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The endoplasmic reticulum (ER) membrane provides infrastructure for intracellular signaling, protein degradation, and communication among the ER lumen, cytosol, and nucleus via transmembrane and membrane-associated proteins. Failure to maintain homeostasis at the ER leads to deleterious conditions in humans, such as protein misfolding-related diseases and neurodegeneration. The ER transmembrane heat shock protein 40 (Hsp40) proteins, including DNAJB12 (JB12) and DNAJB14 (JB14), have been studied for their importance in multiple aspects of cellular events, including degradation of misfolded membrane proteins, proteasome-mediated control of proapoptotic Bcl-2 members, and assembly of multimeric ion channels. This study elucidates a novel facet of JB12 and JB14 in that their expression could be regulated in response to stress caused by the presence of ER stressors and the mitochondrial potential uncoupler CCCP. Furthermore, JB14 overexpression could affect the level of PTEN-induced kinase 1 (PINK1) expression under CCCP-mediated stress. Cells with genetic knockout (KO) of DNAJB12 and DNAJB14 exhibited an altered kinetic of phosphorylated Drp1 in response to the stress caused by CCCP treatment. Surprisingly, JB14-KO cells exhibited a prolonged stabilization of PINK1 during chronic exposure to CCCP. Cells depleted with JB12 or JB14 also revealed an increase in the mitochondrial count and branching. Hence, this study indicates the possible novel functions of JB12 and JB14 involving mitochondria in nonstress conditions and under stress caused by CCCP., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

A rise in temperature triggers a structural change in the human Type I 40 kDa heat shock protein (Hsp40/DnaJ), known as DNAJA1. This change leads to a less compact structure, characterized by an increased presence of solvent-exposed hydrophobic patches and β-sheet-rich regions. This transformation is validated by circular dichroism, thioflavin T binding, and Bis-ANS assays. The formation of this β-sheet-rich conformation, which is amplified in the absence of zinc, leads to protein aggregation. This aggregation is induced not only by high temperatures but also by low ionic strength and high protein concentration. The aggregated conformation exhibits characteristics of an amyloidogenic structure, including a distinctive X-ray diffraction pattern, seeding competence (which stimulates the formation of amyloid-like aggregates), cytotoxicity, resistance to SDS, and fibril formation. Interestingly, the yeast Type I Ydj1 also tends to adopt a similar β-sheet-rich structure under comparable conditions, whereas Type II Hsp40s, whether human or from yeast, do not. Moreover, Ydj1 aggregates were found to be cytotoxic. Studies using DNAJA1- and Ydj1-deleted mutants suggest that the zinc-finger region plays a crucial role in amyloid formation. Our discovery of amyloid aggregation in a C-terminal deletion mutant of DNAJA1, which resembles a spliced homolog expressed in the testis, implies that Type I Hsp40 co-chaperones may generate amyloidogenic species in vivo., (© 2024 Federation of European Biochemical Societies.)

The J-domain proteins (JDP) form the largest protein family among cellular chaperones. In cooperation with the Hsp70 chaperone system, these co-chaperones orchestrate a plethora of distinct functions, including those that help maintain cellular proteostasis and development. JDPs evolved largely through the fusion of a J-domain with other protein subdomains. The highly conserved J-domain facilitates the binding and activation of Hsp70s. How JDPs (re)wire Hsp70 chaperone circuits and promote functional diversity remains insufficiently explained. Here, we discuss recent advances in our understanding of the JDP family with a focus on the regulation built around J-domains to ensure correct pairing and assembly of JDP-Hsp70 machineries that operate on different clientele under various cellular growth conditions., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing non-native proteins to facilitate protein folding, refolding, and degradation. Hsp40 (DnaJ-like proteins) family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. This review describes the mechanisms by which different Hsp40s use specialized sub-domains to direct clients of Hsp70 for triage between folding versus degradation., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

We report on how the endoplasmic reticulum (ER)-associated-autophagy pathway (ERAA) delivers P23H-rhodopsin (P23H-R) to the lysosome. P23H-R accumulates in an ERAD-resistant conformation that is stabilized in a detergent-soluble state by DNAJB12 and Hsp70. P23H-R, DNAJB12, and FIP200 colocalize in discrete foci that punctuate the rim of omegasome rings coated by WIPI1. Loss of DNAJB12 function prevents the association of P23H-R containing ER tubules with omegasomes. P23H-R tubules thread through the wall of WIPI1 rings into their central cavity. Transfer of P23H-R from ER-connected phagophores to lysosomes requires GABARAP and is associated with the transient docking of lysosomes to WIPI1 rings. After departure from WIPI1 rings, new patches of P23H-R are seen in the membranes of lysosomes. The absence of GABARAP prevents transfer of P23H-R from phagophores to lysosomes without interfering with docking. These data identify lysosome docking to omegasomes as an important step in the DNAJB12- and GABARAP-dependent autophagic disposal of dominantly toxic P23H-R.

