C-terminus Of Hsp70-interacting Protein Research Articles

Loss of regulation of protein synthesis and turnover underpins an attenuated stress response in senescent human mesenchymal stem cells

Cells respond to stress by synthesizing chaperone proteins that seek to correct protein misfolding and maintain function. However, abrogation of protein homeostasis is a hallmark of aging, leading to loss of function and the formation of proteotoxic aggregates characteristic of pathology. Consequently, discovering the underlying molecular causes of this deterioration in proteostasis is key to designing effective interventions to disease or to maintaining cell health in regenerative medicine strategies. Here, we examined primary human mesenchymal stem cells, cultured to a point of replicative senescence and subjected to heat shock, as an invitro model of the aging stress response. Multi -omics analysis showed how homeostasis components were reduced in senescent cells, caused by dysregulation of a functional network of chaperones, thereby limiting proteostatic competence. Time-resolved analysis of the primary response factors, including those regulating heat shock protein 70 kDa (HSPA1A), revealed that regulatory control is essentially translational. Senescent cells have a reduced capacity for chaperone protein translation and misfolded protein (MFP) turnover, driven by downregulation of ribosomal proteins and loss of the E3 ubiquitin ligase CHIP (C-terminus of HSP70 interacting protein) which marks MFPs for degradation. This limits the cell's stress response and subsequent recovery. A kinetic model recapitulated these reduced capacities and predicted an accumulation of MFP, a hypothesis supported by evidence of systematic changes to the proteomic fold state. These results thus establish a specific loss of regulatory capacity at the protein, rather than transcript, level and uncover underlying systematic links between aging and loss of protein homeostasis.

Hesperetin induces apoptosis in A549 cells via the Hsp70‑mediated activation of Bax.

Hesperetin, a predominant flavonoid found in citrus fruits, has received considerable attention for its potential anticancer activity through the reduction of cell viability and the induction of apoptosis. Several effector mechanisms have been demonstrated underlying the antitumor properties of hesperetin but its specific mechanisms have not yet been fully elucidated. In the present study, how hesperetin affects the proliferation of A549 cells and the related cell proliferation regulatory mechanisms, were inevstigated. To elucidate the mechanisms underlying the effects of hesperetin on A549 cells, MTT assay, colony formation assay, flow cytometry, immunoblotting, reverse transcription‑quantitative PCR and JC‑1 staining were performed. The data revealed that hesperetin inhibited cell proliferation and induced apoptosis in these cells. Hesperetin also decreased the level of heat shock protein70 (Hsp70), a negative regulator of the mitochondrial apoptosis pathway, often overexpressed in various cancer cells and suspected to contribute to tumor development. Hesperetin‑induced Hsp70 suppression was associated with reduced cytosolic Bax and increased mitochondrial Bax levels, leading to the enhancement of the mitochondrial apoptotic cascade. The Hsp70 overexpression‑induced reduction in the level of hesperetin‑induced apoptosis provides evidence to hesperetin‑induced apoptosis being mediated by affecting Hsp70. Furthermore, it was demonstrated that hesperetin reduced Hsp70 expression by inducing a proteasome‑mediated degradation via the upregulation of E3‑ligase, C‑terminus of Hsp70‑interacting protein (CHIP). The present study highlighted the importance of the Bax activation‑triggered mitochondria‑mediated pathway for hesperetin‑induced apoptosis and demonstrated a novel mechanism of how Hsp70 played a critical role in the negative regulation of this apoptotic network in cancer cells.

A Severe Dementia Syndrome Caused by Intron Retention and Cryptic Splice Site Activation in STUB1 and Exacerbated by TBP Repeat Expansions.

