals is largely dependent upon the induction of responsive protein kinase signaling pathways. Within these pathways, distinct protein-protein interactions play a role in determining the specificity of the response through regulation of kinase function. The interferon-induced serine/threonine protein kinase, PKR, is activated in response to various environmental stimuli. Like many protein kinases, PKR is regulated through direct interactions with activator and inhibitory molecules, including P58^IPK, a cellular PKR inhibitor. P58^IPK functions to represses PKR-mediated phosphorylation of the eukaryotic initiation factor 2α subunit (eIF-2α) through a direct interaction, thereby relieving the PKR-imposed block on mRNA translation and cell growth. To further define the molecular mechanism underlying regulation of PKR, we have utilized an interaction cloning strategy to identify a novel cDNA encoding a P58^IPK-interacting protein. This protein, designated P52^rIPK, possesses limited homology to the charged domain of Hsp90 and is expressed in a wide range of cell lines. P52^rIPK and P58^IPK interacted in a yeast two-hybrid assay and were recovered as a complex from mammalian cell extracts. When coexpressed with PKR in yeast, P58^IPK repressed PKR-mediated eIF-2α phosphorylation, inhibiting the normally toxic and growth-suppressive effects associated with PKR function. Conversely, introduction of P52^rIPK into these strains resulted in restoration of both PKR activity and eIF-2α phosphorylation, concomitant with growth suppression due to inhibition of P58^IPK function. Furthermore, P52^rIPK inhibited P58^IPK function in a reconstituted in vitro PKR-regulatory assay. Our results demonstrate that P58^IPK is inhibited through a direct interaction with P52^rIPK which, in turn, results in upregulation of PKR activity. Taken together, our data describe a novel protein kinase-regulatory system which encompasses an intersection of interferon-, stress-, and growth-regulatory pathways.     

The crystal structure of the human co-chaperone P58(IPK)

P58^IPK is one of the endoplasmic reticulum- (ER-) localised DnaJ (ERdj) proteins which interact with the chaperone BiP, the mammalian ER ortholog of Hsp70, and are thought to contribute to the specificity and regulation of its diverse functions. P58^IPK, expression of which is upregulated in response to ER stress, has been suggested to act as a co-chaperone, binding un- or misfolded proteins and delivering them to BiP. In order to give further insights into the functions of P58^IPK, and the regulation of BiP by ERdj proteins, we have determined the crystal structure of human P58^IPK to 3.0 Å resolution using a combination of molecular replacement and single wavelength anomalous diffraction. The structure shows the human P58^IPK monomer to have a very elongated overall shape. In addition to the conserved J domain, P58^IPK contains nine N-terminal tetratricopeptide repeat motifs, divided into three subdomains of three motifs each. The J domain is attached to the C-terminal end via a flexible linker, and the structure shows the conserved Hsp70-binding histidine-proline-aspartate (HPD) motif to be situated on the very edge of the elongated protein, 100 Å from the putative binding site for unfolded protein substrates. The residues that comprise the surface surrounding the HPD motif are highly conserved in P58^IPK from other organisms but more varied between the human ERdj proteins, supporting the view that their regulation of different BiP functions is facilitated by differences in BiP-binding.     

Double-Stranded RNA-Independent Dimerization of Interferon-Induced Protein Kinase PKR and Inhibition of Dimerization by the Cellular P58IPK Inhibitor