The endoplasmic reticulum (ER) fills the cell with a continuous network of sealed membrane tubules and sheets. The ER is subdivided into microdomains mediating one-third of total protein biosynthesis, oxidative protein folding, secretion, protein quality control, calcium signaling, marcoautophagy/autophagy, stress sensing, and apoptosis. Defects in ER-calcium homeostasis underlie several diseases. Damage to the ER by misfolded membrane proteins is suppressed by specific HSPA/Hsp70 and DNAJ/Hsp40 chaperone pairs that select intermediates for ubiquitination and ER-associated degradation (ERAD) via the proteasome. The ER-transmembrane Hsp40 chaperone DNAJB12 and HSPA/Hsp70 also target toxic intermediates of misfolded membrane proteins for ER-associated autophagy (ERAA). DNAJB12-HSPA/Hsp70 maintain membrane protein degradation intermediates in detergent-soluble and degradation-competent states. DNAJB12-HSPA/Hsp70 also interact with the autophagy initiation kinase ULK1 on ER tubules containing ERAD-resistant misfolded membrane proteins (ERAD-RMPs). Omegasomes are ER microdomains where the autophagosome precursor or phagophore (PG) forms. ER tubules loaded with ERAD-RMPs enter omegasomes where they are converted into ER-connected PG (ER-PG). The Atg8 (autophagy related 8)-family member GABARAP (GABA type A receptor-associated protein) facilitates transfer of ERAD-RMPs from ER-PGs to autolysosomes (AL) that dock transiently with omegasomes. This article describes a model for DNAJB12-HSPA/Hsp70 action during the conformation-dependent triage in the ER of misfolded membrane proteins for folding versus proteasomal or AL degradation., Competing Interests: Disclosure Statement We have no competing financial interests.

The transmembrane Hsp40 DNAJB12 and cytosolic Hsp70 cooperate on the endoplasmic reticulum's (ER) cytoplasmic face to facilitate the triage of nascent polytopic membrane proteins for folding versus degradation. N1303K is a common mutation that causes misfolding of the ion channel CFTR, but unlike F508del-CFTR, biogenic and functional defects in N1303K-CFTR are resistant to correction by folding modulators. N1303K is reported to arrest CFTR folding at a late stage after partial assembly of its N-terminal domains. N1303K-CFTR intermediates are clients of JB12-Hsp70 complexes, maintained in a detergent-soluble state, and have a relatively long 3-h half-life. ER-associated degradation (ERAD)-resistant pools of N1303K-CFTR are concentrated in ER tubules that associate with autophagy initiation sites containing WIPI1, FlP200, and LC3. Destabilization of N1303K-CFTR or depletion of JB12 prevents entry of N1303K-CFTR into the membranes of ER-connected phagophores and traffic to autolysosomes. In contrast, the stabilization of intermediates with the modulator VX-809 promotes the association of N1303K-CFTR with autophagy initiation machinery. N1303K-CFTR is excluded from the ER-exit sites, and its passage from the ER to autolysosomes does not require ER-phagy receptors. DNAJB12 operates in biosynthetically active ER microdomains to triage membrane protein intermediates in a conformation-specific manner for secretion versus degradation via ERAD or selective-ER-associated autophagy.

DNAJB12 (JB12) is an endoplasmic reticulum (ER)-associated Hsp40 family protein that recruits Hsp70 to the ER surface to coordinate the function of ER-associated and cytosolic chaperone systems in protein quality control. Hsp70 is stress-inducible, but paradoxically, we report here that JB12 was degraded by the proteasome during severe ER stress. Destabilized JB12 was degraded by ER-associated degradation complexes that contained HERP, Sel1L, and gp78. JB12 was the only ER-associated chaperone that was destabilized by reductive stress. JB12 knockdown by siRNA led to the induction of caspase processing but not the unfolded protein response. ER stress-induced apoptosis is regulated by the highly labile and ER-associated BCL-2 family member BOK, which is controlled at the level of protein stability by ER-associated degradation components. We found that JB12 was required in human hepatoma cell line 7 (Huh-7) liver cancer cells to maintain BOK at low levels, and BOK was detected in complexes with JB12 and gp78. Depletion of JB12 during reductive stress or by shRNA from Huh-7 cells was associated with accumulation of BOK and activation of Caspase 3, 7, and 9. The absence of JB12 sensitized Huh-7 to death caused by proteotoxic agents and the proapoptotic chemotherapeutic LCL-161. In summary, JB12 is a stress-sensitive Hsp40 whose degradation during severe ER stress provides a mechanism to promote BOK accumulation and induction of apoptosis., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Cystic fibrosis (CF) is a lethal recessive genetic disease caused primarily by the F508del mutation in the CF transmembrane conductance regulator (CFTR). The potentiator VX-770 was the first CFTR modulator approved by the FDA for treatment of CF patients with the gating mutation G551D. Orkambi is a drug containing VX-770 and corrector VX809 and is approved for treatment of CF patients homozygous for F508del, which has folding and gating defects. At least 30% of CF patients are heterozygous for the F508del mutation with the other allele encoding for one of many different rare CFTR mutations. Treatment of heterozygous F508del patients with VX-809 and VX-770 has had limited success, so it is important to identify heterozygous patients that respond to CFTR modulator therapy. R117H is a more prevalent rare mutation found in over 2,000 CF patients. In this study we investigated the effectiveness of VX-809/VX-770 therapy on restoring CFTR function in human bronchial epithelial (HBE) cells from R117H/F508del CF patients. We found that VX-809 stimulated more CFTR activity in R117H/F508del HBEs than in F508del/F508del HBEs. R117H expressed exclusively in immortalized HBEs exhibited a folding defect, was retained in the ER, and degraded prematurely. VX-809 corrected the R117H folding defect and restored channel function. Because R117 is involved in ion conductance, VX-770 acted additively with VX-809 to restore CFTR function in chronically treated R117H/F508del cells. Although treatment of R117H patients with VX-770 has been approved, our studies indicate that Orkambi may be more beneficial for rescue of CFTR function in these patients., (Copyright © 2016 the American Physiological Society.)