Heterozygous pathogenic variants in the STIP1 homologous and U-box containing protein 1 (STUB1) gene have been identified as causes of autosomal dominant inherited spinocerebellar ataxia type 48 (SCA48). SCA48 is characterized by an ataxic movement disorder that is often, but not always, accompanied by a cognitive affective syndrome. We report a severe early onset dementia syndrome that mimics frontotemporal dementia and is caused by the intronic splice donor variant c.524+1G>A in STUB1. Impaired splicing was demonstrated by RNA analysis and in minigene assays of mutated and wild-type constructs of STUB1. The most striking consequence of this splicing impairment was retention of intron 3 in STUB1, which led to an in-frame insertion of 63 amino acids (aa) (p.Arg175_Glu176ins63) into the highly conserved coiled-coil domain of its encoded protein, C-terminus of HSP70-interacting protein (CHIP). To a lesser extent, activation of two cryptic splice sites in intron 3 was observed. The almost exclusively used one, c.524+86, was not predicted by in silico programs. Variant c.524+86 caused a frameshift (p.Arg175fs*93) that resulted in a truncated protein and presumably impairs the C-terminal U-box of CHIP, which normally functions as an E3 ubiquitin ligase. The cryptic splice site c.524+99 was rarely used and led to an in-frame insertion of 33 aa (p.Arg175_Glu176ins33) that resulted in disruption of the coiled-coil domain, as has been previously postulated for complete intron 3 retention. We additionally detected repeat expansions in the range of reduced penetrance in the TATA box-binding protein (TBP) gene by excluding other genes associated with dementia syndromes. The repeat expansion was heterozygous in one patient but compound heterozygous in the more severely affected patient. Therefore, we concluded that the observed severe dementia syndrome has a digenic background, making STUB1 and TBP important candidate genes responsible for early onset dementia syndromes.

C-terminus of Hsp70 Interacting Protein (CHIP) and Neurodegeneration: Lessons from the Bench and Bedside.

Neurodegenerative diseases are characterized by the increasing dysfunction and death of neurons, resulting in progressive impairment of a person’s mobility and/or cognition. Protein misfolding and aggregation are commonly hypothesized to cause neurotoxicity and, eventually, neuronal degeneration that are associated with these diseases. Emerging experimental evidence, as well as recent findings from human studies, reveal that the C-terminus of Hsp70 Interacting Protein (CHIP), or STIP1 Homology and U-box containing Protein 1 (STUB1), is a quality control protein involved in neurodegeneration. Here, we review evidence that CHIP interacts with and plays a role in regulating proteins implicated in the pathogenesis of Parkinson’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis, and polyglutamine diseases, including Huntington’s disease and spinocerebellar ataxias. We also review clinical findings identifying mutations in STUB1 as a cause of both autosomal recessive and autosomal dominant forms of cerebellar ataxia. We propose that CHIP modulation may have therapeutic potential for the treatment of multiple neurodegenerative diseases.

CHIP and BAP1 Act in Concert to Regulate INO80 Ubiquitination and Stability for DNA Replication.

The INO80 chromatin remodeling complex has roles in many essential cellular processes, including DNA replication. However, the mechanisms that regulate INO80 in these processes remain largely unknown. We previously reported that the stability of Ino80, the catalytic ATPase subunit of INO80, is regulated by the ubiquitin proteasome system and that BRCA1-associated protein-1 (BAP1), a nuclear deubiquitinase with tumor suppressor activity, stabilizes Ino80 via deubiquitination and promotes replication fork progression. However, the E3 ubiquitin ligase that targets Ino80 for proteasomal degradation was unknown. Here, we identified the C-terminus of Hsp70-interacting protein (CHIP), the E3 ubiquitin ligase that functions in cooperation with Hsp70, as an Ino80-interacting protein. CHIP polyubiquitinates Ino80 in a manner dependent on Hsp70. Contrary to our expectation that CHIP degrades Ino80, CHIP instead stabilizes Ino80 by extending its half-life. The data suggest that CHIP stabilizes Ino80 by inhibiting degradative ubiquitination. We also show that CHIP works together with BAP1 to enhance the stabilization of Ino80, leading to its chromatin binding. Interestingly, both depletion and overexpression of CHIP compromise replication fork progression with little effect on fork stalling, as similarly observed for BAP1 and Ino80, indicating that an optimal cellular level of Ino80 is important for replication fork speed but not for replication stress suppression. This work therefore idenitifes CHIP as an E3 ubiquitin ligase that stabilizes Ino80 via nondegradative ubiquitination and suggests that CHIP and BAP1 act in concert to regulate Ino80 ubiquitination to fine-tune its stability for efficient DNA replication.

Hsp70:CHIP Ubiquitinates Dysfunctional but Not Native Neuronal NO Synthase.