The interferon (IFN)-induced, double-stranded RNA-activated protein kinase (PKR) mediates the antiviral and antiproliferative actions of IFN, in part, via its translational inhibitory properties. Previous studies have demonstrated that PKR forms dimers and that dimerization is likely to be required for activation and/or function. In the present study we used multiple approaches to examine the modulation of PKR dimerization. Deletion analysis with the λ repressor fusion system identified a previously unrecognized site involved in PKR dimerization. This site comprised amino acids (aa) 244 to 296, which span part of the third basic region of PKR and the catalytic subdomains I and II. Using the yeast two-hybrid system and far-Western analysis, we verified the importance of this region for dimerization. Furthermore, coexpression of the 52-aa region alone inhibited the formation of full-length PKR dimers in the λ repressor fusion and two-hybrid systems. Importantly, coexpression of aa 244 to 296 exerted a dominant-negative effect on wild-type kinase activity in a functional assay. Due to its role as a mediator of IFN-induced antiviral resistance, PKR is a target of viral and cellular inhibitors. Curiously, PKR aa 244 to 296 contain the binding site for a select group of specific inhibitors, including the cellular protein P58^IPK. We demonstrated, utilizing both the yeast and λ systems, that P58^IPK, a member of the tetratricopeptide repeat protein family, can block kinase activity by preventing PKR dimerization. In contrast, a nonfunctional form of P58^IPK lacking a TPR motif did not inhibit kinase activity or perturb PKR dimers. These results highlight a potential mechanism of PKR inhibition and define a novel class of PKR inhibitors. Finally, the data document the first known example of inhibition of protein kinase dimerization by a cellular protein inhibitor. On the basis of these results we propose a model for the regulation of PKR dimerization.Cellular protein kinases play crucial roles in propagating, regulating, and coordinating signals necessary for many seminal biological processes, including metabolism, gene expression, cell growth, differentiation, and development. As a result, protein kinases are subjected to elaborate control mechanisms, including association with domains or subunits that inhibit kinase activity by an autoregulatory process (40, 44) or domains that target the kinase to different subcellular localizations and/or substrates (23, 36). In addition, association with activating or inhibitory proteins (21, 86), reversible protein phosphorylation (19, 32), and multimerization (31, 76) also may regulate kinase activity. While dimerization is a common regulatory mechanism for receptor protein kinases, it is less so for cytosolic nonreceptor protein kinases. The latter class of protein kinases, whose dimerization is implicated in their activation and/or function, includes the cGMP- and cAMP-dependent kinases (81), casein kinase 2 (9), Mst1 kinase (17), Raf-1 kinase (22), and the interferon (IFN)-induced, double-stranded (ds)-RNA-activated kinase (PKR) (60). PKR is novel in that it also regulates its own protein synthesis at the translational level (7, 82).PKR is a pivotal component of the host antiviral defense system because of its translational inhibitory properties (58, 74). Viral replication produces dsRNA that can bind PKR via two dsRNA-binding motifs (DSRMs) located in the N-terminal portion of the kinase, resulting in autophosphorylation and consequently activation of the enzyme. Activated PKR, in turn, phosphorylates the α subunit of eukaryotic initiation factor-2 (eIF-2α), leading to a complex series of biochemical events that culminate in a dramatic decrease in the initiation of protein synthesis (15, 59). This disables the use of the translational machinery for the production of viral proteins, and hence restricts viral replication within the cell. Due to its function in antiviral defense, PKR is a target of viral and cellular inhibitors (42, 51). The best-characterized cellular protein inhibitor of PKR is P58^IPK, which is activated upon influenza virus infection (53, 54). P58^IPK appears to be a member of a potential new class of molecular chaperones containing tetratricopeptide repeat motifs and the “J region” of the DnaJ family (52, 62). The non-enzymatic P58^IPK protein inhibits both the auto- and trans-phosphorylation activities of PKR (53, 54). However, the exact mode of P58^IPK action is not fully understood, although it likely involves direct physical interaction with PKR (25, 69).In addition to its role in interferon-induced antiviral resistance, there is growing evidence that PKR is involved in the control of cell growth and proliferation. Overexpression of PKR in mouse (46), insect (4), and yeast (14) cells results in severe inhibition of growth due to increased eIF-2α phosphorylation. Furthermore, expression of catalytically inactive mutants of PKR elicits fibroblast transformation and tumor formation upon injection of the cells into nude mice, suggesting that PKR has tumor suppressor properties (6, 46, 61). In support of this view, the P58^IPK protein exhibits oncogenic potential; overexpression of the cellular PKR inhibitor causes a transformed phenotype and rapid tumor formation in nude mice (3).The mechanism(s) by which the functionally defective PKR mutants or wild-type P58^IPK induce malignant transformation is not known. One hypothesis is that the PKR mutants inhibit kinase function by forming inactive heterodimers with endogenous wild-type PKR (6, 46). This also raises a fundamental question concerning the role of dimerization on PKR function. Indeed, evidence for PKR dimerization dependent on the DSRMs has been reported (16, 67), although its role in activation and/or function remains unclear (71, 85). Moreover, the role of P58^IPK in modulating PKR dimer formation has not been investigated.Recent efforts to identify the region and mechanism responsible for PKR dimerization have led to conflicting results (16, 66, 67). This controversy can be explained, at least in part, by our demonstration herein that PKR can dimerize through a previously unrecognized region independent of the DSRMs. In addition, we show that P58^IPK, but not a nonfunctional form of the protein, prevents PKR dimer formation, suggesting that P58^IPK inhibits PKR by converting PKR dimers into stable monomers. To our knowledge, our findings present the first known example of a cytosolic kinase whose activity may be modulated at the level of dimerization through association with a nonenzymatic cellular protein inhibitor.     