More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients., (© 2016 Veit et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Cellular homeostasis and stress survival requires maintenance of the proteome and suppression of proteotoxicity. Molecular chaperones promote cell survival through repair of misfolded proteins and cooperation with protein degradation machines to discard terminally damaged proteins. Hsp70 family members play an essential role in cellular protein metabolism by binding and releasing nonnative proteins to facilitate protein folding, refolding and degradation. Hsp40 family members are Hsp70 co-chaperones that determine the fate of Hsp70 clients by facilitating protein folding, assembly, and degradation. Hsp40s select substrates for Hsp70 via use of an intrinsic chaperone activity to bind non-native regions of proteins. During delivery of bound cargo Hsp40s employ a conserved J-domain to stimulate Hsp70 ATPase activity and thereby stabilize complexes between Hsp70 and non-native proteins. Type I and Type II Hsp40s direct Hsp70 to preform multiple functions in protein homeostasis. This review describes the mechanisms by which Type I and Type II sub-types of Hsp40 bind and deliver substrates to Hsp70.

Protein conformational maladies such as Huntington Disease are characterized by accumulation of intracellular and extracellular protein inclusions containing amyloid-like proteins. There is an inverse correlation between proteotoxicity and aggregation, so facilitated protein aggregation appears cytoprotective. To define mechanisms for protective protein aggregation, a screen for suppressors of nuclear huntingtin (Htt103Q) toxicity was conducted. Nuclear Htt103Q is highly toxic and less aggregation prone than its cytosolic form, so we identified suppressors of cytotoxicity caused by Htt103Q tagged with a nuclear localization signal (NLS). High copy suppressors of Htt103Q-NLS toxicity include the polyQ-domain containing proteins Nab3, Pop2, and Cbk1, and each suppresses Htt toxicity via a different mechanism. Htt103Q-NLS appears to inactivate the essential functions of Nab3 in RNA processing in the nucleus. Function of Pop2 and Cbk1 is not impaired by nuclear Htt103Q, as their respective polyQ-rich domains are sufficient to suppress Htt103Q toxicity. Pop2 is a subunit of an RNA processing complex and is localized throughout the cytoplasm. Expression of just the Pop2 polyQ domain and an adjacent proline-rich stretch is sufficient to suppress Htt103Q toxicity. The proline-rich domain in Pop2 resembles an aggresome targeting signal, so Pop2 may act in trans to positively impact spatial quality control of Htt103Q. Cbk1 accumulates in discrete perinuclear foci and overexpression of the Cbk1 polyQ domain concentrates diffuse Htt103Q into these foci, which correlates with suppression of Htt toxicity. Protective action of Pop2 and Cbk1 in spatial quality control is dependent upon the Hsp70 co-chaperone Sti1, which packages amyloid-like proteins into benign foci. Protein:protein interactions between Htt103Q and its intracellular neighbors lead to toxic and protective outcomes. A subset of polyQ-rich proteins buffer amyloid toxicity by funneling toxic aggregation intermediates to the Hsp70/Sti1 system for spatial organization into benign species.

Molecular chaperones triage misfolded proteins via action as substrate selectors for quality control (QC) machines that fold or degrade clients. Herein, the endoplasmic reticulum (ER)-associated Hsp40 JB12 is reported to participate in partitioning mutant conformers of gonadotropin-releasing hormone receptor (GnRHR), a G protein-coupled receptor, between ER-associated degradation (ERAD) and an ERQC autophagy pathway. ERQC autophagy degrades E90K-GnRHR because pools of its partially folded and detergent-soluble degradation intermediates are resistant to ERAD. S168R-GnRHR is globally misfolded and disposed of via ERAD, but inhibition of p97, the protein retrotranslocation motor, shunts S168R-GnRHR from ERAD to ERQC autophagy. Partially folded and grossly misfolded forms of GnRHR associate with JB12 and Hsp70. Elevation of JB12 promotes ERAD of S168R-GnRHR, with E90K-GnRHR being resistant. E90K-GnRHR elicits association of the Vps34 autophagy initiation complex with JB12. Interaction between ER-associated Hsp40s and the Vps34 complex permits the selective degradation of ERAD-resistant membrane proteins via ERQC autophagy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

N-glycosylation of proteins in endoplasmic reticulum is critical for protein quality control. We showed here a post-translational N-glycosylation affected by the HRD1 E3 ubiquitin ligase. Both WT- and E3-defective C329S-HRD1 decreased the level of high mannose form of ABCG8, a protein that heterodimerizes with ABCG5 to control sterol balance. Meanwhile, HRD1 increased the non-glycosylated ABCG8 regardless of its E3 activity, thereby suppressing full maturation of ABCG5/8 transporter. Pulse chase and mutational analysis indicated that HRD1 inhibits STT3B-dependent post-translational N-glycosylation of ABCG8. Whereas, HRD1 had only slight effect on the N-glycosylation status of ABCG5; rather it accelerated ABCG5 degradation in an E3 activity-dependent manner. Finally, RMA1, another E3 ubiquitin ligase, accelerated the degradation of both ABCG5 and ABCG8 via E3 activity-dependent manner. HRD1 and RMA1 may therefore be negative regulators of disease-associated transporter ABCG5/ABCG8. The findings also highlight the unexpected E3 activity-independent role of HRD1 in the regulation of N-glycosylation.