Heat shock protein (Hsp) 70 modulators are being developed to enhance the removal of toxic proteins in a variety of protein misfolding diseases. In the course of our studies on neuronal nitric oxide synthase (nNOS), a client of the Hsp90 and Hsp70 chaperone system, we have established that inactivation of nNOS by heme or tetrahydrobiopterin (BH4) alteration and loss triggers ubiquitination by the Hsp70-associated E3 ligase c-terminus of Hsp70-interacting protein (CHIP) and subsequent degradation in cells. Although in cells Hsp90 and Hsp70 work together to maintain protein quality control, in this study, we specifically developed an assay to assess the selectivity of the Hsp70:CHIP complex for inactivated nNOS. We developed a highly sensitive ELISA to measure Hsp70:CHIP-dependent nNOS ubiquitination without interference from direct ubiquitination by CHIP, as evidenced by Bcl-2 associated athanogene 1-M completely abolishing ubiquitination. To further validate the assay we demonstrated, JG-98, a rhodocyanin compound that acts on Hsp70 but not its inactive structural analog JG-258, enhances the ubiquitination of nNOS 3-fold. Utilizing this assay, we have shown that the Hsp70:CHIP complex preferentially ubiquitinates heme-deficient nNOS (apo-nNOS) over heme-containing nNOS (holo-nNOS). Moreover, depletion of nNOS-bound BH4 triggers ubiquitination of holo-nNOS by the Hsp70:CHIP complex. Most importantly, JG-98 was shown to enhance the ubiquitination of only dysfunctional nNOS while leaving the native functional nNOS untouched. Thus, the finding that enhancing Hsp70:CHIP-mediated ubiquitination does not affect native proteins has important pharmacological implications. Moreover, development of a facile in vitro method for Hsp70:CHIP-mediated ubiquitination will be beneficial for testing other Hsp70 modulators. SIGNIFICANCE STATEMENT: The heat shock protein 70 (Hsp70):c-terminus of Hsp70-interacting protein (CHIP) complex facilitates the ubiquitination and subsequent degradation of several hundred-client proteins, and activation of Hsp70 has been suggested as a therapeutic strategy to enhance the degradation of disease-causing proteins. The current study shows that the pharmacological activation of Hsp70 enhances the ubiquitination of dysfunctional but not native nNOS, and it suggests that this therapeutic strategy will likely be highly selective.

Aurora B Kinase Promotes CHIP-Dependent Degradation of HIF1α in Prostate Cancer Cells.

Hypoxia is a major factor in tumor progression and resistance to therapies, which involves elevated levels of the transcription factor HIF1α. Here, we report that prostate tumor xenografts express high levels of HIF1α and show greatly enhanced growth in response to knockdown of the E3 ligase CHIP (C-terminus of Hsp70-interacting protein). In multiple human prostate cancer cell lines under hypoxia, taxol treatment induces the degradation of HIF1α, and this response is abrogated by knockdown of CHIP, but not by E3 ligase VHL or RACK1. HIF1α degradation is accompanied by loss of function, evidenced by reduced expression of HIF1α-dependent genes. CHIP-dependent HIF1α degradation also occurs in cells arrested in mitosis by nocodazole instead of taxol. Mitotic kinase Aurora B activity is required for taxol-induced HIF1α degradation. Purified Aurora B directly phosphorylates HIF1α at multiple sites, and these modifications enhance its polyubiquitination by CHIP in a purified reconstituted system. Our results show how activation of Aurora B promotes CHIP-dependent degradation of HIF1α in prostate cancer cells. This new knowledge may affect the use of mitotic kinase inhibitors and open new approaches for treatment of hypoxic prostate tumors.

Rapid Real‐Time Ubiquitination Assays as Alternatives to Western Blotting

The refolding and degradative pathways of the protein quality control system converge through the chaperoned ubiquitination complex formed by the interaction of the E3 ubiquitin ligase CHIP ( C‐terminus of Hsp70 Interacting Protein) and the ATP‐dependent chaperone Hsp70 (70 kilodalton heat shock protein). We have applied the CHIP/Hsp70 chaperoned ubiquitination system as a test for our newly developed ubiquitination assay, presenting a viable alternative to traditional western blots for detection of ubiquitination. Here we describe the cutting‐edge biolayer interferometry ubiquitination assay to rapidly assay ubiquitination in real time. Our BLI ubiquitination assay is recorded with 1‐second resolution and is the first assay to allow for determination of rate and extent of ubiquitination in real‐time. This assay detects formation of polyubiquitin chains directly on a biosensor‐bound target. Polyubiquitination of substrates using wild‐type components produces a robust signal. Reagent concentrations and quantities are similar to those used by traditional western blot‐based ubiquitination assays. Results are returned instantaneously and result in substantial time savings.Support or Funding InformationNIGMS R35 GM128595