Active regulator of SIRT1 is required for ribosome biogenesis and function

Active regulator of SIRT1 (AROS) binds and upregulates SIRT1, an NAD⁺-dependent deacetylase. In addition, AROS binds RPS19, a structural ribosomal protein, which also functions in ribosome biogenesis and is implicated in multiple disease states. The significance of AROS in relation to ribosome biogenesis and function is unknown. Using human cells, we now show that AROS localizes to (i) the nucleolus and (ii) cytoplasmic ribosomes. Co-localization with nucleolar proteins was verified by confocal immunofluorescence of endogenous protein and confirmed by AROS depletion using RNAi. AROS association with cytoplasmic ribosomes was analysed by sucrose density fractionation and immunoprecipitation, revealing that AROS selectively associates with 40S ribosomal subunits and also with polysomes. RNAi-mediated depletion of AROS leads to deficient ribosome biogenesis with aberrant precursor ribosomal RNA processing, reduced 40S subunit ribosomal RNA and 40S ribosomal proteins (including RPS19). Together, this results in a reduction in 40S subunits and translating polysomes, correlating with reduced overall cellular protein synthesis. Interestingly, knockdown of AROS also results in a functionally significant increase in eIF2α phosphorylation. Overall, our results identify AROS as a factor with a role in both ribosome biogenesis and ribosomal function.     

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.     

Ca2+/S100 Proteins Act as Upstream Regulators of the Chaperone-associated Ubiquitin Ligase CHIP (C Terminus of Hsc70-interacting Protein)

The U-box E3 ubiquitin ligase CHIP (C terminus of Hsc70-interacting protein) binds Hsp90 and/or Hsp70 via its tetratricopeptide repeat (TPR), facilitating ubiquitination of the chaperone-bound client proteins. Mechanisms that regulate the activity of CHIP are, at present, poorly understood. We previously reported that Ca²⁺/S100 proteins directly associate with the TPR proteins, such as Hsp70/Hsp90-organizing protein (Hop), kinesin light chain, Tom70, FKBP52, CyP40, and protein phosphatase 5 (PP5), leading to the dissociation of the interactions of the TPR proteins with their target proteins. Therefore, we have hypothesized that Ca²⁺/S100 proteins can interact with CHIP and regulate its function. GST pulldown assays indicated that Ca²⁺/S100A2 and S100P bind to the TPR domain and lead to interference with the interactions of CHIP with Hsp70, Hsp90, HSF1, and Smad1. In vitro ubiquitination assays indicated that Ca²⁺/S100A2 and S100P are efficient and specific inhibitors of CHIP-mediated ubiquitination of Hsp70, Hsp90, HSF1, and Smad1. Overexpression of S100A2 and S100P suppressed CHIP-chaperone complex-dependent mutant p53 ubiquitination and degradation in Hep3B cells. The association of the S100 proteins with CHIP provides a Ca²⁺-dependent regulatory mechanism for the ubiquitination and degradation of intracellular proteins by the CHIP-proteasome pathway.     

Interaction of the interferon-induced PKR protein kinase with inhibitory proteins P58IPK and vaccinia virus K3L is mediated by unique domains: implications for kinase regulation.

Expression of the double-stranded RNA-activated protein kinase (PKR) is induced by interferons, with PKR activity playing a pivotal role in establishing the interferon-induced antiviral and antiproliferative states. PKR is directly regulated by physical association with the specific inhibitor, P58IPK, a cellular protein of the tetratricopeptide repeat (TPR) family, and K3L, the product of the corresponding vaccinia virus gene. P58IPK and K3L repress PKR activation and activity. To investigate the mechanism of P58IPK- and K3L-mediated PKR inhibition, we have used a combination of in vitro and in vivo binding assays to identify the interactive regions of these proteins. The P58IPK-interacting site of PKR was mapped to a 52-amino-acid aa segment (aa 244 to 296) spanning the ATP-binding region of the protein kinase catalytic domain. The interaction with PKR did not require the C-terminal DNA-J homology region of P58IPK but was dependent on the presence of the eukaryotic initiation factor 2-alpha homology region, mapping to the 34 aa within the sixth P58IPK TPR motif. Consistent with other TPR proteins, P58IPK formed multimers in vivo: the N-terminal 166 aa were both necessary and sufficient for complex formation. A parallel in vivo analysis to map the K3L-binding region of PKR revealed that like P58IPK , K3L interacted exclusively with the PKR protein kinase catalytic domain. In contrast, however, the K3L-binding region of PKR was localized to within aa 367 to 551, demonstrating that each inhibitor bound PKR in unique, nonoverlapping domains. These data, taken together, suggest that P58IPK and K3L may mediate PKR inhibition by distinct mechanisms. Finally, we will propose a model of PKR inhibition in which P58IPK or a P58IPK complex binds PKR and interferes with nucleotide binding and autoregulation, while formation of a PKR-K3L complex interferes with active-site function and/or substrate association.     