Conformational diseases are associated with the conversion of normal proteins into aggregation-prone toxic conformers with structures similar to that of β-amyloid. Spatial distribution of amyloid-like proteins into intracellular quality control centers can be beneficial, but cellular mechanisms for protective aggregation remain unclear. We used a high-copy suppressor screen in yeast to identify roles for the Hsp70 system in spatial organization of toxic polyglutamine-expanded Huntingtin (Huntingtin with 103Q glutamine stretch [Htt103Q]) into benign assemblies. Under toxic conditions, Htt103Q accumulates in unassembled states and speckled cytosolic foci. Subtle modulation of Sti1 activity reciprocally affects Htt toxicity and the packaging of Htt103Q into foci. Loss of Sti1 exacerbates Htt toxicity and hinders foci formation, whereas elevation of Sti1 suppresses Htt toxicity while organizing small Htt103Q foci into larger assemblies. Sti1 also suppresses cytotoxicity of the glutamine-rich yeast prion [RNQ+] while reorganizing speckled Rnq1-monomeric red fluorescent protein into distinct foci. Sti1-inducible foci are perinuclear and contain proteins that are bound by the amyloid indicator dye thioflavin-T. Sti1 is an Hsp70 cochaperone that regulates the spatial organization of amyloid-like proteins in the cytosol and thereby buffers proteotoxicity caused by amyloid-like proteins.

Cystic fibrosis (CF) is a fatal genetic disorder associated with defective hydration of lung airways due to the loss of chloride transport through the CF transmembrane conductance regulator protein (CFTR). CFTR contains two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBDs), and a regulatory domain, and its channel assembly requires multiple interdomain contacts. The most common CF-causing mutation, F508del, occurs in NBD1 and results in misfolding and premature degradation of F508del-CFTR. VX-809 is an investigational CFTR corrector that partially restores CFTR function in people who are homozygous for F508del-CFTR. To identify the folding defect(s) in F508del-CFTR that must be repaired to treat CF, we explored the mechanism of VX-809 action. VX-809 stabilized an N-terminal domain in CFTR that contains only MSD1 and efficaciously restored function to CFTR forms that have missense mutations in MSD1. The action of VX-809 on MSD1 appears to suppress folding defects in F508del-CFTR by enhancing interactions among the NBD1, MSD1, and MSD2 domains. The ability of VX-809 to correct F508del-CFTR is enhanced when combined with mutations that improve F508del-NBD1 interaction with MSD2. These data suggest that the use of VX-809 in combination with an additional CFTR corrector that suppresses folding defects downstream of MSD1 may further enhance CFTR function in people with F508del-CFTR.

Protein quality control and metabolic homeostasis are integral to maintaining cardiac function during stress; however, little is known about if or how these systems interact. Here we demonstrate that C terminus of HSC70-interacting protein (CHIP), a regulator of protein quality control, influences the metabolic response to pressure overload by direct regulation of the catalytic α subunit of AMPK. Induction of cardiac pressure overload in Chip-/- mice resulted in robust hypertrophy and decreased cardiac function and energy generation stemming from a failure to activate AMPK. Mechanistically, CHIP promoted LKB1-mediated phosphorylation of AMPK, increased the specific activity of AMPK, and was necessary and sufficient for stress-dependent activation of AMPK. CHIP-dependent effects on AMPK activity were accompanied by conformational changes specific to the α subunit, both in vitro and in vivo, identifying AMPK as the first physiological substrate for CHIP chaperone activity and establishing a link between cardiac proteolytic and metabolic pathways.

Mechanisms for cooperation between the cytosolic Hsp70 system and the ubiquitin proteasome system during protein triage are not clear. Herein, we identify new mechanisms for selection of misfolded cytosolic proteins for degradation via defining functional interactions between specific cytosolic Hsp70/Hsp40 pairs and quality control ubiquitin ligases. These studies revolved around the use of S. cerevisiae to elucidate the degradation pathway of a terminally misfolded reporter protein, short-lived GFP (slGFP). The Type I Hsp40 Ydj1 acts with Hsp70 to suppress slGFP aggregation. In contrast, the Type II Hsp40 Sis1 is required for proteasomal degradation of slGFP. Sis1 and Hsp70 operate sequentially with the quality control E3 ubiquitin ligase Ubr1 to target slGFP for degradation. Compromise of Sis1 or Ubr1 function leads slGFP to accumulate in a Triton X-100-soluble state with slGFP degradation intermediates being concentrated into perinuclear and peripheral puncta. Interestingly, when Sis1 activity is low the slGFP that is concentrated into puncta can be liberated from puncta and subsequently degraded. Conversely, in the absence of Ubr1, slGFP and the puncta that contain slGFP are relatively stable. Ubr1 mediates proteasomal degradation of slGFP that is released from cytosolic protein handling centers. Pathways for proteasomal degradation of misfolded cytosolic proteins involve functional interplay between Type II Hsp40/Hsp70 chaperone pairs, PQC E3 ligases, and storage depots for misfolded proteins.