Modulation of CHIP‐mediated degradation through posttranslational modifications

Interactions between CHIP (C-terminus of Hsp70 Interacting Protein) and Hsp70 (70-kilodalton heat shock protein) promote CHIP-mediated degradation of misfolded proteins (clients) via the ubiquitin proteasome pathway. Modulating interactions of Hsp70 with either CHIP or HOP (Hsp Organizing Protein) controls misfolded protein turnover by shunting misfolded client proteins to either the degradative pathway or the refolding pathway. Differential affinity of Hsp70 for the tetratricopeptide repeat (TPR) domains of CHIP and HOP, which is responsible for the observed modulation, is achieved through interactions with the six residues preceding the carboxy-terminal Asp641 of Hsp70. In particular, phosphorylation of Hsp70 Thr636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Thus, phosphorylation of Hsp70 Thr636 acts as a switch that modulates the response to protein misfolding and the ability for CHIP to degrade misfolded proteins via the ubiquitin-proteasome system. Herein, through biophysical assays and new structures of CHIP-Hsp70 and HOP-Hsp70 we identify the molecular mechanism that enables phosphorylation-driven switching that regulates protein degradation through CHIP-mediated ubiquitination. We also present a structure-guided mutation of CHIP that bypasses Thr636 phosphorylation-driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP and directing misfolded protein clients toward degradation. This structure-guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP-Hsp70/HOP-Hsp70 complexes toward chaperoned ubiquitination via CHIP. Support or Funding Information NIH R35 GM128595, NSF 15–52113 A structure-guided mutation restores affinity of CHIP for pThr636Hsp70 This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sulforaphane Upregulates the Heat Shock Protein Co-Chaperone CHIP and Clears Amyloid-β and Tau in a Mouse Model of Alzheimer's Disease.

Sulforaphane is an herbal isothiocyanate enriched in cruciferous vegetables. Here, the authors investigate whether sulforaphane modulates the production of amyloid-β (Aβ) and tau, the two main pathological factors in Alzheimer's disease (AD). A triple transgenic mouse model of AD (3 × Tg-AD) is used to study the effect of sulforaphane. Oral gavage of sulforaphane reduces protein levels of monomeric and polymeric forms of Aβ as well as tau and phosphorylated tau in 3 × Tg-AD mice. However, sulforaphane treatment do not affect mRNA expression of amyloid precursor protein or tau. As previous studies show that Aβ and tau metabolism are influenced by a heat shock protein (HSP) co-chaperone, C-terminus of HSP70-interacting protein (CHIP), the authors examine whether sulforaphane can modulate CHIP. The authors find that sulforaphane treatment increase levels of CHIP and HSP70. Furthermore, observations of CHIP-deficient primary neurons derived from 3 × Tg-AD mice suggest that sulforaphane treatment increase CHIP level and clear the accumulation of Aβ and tau. Finally, sulforaphane ameliorated memory deficits in 3 × Tg-AD mice as reveal by novel object/location recognition tests and contextual fear conditioning tests. These results demonstrate that sulforaphane treatment upregulates CHIP and has the potential to decrease the accumulation of Aβ and tau in patients with AD.

Angiotensin II Promotes KV7.4 Channels Degradation Through Reduced Interaction With HSP90 (Heat Shock Protein 90).

Voltage-gated Kv7.4 channels have been implicated in vascular smooth muscle cells' activity because they modulate basal arterial contractility, mediate responses to endogenous vasorelaxants, and are downregulated in several arterial beds in different models of hypertension. Angiotensin II (Ang II) is a key player in hypertension that affects the expression of several classes of ion channels. In this study, we evaluated the effects of Ang II on the expression and function of vascular Kv7.4. Western blot and quantitative polymerase chain reaction revealed that in whole rat mesenteric artery, Ang II incubation for 1 to 7 hours decreased Kv7.4 protein expression without reducing transcript levels. Moreover, Ang II decreased XE991 (Kv7)-sensitive currents and attenuated membrane potential hyperpolarization and relaxation induced by the Kv7 activator ML213. Ang II also reduced Kv7.4 staining at the plasma membrane of vascular smooth muscle cells. Proteasome inhibition with MG132 prevented Ang II-induced decrease of Kv7.4 levels and counteracted the functional impairment of ML213-induced relaxation in myography experiments. Proximity ligation assays showed that Ang II impaired the interaction of Kv7.4 with the molecular chaperone HSP90 (heat shock protein 90), enhanced the interaction of Kv7.4 with the E3 ubiquitin ligase CHIP (C terminus of Hsp70-interacting protein), and increased Kv7.4 ubiquitination. Similar alterations were found in mesenteric vascular smooth muscle cells isolated from Ang II-infused mice. The effect of Ang II was emulated by 17-AAG (17-demethoxy-17-(2-propenylamino) geldanamycin) that inhibits HSP90 interactions with client proteins. These results show that Ang II downregulates Kv7.4 by altering protein stability through a decrease of its interaction with HSP90. This leads to the recruitment of CHIP and Kv7.4 ubiquitination and degradation via the proteasome.