Attenuation of 40S Ribosomal Subunit Abundance Differentially Affects Host and HCV Translation and Suppresses HCV Replication

For Hepatitis C virus (HCV), initiation of translation is cap-independently mediated by its internal ribosome entry site (IRES). Unlike other IRES-containing viruses that shut off host cap-dependent translation, translation of HCV coexists with that of the host. How HCV IRES-mediated translation is regulated in the infected cells remains unclear. Here, we show that the intracellular level of 40S ribosomal subunit plays a key role in facilitating HCV translation over host translation. In a loss-of-function screen, we identified small subunit ribosomal protein 6 (RPS6) as an indispensable host factor for HCV propagation. Knockdown of RPS6 selectively repressed HCV IRES-mediated translation, but not general translation. Such preferential suppression of HCV translation correlated well with the reduction of the abundance of 40S ribosomal subunit following knockdown of RPS6 or other RPS genes. In contrast, reduction of the amount of ribosomal proteins of the 60S subunit did not produce similar effects. Among the components of general translation machineries, only knockdowns of RPS genes caused inhibitory effects on HCV translation, pointing out the unique role of 40S subunit abundance in HCV translation. This work demonstrates an unconventional notion that the translation initiation of HCV and host possess different susceptibility toward reduction of 40S ribosomal subunit, and provides a model of selective modulation of IRES-mediated translation through manipulating the level of 40S subunit.     

Ribosomal proteins Rps0 and Rps21 of Saccharomyces cerevisiae have overlapping functions in the maturation of the 3' end of 18S rRNA

The Rps0 proteins of Saccharomyces cerevisiae are components of the 40S ribosomal subunit required for maturation of the 3′ end of 18S rRNA. Drosophila and human homologs of the Rps0 proteins physically interact with Rps21 proteins, and decreased expression of both proteins in Drosophila impairs control of cellular proliferation in hematopoietic organs during larval development. Here, we characterize the yeast RPS21A/B genes and show that strains where both genes are disrupted are not viable. Relative to the wild type, cells with disrupted RPS21A or RPS21B genes exhibit a reduction in growth rate, a decrease in free 40S subunits, an increase in the amount of free 60S subunits, and a decrease in polysome size. Ribosomal RNA processing studies reveal RPS21 and RPS0 mutants have virtually identical processing defects. The pattern of processing defects observed in RPS0 and RPS21 mutants is not a general characteristic of strains with suboptimal levels of small subunit ribosomal proteins, since disruption of the RPS18A or RPS18B genes results in related but distinct processing defects. Together, these data link the Rps0 and Rps21 proteins together functionally in promoting maturation of the 3′ end of 18S rRNA and formation of active 40S ribosomal subunits.     

Fibroblast growth factor-2 interacts with free ribosomal protein S19.

Exogenous FGF-2 added to cells is internalized and part of it translocates to the nucleus of the cells. To get a better understanding of the FGF-2-induced signaling pathway, we looked for proteins associated with FGF-2 in the cytoplasm of the target cells. We first used the GST-FGF-2 to isolate cytoplasmic proteins complexes containing FGF-2 from S100 extract (supernatant 100,000g). Among the retrieved proteins, we focused our studies on RPS19, a protein of the 40S small ribosomal subunit. We showed that FGF-2 interacts directly with RPS19 in vitro. Second, we coimmunoprecipitated RPS19 and FGF-2 from a S240 extract (240,000g supernatant) prepared from FGF-2-stimulated cells and devoid of 40S ribosomal subunit. The result of these experiments suggest that a pool of free RPS19 exists in cells and that FGF-2 interacts in vivo with free RPS19.     

Interaction of Hsp40 with influenza virus M2 protein: implications for PKR signaling pathway

Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58^IPK that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58^IPK by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58^IPKin vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58^IPK, and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58^IPK-mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.     