To prevent the accumulation of misfolded and aggregated proteins, the cell has developed a complex network of cellular quality control (QC) systems to recognize misfolded proteins and facilitate their refolding or degradation. The cell faces numerous obstacles when performing quality control on transmembrane proteins. Transmembrane proteins have domains on both sides of a membrane and QC systems in distinct compartments must coordinate to monitor the folding status of the protein. Additionally, transmembrane domains can have very complex organization and QC systems must be able to monitor the assembly of transmembrane domains in the membrane. In this review, we will discuss the QC systems involved in repair and degradation of misfolded transmembrane proteins. Also, we will elaborate on the factors that recognize folding defects of transmembrane domains and what happens when misfolded transmembrane proteins escape QC and aggregate. This article is part of a Special Issue entitled: Protein Folding in Membranes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Maintenance of the proteome is a major homeostatic task of the cell and disregulation of protein homeostasis can be deadly. The accumulation of different forms of misfolded protein can perturb protein homeostasis and cause extensive cell and tissue damage. The cell has various quality control systems to help prevent the accumulation of misfolded proteins and the complexity of the different mechanisms that have evolved is bewildering. The first order of business for all quality control systems is recognition of misfolded proteins, which is followed by a triage decision. In many cases, modular molecular chaperones function in different assemblies with degradatory or folding co-factors to direct a misfolded protein toward continued life or death. Herein, an overview of quality control mechanisms that triage soluble cytosolic proteins, protein aggregates, and ER-associated proteins is presented.

Protein folding, refolding and degradation are essential for cellular life and are regulated by protein homeostatic processes such those that involve the molecular chaperone DnaK/Hsp70 and its co-chaperone DnaJ. Hsp70 action is initiated when proteins from the DnaJ family bind an unfolded protein for delivery purposes. In eukaryotes, the DnaJ family can be divided into two main groups, Type I and Type II, represented by yeast cytosolic Ydj1 and Sis1, respectively. Although sharing some unique features both members of the DnaJ family, Ydj1 and Sis1 are structurally and functionally distinct as deemed by previous studies, including the observation that their central domains carry the structural and functional information even in switched chimeras. In this study, we combined several biophysical tools for evaluating the stability of Sis1 and mutants that had the central domains (named Gly/Met rich domain and C-terminal Domain I) deleted or switched to those of Ydj1 to gain insight into the role of these regions in the structure and function of Sis1. The mutants retained some functions similar to full length wild-type Sis1, however they were defective in others. We found that: 1) Sis1 unfolds in at least two steps as follows: folded dimer to partially folded monomer and then to an unfolded monomer. 2) The Gly/Met rich domain had intrinsically disordered characteristics and its deletion had no effect on the conformational stability of the protein. 3) The deletion of the C-terminal Domain I perturbed the stability of the dimer. 4) Exchanging the central domains perturbed the conformational stability of the protein. Altogether, our results suggest the existence of two similar subdomains in the C-terminal domain of DnaJ that could be important for stabilizing each other in order to maintain a folded substrate-binding site as well as the dimeric state of the protein.

Background: Ydj1 and Sis1 are structurally and functionally distinct Hsp40 proteins of the yeast cytosol. Sis1 is an essential gene whereas the ydj1 gene is essential for growth at elevated temperatures and cannot complement sis1 gene deletion. Truncated polypeptides capable of complementing the sis1 gene deletion comprise the J-domain of either Sis1 or Ydj1 connected to the G/F region of Sis1 (but not Ydj1). Sis1 mutants in which the G/F was deleted but G/M maintained were capable of complementing the sis1 gene deletion., Results: To investigate the relevance of central domains on the structure and function of Ydj1 and Sis1 we prepared Sis1 constructs deleting specific domains. The mutants had decreased affinity for heated luciferase but were equally capable of stimulating ATPase activity of Hsp70. Detailed low resolution structures were obtained and the overall flexibility of Hsp40 and its mutants were assessed using SAXS methods. Deletion of either the G/M or the G/M plus CTDI domains had little impact on the quaternary structure of Sis1 analyzed by the SAXS technique. However, deletion of the ZFLR-CTDI changed the relative position of the J-domains in Ydj1 in such a way that they ended up resembling that of Sis1. The results revealed that the G/F and G/M regions are not the only flexible domains. All model structures exhibit a common clamp-like conformation., Conclusions: Our results suggest that the central domains, previously appointed as important features for substrate binding, are also relevant keeping the J-domains in their specific relative positions. The clamp-like architecture observed seems also to be favorable to the interactions of Hsp40 with Hsp70.

Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, ΔF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRΔF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl(-) channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl(-) conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to ∼20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTR(inh-172) and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRΔF508 function.