Modulation of the Hsp70 Protein Quality Control System by Phosphorylation of Chaperones and Co‐chaperones

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of the co‐chaperones CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Hsp70 can only interact with one of the co‐chaperones at a time, thus modulating interactions of Hsp70 with either CHIP or HOP controls the Hsp70 protein quality control axis by shunting misfolded client proteins to either the degradative pathway or the refolding pathway. Interactions of Hsp70 with CHIP promote CHIP‐mediated degradation via the ubiquitin proteasome pathway. Interactions of Hsp70 with HOP promote protein refolding since HOP connect Hsp70 with the 90 kilodalton heat shock protein Hsp90. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp641 of Hsp70. In particular, phosphorylation of Hsp70 Thr636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Thus, phosphorylation of Hsp70 Thr636 acts as a switch that directs the output and function of cellular protein quality control. Phosphorylation of CHIP Ser20 or CHIP Ser24 also modulates the interactions between CHIP and Hsp70, providing yet another level of regulation for the Hsp70 protein quality control system. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and HOP‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex.Support or Funding InformationNSF 15‐52113This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphoproteomics analyses reveal that phosphorylation of heat shock protein 70 (hsp70) by protein kinase B (Akt1) on ser631 controls activity of mitochondrial superoxide‐2 (SOD2).

BackgroundSOD2 is a nuclear encoded gene whose product must be imported into the mitochondria for activation. Previously, we showed that hsp70 chaperones SOD2 to the mitochondria. The chaperone activity of hsp70 is negatively regulated by the C‐terminus of hsp70‐interacting protein (CHIP). However, the mechanism that controls SOD2 activity proportional to activating signals to maintain mitochondrial redox balance is unknown.MethodsTo identify the signaling protein that regulates activity of hsp70, HEK‐293T cells were transfected with GST‐tagged‐hsp70. The GST epitope was immuno‐precipitated and the differential band was sent for mass spectrometry and Akt1 was identified. To examine the contributions of Akt1 to SOD2 induction in response to stimuli, we used PI3K inhibitor, Ly294002 treatment as well as Akt1‐siRNA knockdown methods. Pulmonary artery endothelial cells (PAECs) were transfected with Akt1‐siRNA and we re‐expressed myristoylated Akt1 or dominant negative Akt1. We then quantified SOD2 protein and activity in isolated mitochondria using Western blots and colorimetric assays. To identify the amino acid residue (s) in hsp70 that are phosphorylated by Akt1, we incubated recombinant Akt1 proteins with purified hsp70 in an in‐vitro kinase reaction and the band containing phosphorylated hsp70 was sent for phosphoproteomics analysis and site identification using LC‐MS/MS. We characterized the newly identified residue by point mutation and assessed the effect of mutations on SOD2 localization to the mitochondria by hsp70 using immuno‐fluorescent staining. To examined the effect of hsp70 phosphorylation on SOD2 activity in response to stimuli, we knockeddown Akt1 using siRNA and generated global akt1 null mice and measured SOD2 import, and activity and rates of superoxide and hydrogen peroxide levels in cells and lung lysates following stimulation using LC‐MS/MS following mitoSOX/mito‐B stainings. We determined the effect of hsp70 phosphorylation on the interaction between CHIP and hsp70, and the functional relevance of CHIP interaction on SOD2 function using CHIP overexpression, ubiquitin assays and immunoprecipitation methods.ResultsInduction of Akt1 activity enhanced SOD2 activity in PAECs within minutes following PDGF stimulation, reaching a peak at 20 minutes and is then downregulated. Conversely, inhibition of Akt1 activity robustly decreased SOD2 protein and activity in isolated mitochondria and increased mitochondrial O2− formation compared to controls. Re‐expression of myr‐Akt1 restored SOD2 activity whereas DN‐Akt1 failed to improve SOD2 activity. Mechanistically, we demonstrated that Akt1 binds to and phosphorylates hsp70 at ser‐631. Substitution of ser for Ala at position 631 in hsp70 blocked the PDGF mediated induction of SOD2 accumulation in the mitochondria. Phosphorylated hsp70 inhibited the chaperone interaction with CHIP, and conversely, dephosphorylated hsp70 preferentially recruited CHIP and facilitated SOD2 degradation. Treatment with mito‐TEMPO induced PP2A/2C protein expression, inactivated Akt1 and dephosphorylated hsp70‐ser‐631, leading to the eventual termination of SOD2 import.ConclusionAkt1 integrates activation and inhibition signals, links them to hsp70 to regulates SOD2 activity.Support or Funding InformationSupported by NIHKO8 (AJA), Children Research Institute (CRI) and Department of Pediatrics.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 085: CHIP: E3 Ubiquitin Ligase Mediates Proteasomal Degradation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of Rats with Heart Failure