Analysis of the interactome of ribosomal protein S19 mutants

Diamond–Blackfan anemia, characterized by defective erythroid progenitor maturation, is caused in one‐fourth of cases by mutations of ribosomal protein S19 (RPS19), which is a component of the ribosomal 40S subunit. Our previous work described proteins interacting with RPS19 with the aim to determine its functions. Here, two RPS19 mutants, R62W and R101H, have been selected to compare their interactomes versus the wild‐type protein one, using the same functional proteomic approach that we employed to characterize RPS19 interactome. Mutations R62W and R101H impair RPS19 ability to associate with the ribosome. Results presented in this paper highlight the striking differences between the interactomes of wild‐type and mutant RPS19 proteins. In particular, mutations abolish interactions with proteins having splicing, translational and helicase activity, thus confirming the role of RPS19 in RNA processing/metabolism and translational control. The data have been deposited to the ProteomeXchange with identifier PXD000640 ( http://proteomecentral.proteomexchange.org/dataset/PXD000640 ).     

Inactivation of the PKR protein kinase and stimulation of mRNA translation by the cellular co-chaperone P58(IPK) does not require J domain function

P58(IPK) was discovered as an inhibitor of the interferon-induced, protein kinase, PKR. Upon virus infection, PKR can, as part of the host defense system, inhibit mRNA translation by phosphorylating the alpha subunit of protein synthesis eukaryotic initiation factor 2 (eIF-2alpha). We previously found that influenza virus recruits the cellular P58(IPK) co-chaperone to inhibit PKR activity and thus facilitate viral protein synthesis. P58(IPK) contains nine tetratricopeptide repeat (TPR) motifs in addition to the highly conserved J domain found in all DnaJ chaperone family members. To define the role of molecular chaperones in regulating cell growth in addition to PKR regulation, we performed a detailed analysis of the P58(IPK) J domain. Using growth rescue assays, we found that the P58(IPK) J domain substituted for the J domains of other DnaJ proteins, including DnaJ in Escherichia coli and Ydj1 in Saccharomyces cerevisiae. This is the first time a cellular J domain from a mammalian DnaJ family member was shown to be functional in both prokaryotic DnaJ and eukaryotic Ydj1 constructs. Furthermore, point mutations within the conserved HPD residue cluster of the P58(IPK) J domain disrupted P58(IPK) J function including stimulation of ATPase activity of Hsp70. However, the P58(IPK) HPD mutants still inhibited PKR activity and thus supported cell growth in a yeast rescue assay. Overexpression of the HPD mutants of P58(IPK), similar to their wild-type counterpart, also stimulated mRNA translation in a mammalian cell system. Taken together, our data necessitate a model of P58(IPK) inhibition of PKR kinase activity and stimulation of mRNA translation, which does not require classical J domain function found in the DnaJ molecular chaperone family.     

Endoplasmic Reticulum Protein Quality Control Is Determined by Cooperative Interactions between Hsp/c70 Protein and the CHIP E3 Ligase

The C terminus of Hsp70 interacting protein (CHIP) E3 ligase functions as a key regulator of protein quality control by binding the C-terminal (M/I)EEVD peptide motif of Hsp/c70(90) with its N-terminal tetratricopeptide repeat (TPR) domain and facilitating polyubiquitination of misfolded client proteins via its C-terminal catalytic U-box. Using CFTR as a model client, we recently showed that the duration of the Hsc70-client binding cycle is a primary determinant of stability. However, molecular features that control CHIP recruitment to Hsp/c70, and hence the fate of the Hsp/c70 client, remain unknown. To understand how CHIP recognizes Hsp/c70, we utilized a dominant negative mutant in which loss of a conserved proline in the U-box domain (P269A) eliminates E3 ligase activity. In a cell-free reconstituted ER-associated degradation system, P269A CHIP inhibited Hsc70-dependent CFTR ubiquitination and degradation in a dose-dependent manner. Optimal inhibition required both the TPR and the U-box, indicating cooperativity between the two domains. Neither the wild type nor the P269A mutant changed the extent of Hsc70 association with CFTR nor the dissociation rate of the Hsc70-CFTR complex. However, the U-box mutation stimulated CHIP binding to Hsc70 while promoting CHIP oligomerization. CHIP binding to Hsc70 binding was also stimulated by the presence of an Hsc70 client with a preference for the ADP-bound state. Thus, the Hsp/c70 (M/I)EEVD motif is not a simple anchor for the TPR domain. Rather CHIP recruitment involves reciprocal allosteric interactions between its TPR and U-box domains and the substrate-binding and C-terminal domains of Hsp/c70.