Accumulation of amyloid-like aggregates is a hallmark of numerous neurodegenerative disorders such as Alzheimer's and polyglutamine disease. Yet, whether the amyloid inclusions found in these diseases are toxic or cytoprotective remains unclear. Various studies suggest that the toxic culprit in the amyloid folding pathway is actually a soluble oligomeric species which might interfere with normal cellular function by a multifactorial mechanism including aberrant protein-protein interactions. Molecular chaperones suppress toxicity of amyloidogenic proteins by inhibiting aggregation of non-native disease substrates and targeting them for refolding or degradation. Paradoxically, recent studies also suggest a protective action of chaperones in their promotion of the assembly of large, tightly packed, benign aggregates that sequester toxic protein species., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The formation of amyloid-like fibrils is a hallmark of several neurodegenerative diseases. How the assembly of amyloid-like fibrils contributes to cell death is a major unresolved question in the field. The budding yeast Saccharomyces cerevisiae is a powerful model organism to study basic mechanisms for how cellular pathways regulate amyloid assembly and proteotoxicity. For example, studies of the amyloidogenic yeast prion [RNQ(+)] have revealed novel roles by which molecular chaperones protect cells from the accumulation of cytotoxic protein species. In budding yeast there are a variety of cellular assays that can be employed to analyze the assembly of amyloid-like aggregates and mechanistically dissect how cellular pathways influence proteotoxicity. In this review, we describe several assays that are routinely used to investigate aggregation and toxicity of the [RNQ(+)] prion in yeast., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Relative contributions of folding kinetics versus protein quality control (QC) activity in the partitioning of non-native proteins between life and death are not clear. Cystic fibrosis transmembrane conductance regulator (CFTR) biogenesis serves as an excellent model to study this question because folding of nascent CFTR is inefficient and deletion of F508 causes accumulation of CFTRΔF508 in a kinetically trapped, but foldable state. Herein, a novel endoplasmic reticulum (ER)-associated Hsp40, DNAJB12 (JB12) is demonstrated to play a role in control of CFTR folding efficiency. JB12 cooperates with cytosolic Hsc70 and the ubiquitin ligase RMA1 to target CFTR and CFTRΔF508 for degradation. Modest elevation of JB12 decreased nascent CFTR and CFTRΔF508 accumulation while increasing association of Hsc70 with ER forms of CFTR and the RMA1 E3 complex. Depletion of JB12 increased CFTR folding efficiency up to threefold and permitted a pool of CFTRΔF508 to fold and escape the ER. Introduction of the V510D misfolding suppressor mutation into CFTRΔF508 modestly increased folding efficiency, whereas combined inactivation of JB12 and suppression of intrinsic folding defects permitted CFTRΔF508 to fold at 50% of wild-type efficiency. Therapeutic correction of CFTRΔF508 misfolding in cystic fibrosis patients may require repair of defective folding kinetics and suppression of ER QC factors, such as JB12.

Human breast cancer resistance protein (BCRP)/MXR/ABCG2 is a well-recognized ABC half-transporter that is highly expressed at the apical membrane of many normal tissues and cancer cells. BCRP facilitates disposition of endogenous and exogenous harmful xenobiotics to protect cells/tissues from xenobiotic-induced toxicity. Despite the enormous impact of BCRP in the physiological and pathophysiological regulation of the transport of a wide variety of substrates, little is known about the factors that regulate posttranslational expression of BCRP. Here, we identified Derlin-1, a member of a family of proteins that bears homology to yeast Der1p, as a posttranslational regulator of BCRP expression. Overexpression of Derlin-1 suppressed ER to Golgi transport of wild-type (WT) BCRP that is known to be efficiently trafficked to the plasma membrane. On the other hand, protein expression of N596Q variant of BCRP, N-linked glycosylation-deficient mutant that preferentially undergoes ubiquitin-mediated ER-associated degradation (ERAD), was strongly suppressed by the overexpression of Derlin-1, whereas knockdown of Derlin-1 stabilized N596Q protein, suggesting a negative regulatory role of Derlin-1 for N596Q protein expression. Notably, knockdown of Derlin-1 also stabilized the expression of tunicamycin-induced deglycosylated WT BCRP protein, implying the importance of glycosylation state for the recognition of BCRP by Derlin-1. Thus, our data demonstrate that Derlin-1 is a negative regulator for both glycosylated and non-glycosylated BCRP expression and provide a novel posttranslational regulatory mechanism of BCRP by Derlin-1., (Copyright © 2010 Elsevier Inc. All rights reserved.)

CHIP, the carboxyl-terminus of Hsp70 interacting protein, is both an E3 ubiquitin ligase and an Hsp70 co-chaperone and is implicated in the degradation of cytosolic quality control and numerous disease substrates. CHIP has been shown to monitor the folding status of the CFTR protein, and we have successfully reconstituted this activity using a recombinant CFTR fragment consisting of the cytosolic NBD1 and R domains. We have found that efficient ubiquitination of substrates requires chaperone activity to either deliver the substrate to CHIP or to maintain the substrate in a ubiquitination-competent conformation. This chaperone activity can be provided by the Hsp70/Hsp40 molecular chaperone system as seen in the NBD1-R ubiquitination assay. Alternatively, heat treatment of CHIP can activate its own innate substrate-binding activity and allow for efficient ubiquitination of model substrates, such as denatured luciferase. Here, we describe methods for purifying the recombinant proteins necessary for in vitro reconstitution of CHIP ubiquitin ligase activity, as well as two methods used to monitor CHIP ligase activity. One method allows for the measurement of the Hsp70- and Hsp40-dependent CHIP activity while the other measures the Hsp40- and Hsp70-independent activity of heat-activated CHIP.

Misfolding and premature degradation of F508del-CFTR is the major cause of cystic fibrosis. Components of the ubiquitin-proteasome system function on the surface of the endoplasmic reticulum to select misfolded proteins for degradation. The folding status of F508del-CFTR is monitored by at least two ER quality control checkpoints. The ER-associated Derlin-1/RMA1 E3 complex appears to recognize folding defects in CFTR that involve misassembly of NBD1 into a complex with the R-domain. In contrast, the cytosolic Hsp70/CHIP E3 complex appears to sense folding defects that occur after synthesis of NBD2. Herein we describe methods that allow for the study of how modulation of these ER quality control factors by siRNA impacts CFTR folding and degradation. The experimental system described employs transiently transfected HEK293 cells and is utilized to monitor the biogenesis of CFTR by both Western blot and pulse chase studies. Methods to detect complexes formed between CFTR folding intermediates and ER quality control factors will also be described.