The exaggerated sympathetic drive is a characteristic of heart failure (HF) due to reduced neuronal nitric oxide synthase (nNOS) within the paraventricular nucleus (PVN). Previously we have shown that there were increased accumulation of nNOS-ubiquitin (nNOS-Ub) conjugates in the PVN of rats with HF (1.0±0.05 Sham vs. 1.29±0.06 HF) due to the increased levels of PIN (a protein inhibitor of nNOS, known to dissociate nNOS dimers into monomers) (0.76±0.10 Sham vs. 1.12±0.09 HF) and decreased levels of tetrahydrobiopterin (BH4): a cofactor required for stabilization of nNOS dimers (0.62±0.02 Sham vs. 0.44±0.03 HF). We also showed that there is blunted nitric oxide-mediated inhibition of sympathetic tone via the PVN in HF. Here we examined whether CHIP(C-terminus of Hsp70 -interacting protein), a chaperone-dependent E3 ubiquitin-protein isopeptide ligase known to ubiquitylate Hsp90-chaperoned proteins could act as an ubiquitin ligase for nNOS in the PVN. Immunofluorescence studies revealed colocalization of nNOS and CHIP in the PVN indicating their possible interaction. CHIP expression was increased by 50% in the PVN of rats with HF(0.96±0.08 Sham vs.1.44±0.10* HF). It is shown that Hsp90 protects nNOS from ubiquitination while Hsp70 promotes the ubiquitination and degradation. We observed significant upregulation of Hsp70 (0.49±0.03 Sham vs. 0.65±0.02* HF) with a trend toward the decrease in Hsp90 expression (0.90±0.07 Sham vs. 0.71±0.06 HF). The opposing effects of the two chaperones could account for the increased CHIP-mediated ubiquitination and degradation of dysfunctional nNOS monomers in the PVN of rats with HF. Furthermore, neuronal NG108-15 cell line transfected with the pCMV3-CHIP-GFP spark (CHIP overexpression plasmid) showed approximately 74% increase in CHIP with concomitant 49% decrease in nNOS expression. In vitro ubiquitination assay in NG108 cells transfected with pCMV-(HA-Ub) 8 and pCMV3-CHIP-GFP spark plasmid reveal increased HA-Ub-nNOS conjugates (1.13 ± 0.09 Scramble vs. 1.65 ± 0.12* CHIP plasmid). Taken together, our results identify CHIP as an E3 ligase for ubiquitination of dysfunctional nNOS and CHIP expression is augmented during HF leading to increased proteasomal degradation of nNOS in the PVN.

Differential HspBP1 expression accounts for the greater vulnerability of neurons than astrocytes to misfolded proteins

Although it is well known that astrocytes are less vulnerable than neurons in neurodegenerative diseases, the mechanism behind this differential vulnerability is unclear. Here we report that neurons and astrocytes show markedly different activities in C terminus of Hsp70-interacting protein (CHIP), a cochaperone of Hsp70. In astrocytes, CHIP is more actively monoubiquitinated and binds to mutant huntingtin (mHtt), the Huntington's disease protein, more avidly, facilitating its K48-linked polyubiquitination and degradation. Astrocytes also show the higher level and heat-shock induction of Hsp70 and faster CHIP-mediated degradation of various misfolded proteins than neurons. In contrast to astrocytes, neurons express abundant HspBP1, a CHIP inhibitory protein, resulting in the low activity of CHIP. Silencing HspBP1 expression via CRISPR-Cas9 in neurons ameliorated mHtt aggregation and neuropathology in HD knockin mouse brains. Our findings indicate a critical role of HspBP1 in differential CHIP/Hsp70 activities in neuronal and glial cells and the greater neuronal vulnerability to misfolded proteins in neurodegenerative diseases.

The Antitumor Effect of C-terminus of Hsp70-Interacting Protein via Degradation of c-Met in Small Cell Lung Cancer.