Quality control systems facilitate polypeptide folding and degradation to maintain protein homeostasis. Molecular chaperones promote folding, whereas the ubiquitin/proteasome system mediates degradation. We show here that Saccharomyces cerevisiae Ubr1 and Ubr2 ubiquitin ligases promote degradation of unfolded or misfolded cytosolic polypeptides. Ubr1 also catalyzes ubiquitinylation of denatured but not native luciferase in a purified system. This activity is based on the direct interaction of denatured luciferase with Ubr1, although Hsp70 stimulates polyubiquitinylation of the denatured substrate. We also report that loss of Ubr1 and Ubr2 function suppressed the growth arrest phenotype resulting from chaperone mutation. This correlates with increased protein kinase maturation and indicates partitioning of foldable conformers toward the proteasome. Our findings, based on the efficiency of this quality control system, suggest that the cell trades growth potential to avert the potential toxicity associated with accumulation of unfolded or misfolded proteins. Ubr1 and Ubr2 therefore represent E3 components of a novel quality control pathway for proteins synthesized on cytosolic ribosomes.

The misfolding and aggregation of disease proteins is characteristic of numerous neurodegenerative diseases. Particular neuronal populations are more vulnerable to proteotoxicity while others are more apt to tolerate the misfolding and aggregation of disease proteins. Thus, the cellular environment must play a significant role in determining whether disease proteins are converted into toxic or benign forms. The endomembrane network of eukaryotes divides the cell into different subcellular compartments that possess distinct sets of molecular chaperones and protein interaction networks. Chaperones act as agonists and antagonists of disease protein aggregation to prevent the accumulation of toxic intermediates in the aggregation pathway. Interacting partners can also modulate the conformation and localization of disease proteins and thereby influence proteotoxicity. Thus, interplay between these protein homeostasis network components can modulate the self-association of disease proteins and determine whether they elicit a toxic or benign outcome., ((c) 2009 Wiley Periodicals, Inc.)

Onset of proteotoxicity is linked to change in the subcellular location of proteins that cause misfolding diseases. Yet, factors that drive changes in disease protein localization and the impact of residence in new surroundings on proteotoxicity are not entirely clear. To address these issues, we examined aspects of proteotoxicity caused by Rnq1-green fluorescent protein (GFP) and a huntingtin's protein exon-1 fragment with an expanded polyglutamine tract (Htt-103Q), which is dependent upon the intracellular presence of [RNQ+] prions. Increasing heat-shock protein 40 chaperone activity before Rnq1-GFP expression, shifted Rnq1-GFP aggregation from the cytosol to the nucleus. Assembly of Rnq1-GFP into benign amyloid-like aggregates was more efficient in the nucleus than cytosol and nuclear accumulation of Rnq1-GFP correlated with reduced toxicity. [RNQ+] prions were found to form stable complexes with Htt-103Q, and nuclear Rnq1-GFP aggregates were capable of sequestering Htt-103Q in the nucleus. On accumulation in the nucleus, conversion of Htt-103Q into SDS-resistant aggregates was dramatically reduced and Htt-103Q toxicity was exacerbated. Alterations in activity of molecular chaperones, the localization of intracellular interaction partners, or both can impact the cellular location of disease proteins. This, in turn, impacts proteotoxicity because the assembly of proteins to a benign state occurs with different efficiencies in the cytosol and nucleus.

Premature degradation of CFTRDeltaF508 causes cystic fibrosis (CF). CFTRDeltaF508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTRDeltaF508 folding efficiency and the identity of CFTRDeltaF508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTRDeltaF508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTRDeltaF508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTRDeltaF508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTRDeltaF508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTRDeltaF508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTRDeltaF508 after MSD2 synthesis and was ineffective at rescue of DeltaF508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTRDeltaF508 folding and provide a therapeutic approach for CF.

Our previous studies have implicated CHIP (carboxyl terminus of Hsp70-interacting protein) as a co-chaperone/ubiquitin ligase whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP and Daxx (death domain-associated protein). This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP(+/+) but not in CHIP(-/-) heat shocked mouse embryonic fibroblasts. The interaction between CHIP and Daxx results in ubiquitination of Daxx, which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that ubiquitin chain formation utilizes non-canonical lysine linkages associated with resistance to proteasomal degradation. The ubiquitination of Daxx by CHIP utilizes lysines 630 and 631 and competes with the sumoylation machinery of the cell at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters the pro-apoptotic influence of Daxx in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the proteotoxic stress response of the cell with cell cycle pathways that influence cell survival.

Protein aggregation is a hallmark of a large and diverse number of conformational diseases. Molecular chaperones of the Hsp40 family (Escherichia coli DnaJ homologs) recognize misfolded disease proteins and suppress the accumulation of toxic protein species. Type I Hsp40s are very potent at suppressing protein aggregation and facilitating the refolding of damaged proteins. Yet, the molecular mechanism for the recognition of nonnative polypeptides by Type I Hsp40s such as yeast Ydj1 is not clear. Here we computationally identify a unique motif that is selectively recognized by Ydj1p. The motif is characterized by the consensus sequence GX[LMQ]{P}X{P}{CIMPVW}, where [XY] denotes either X or Y and {XY} denotes neither X nor Y. We further verify the validity of the motif by site-directed mutagenesis and show that substrate binding by Ydj1 requires recognition of this motif. A yeast proteome screen revealed that many proteins contain more than one stretch of residues that contain the motif and are separated by varying numbers of amino acids. In light of our results, we propose a 2-site peptide-binding model and a plausible mechanism of peptide presentation by Ydj1p to the chaperones of the Hsp70 family. Based on our results, and given that Ydj1p and its human ortholog Hdj2 are functionally interchangeable, we hypothesize that our results can be extended to understanding human diseases.