BackgroundThe mesenchymal-epithelial transition factor (MET) receptor can be overexpressed in solid tumors, including small cell lung cancer (SCLC). However, the molecular mechanism regulating MET stability and turnover in SCLC remains undefined. One potential mechanism of MET regulation involves the C-terminus of Hsp70-interacting protein (CHIP), which targets heat shock protein 90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, we investigated the functional effects of CHIP expression on MET regulation and the control of SCLC cell apoptosis and invasion.MethodsTo evaluate the expression of CHIP and c-Met, which is a protein that in humans is encoded by the MET gene (the MET proto-oncogene), we examined the expression pattern of c-Met and CHIP in SCLC cell lines by western blotting. To investigate whether CHIP overexpression reduced cell proliferation and invasive activity in SCLC cell lines, we transfected cells with CHIP and performed a cell viability assay and cellular apoptosis assays.ResultsWe found an inverse relationship between the expression of CHIP and MET in SCLC cell lines (n=5). CHIP destabilized the endogenous MET receptor in SCLC cell lines, indicating an essential role for CHIP in the regulation of MET degradation. In addition, CHIP inhibited MET-dependent pathways, and invasion, cell growth, and apoptosis were reduced by CHIP overexpression in SCLC cell lines.ConclusionCHIP is capable of regulating SCLC cell apoptosis and invasion by inhibiting MET-mediated cytoskeletal and cell survival pathways in NCI-H69 cells. CHIP suppresses MET-dependent signaling, and regulates MET-mediated SCLC motility.

Interactions and Dynamics Within CHIP/Hsp70 Complexes

Interactions between the E3 ubiquitin ligase CHIP (C‐terminus of Hsp70 interacting protein) and the chaperone Hsp70 represent the physical intersection of two competing protein quality control efforts: refolding, and degradation. Interaction with CHIP allows for the formation of a productive encounter complex that facilitates ubiquitination of Hsp70‐bound substrates, thereby targeting those proteins for proteasomal degradation. Central to the CHIP/Hsp70 interaction are changes in structure and dynamics that occur upon binding. Although recruitment of Hsp70 to CHIP via interaction with the CHIP tetratricopeptide repeat (TPR) domain is well established, alterations in structure and dynamics of CHIP and Hsp70 upon binding are not well understood. Using NMR we describe residue‐level changes in dynamics to the CHIP‐TPR domain that occur upon binding Hsp70. We also utilize small angle X‐ray scattering (SAXS) and electron paramagnetic resonance (EPR) to identify motions with the CHIP/Hsp70 complex. Independent SAXS and EPR experiments both point to a mobile Hsp70 substrate binding domain that is only partially constrained through interactions with the CHIP‐TPR. Building upon static models of the full‐length CHIP/Hsp70 complex, afforded by our recent crystal structure of the CHIP‐TPR/Hsp70‐lid‐tail complex, we have assembled a dynamic model of the CHIP‐Hsp70 complex. This dynamic model sheds light on the function of the CHIP/Hsp70 chaperoned ubiquitination complex and how CHIP harnesses dynamics of Hsp70 to overcome significant distance separations identified in earlier static models of the CHIP/Hsp70 complex.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Shifting Sands: Modulating Interactions and Outputs of the CHIP‐Hsp70/HOP‐Hsp70 Protein Quality Control Complexes

The 70‐kilodalton heat shock protein (Hsp70) interacts with tetratricopeptide repeat (TPR) domains of CHIP (C‐terminus of Hsp70 Interacting Protein) and HOP (Hsp Organizing Protein) using a two‐carboxylate clamp binding mechanism. Differential affinity for the TPR domains of CHIP and HOP are achieved through interactions with the six residues preceding the carboxy‐terminal Asp641 of Hsp70. In particular, phosphorylation of Hsp70 Thr636 produces a dramatic swing in binding affinities that shift preferential formation of CHIP/Hsp70 complexes to preferential formation of HOP/Hsp70 complexes. Given that CHIP‐Hsp70 complexes promote ubiquitin‐mediated degradation of Hsp70‐chaperoned clients while HOP‐Hsp70 complexes promote refolding of Hsp70‐chaperoned clients, phosphorylation of Hsp70 Thr636 acts as a switch that directs the output and function of cellular protein quality control. Herein, through biophysical assays and new structures of CHIP‐Hsp70 and Hop‐Hsp70 we identify the molecular mechanism that enables phosphorylation‐driven switching that directs interactions within the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes. We also present a structure‐guided mutation of Hsp70 that bypasses Thr636 phosphorylation‐driven switching by recovering affinity of CHIP for phosphorylated Hsp70, thereby redirecting Hsp70 to preferentially bind to CHIP. This structure‐guided mutation corrects a binding defect present in wild type CHIP and provides a system for studying the effect of shifting output of the CHIP‐Hsp70/HOP‐Hsp70 protein quality control complexes toward chaperoned ubiquitination via the CHIP‐Hsp70 complex.Support or Funding InformationThe authors acknowledge financial support from the US National Science Foundation (Award No. MCB 1552113 to RCP), the American Heart Association (Award No. 16SDG26960000 to RCP), the Burroughs Wellcome Fund (Award No. 1014031 to RCP), and institutional support from Miami University through the Robert H. and Nancy J. Blayney Professorship (to RCP). The Advanced Light Source is supported by the US Department of Energy under contract number DE‐AC03‐76SF00098 at Lawrence Berkeley National Laboratory.

Heat Shock Protein 90 Ensures Efficient Mumps Virus Replication by Assisting with Viral Polymerase Complex Formation.

Paramyxoviral RNAs are synthesized by a viral RNA-dependent RNA polymerase (RdRp) consisting of the large (L) protein and its cofactor phosphoprotein (P protein). The L protein is a multifunctional protein that catalyzes RNA synthesis, mRNA capping, and mRNA polyadenylation. Growing evidence shows that the stability of several paramyxovirus L proteins is regulated by heat shock protein 90 (Hsp90). In this study, we demonstrated that Hsp90 activity was important for mumps virus (MuV) replication. The Hsp90 activity was required for L-protein stability and activity because an Hsp90-specific inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), destabilized the MuV L protein and suppressed viral RNA synthesis. However, once the L protein formed a mature polymerase complex with the P protein, Hsp90 activity was no longer required for the stability and activity of the L protein. When the Hsp90 activity was inhibited, the MuV L protein was degraded through the CHIP (C terminus of Hsp70-interacting protein)-mediated proteasomal pathway. High concentrations of 17-AAG showed strong cytotoxicity to certain cell types, but combined use of an Hsp70 inhibitor, VER155008, potentiated degradation of the L protein, allowing a sufficient reduction of 17-AAG concentration to block MuV replication with minimum cytotoxicity. Regulation of the L protein by Hsp90 and Hsp70 chaperones was also demonstrated for another paramyxovirus, the measles virus. Collectively, our data show that the Hsp90/Hsp70 chaperone machinery assists in the maturation of the paramyxovirus L protein and thereby in the formation of a mature RdRp complex and efficient viral replication.IMPORTANCE Heat shock protein 90 (Hsp90) is nearly universally required for viral protein homeostasis. Here, we report that Hsp90 activity is required for efficient propagation of mumps virus (MuV). Hsp90 functions in the maintenance of the catalytic subunit of viral polymerase, the large (L) protein, prior to formation of a mature polymerase complex with the polymerase cofactor of L, phosphoprotein. Hsp70 collaborates with Hsp90 to regulate biogenesis of the MuV L protein. The functions of these chaperones on the viral polymerase may be common among paramyxoviruses because the L protein of measles virus is also similarly regulated. Our data provide important insights into the molecular mechanisms of paramyxovirus polymerase maturation as well as a basis for the development of novel antiviral drugs.

Molecular Chaperone HSP90 Is Necessary to Prevent Cellular Senescence via Lysosomal Degradation of p14ARF.

The tumor suppressor function of p14ARF is regulated at a posttranslational level via mechanisms yet to be fully understood. Here, we report the identification of an unconventional p14ARF degradation pathway induced by the chaperone HSP90 in association with the E3 ubiquitin ligase C-terminus of HSP70-interacting protein (CHIP). The ternary complex of HSP90, CHIP, and p14ARF was required to induce the lysosomal degradation of p14ARF by an ubiquitination-independent but LAMP2A-dependent mechanism. Depletion of HSP90 or CHIP induced p14ARF-dependent senescence in human fibroblasts. Premature senescence observed in cells genetically deficient in CHIP was rescued in cells that were doubly deficient in CHIP and p14ARF. Notably, non-small cell lung cancer cells (NSCLC) positive for p14ARF were sensitive to treatment with the HSP90 inhibitor geldanamycin. Furthermore, overexpression of HSP90 and CHIP with a concomitant loss of p14ARF correlated with poor prognosis in patients with NSCLC. Our findings identify a relationship between p14ARF and its chaperones that suggest new therapeutic strategies in cancers that overexpress HSP90. Cancer Res; 77(2); 343-54. ©2016 AACR.