Neurodegenerative diseases ranging from Alzheimer disease and polyglutamine diseases to transmissible spongiform encephalopathies are associated with the aggregation and accumulation of misfolded proteins. In several cases the intracellular and extracellular protein deposits contain a fibrillar protein species called amyloid. However while amyloid deposits are hallmarks of numerous neurodegenerative diseases, their actual role in disease progression remains unclear. Especially perplexing is the often poor correlation between these deposits and other markers of neurodegeneration. As a result the question remains whether amyloid deposits are the disease-causing species, the consequence of cellular disease pathology or even the result of a protective cellular response to misfolded protein species. Here we highlight studies that suggest that accumulation and sequestration of misfolded protein in amyloid inclusion bodies and plaques can serve a protective function. Furthermore, we discuss how exceeding the cellular capacity for protective deposition of misfolded proteins may contribute to the formation of toxic protein species.

Heat shock protein 40 (Hsp40) co-chaperones assist in cellular protein folding and degradation through the binding and delivery of non-native proteins to heat shock protein 70 (Hsp70). The mechanism for substrate transfer from Hsp40s to Hsp70 is unknown. Two recent studies provide new details that shed light on novel mechanisms for substrate recognition by Hsp40s and a common mechanism for polypeptide transfer to Hsp70.

Molecular chaperones regulate essential steps in the propagation of yeast prions. Yeast prions possess domains enriched in glutamines and asparagines that act as templates to drive the assembly of native proteins into beta-sheet-rich, amyloid-like fibrils. Several recent studies highlight a significant and complex function for Hsp40 co-chaperones in propagation of prion elements in yeast. Hsp40 co-chaperones bind non-native polypeptides and transfer these clients to Hsp70s for refolding or degradation. How Hsp40 co-chaperones bind amyloid-like prion conformers that are enriched in hydrophilic residues such as glutamines and asparagines is a significant question in the field. Interestingly, selective recognition of amyloid-like conformers by distinct Hsp40s appears to confer opposing actions on prion assembly. For example, the Type I Hsp40 Ydj1 and Type II Hsp40 Sis1 bind different regions within the prion protein Rnq1 and function respectively to inhibit or promote [RNQ(+)] prion assembly. Thus, substrate selectivity enables distinct Hsp40s to act at unique steps in prion propagation.

The self-association of misfolded or damaged proteins into ordered amyloid-like aggregates characterizes numerous neurodegenerative disorders. Insoluble amyloid plaques are diagnostic of many disease states. Yet soluble, oligomeric intermediates in the aggregation pathway appear to represent the toxic culprit. Molecular chaperones regulate the fate of misfolded proteins and thereby influence their aggregation state. Chaperones conventionally antagonize aggregation of misfolded, disease proteins and assist in refolding or degradation pathways. Recent work suggests that chaperones may also suppress neurotoxicity by converting toxic, soluble oligomers into benign aggregates. Chaperones can therefore suppress or promote aggregation of disease proteins to ameliorate the proteotoxic accumulation of soluble, assembly intermediates.

Type I Hsp40s are molecular chaperones that protect neurons from degeneration by modulating the aggregation state of amyloid-forming proteins. How Type I Hsp40s recognize beta-rich, amyloid-like substrates is currently unknown. Thus, we examined the mechanism for binding between the Type I Hsp40 Ydj1 and the yeast prion [RNQ+]. Ydj1 recognized the Gln/Asn-rich prion domain from Rnq1 specifically when it assembled into the amyloid-like [RNQ+] prion state. Upon deletion of YDJ1, overexpression of the Rnq1 prion domain killed yeast. Surprisingly, binding and suppression of prion domain toxicity by Ydj1 was dependent upon farnesylation of its C-terminal CAAX box and action of a zinc finger-like region. In contrast, folding of luciferase was independent of farnesylation, yet required the zinc finger-like region of Ydj1 and a conserved hydrophobic peptide-binding pocket. Type I Hsp40s contain at least three different domains that work in concert to bind different protein conformers. The combined action of a farnesyl moiety and zinc finger-like region enable Type I Hsp40s to recognize amyloid-like substrates and prevent formation of cytotoxic protein species.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a polytopic membrane protein that functions as a Cl(-) channel and consists of two membrane spanning domains (MSDs), two cytosolic nucleotide binding domains (NBDs), and a cytosolic regulatory domain. Cytosolic 70-kDa heat shock protein (Hsp70), and endoplasmic reticulum-localized calnexin are chaperones that facilitate CFTR biogenesis. Hsp70 functions in both the cotranslational folding and posttranslational degradation of CFTR. Yet, the mechanism for calnexin action in folding and quality control of CFTR is not clear. Investigation of this question revealed that calnexin is not essential for CFTR or CFTRDeltaF508 degradation. We identified a dependence on calnexin for proper assembly of CFTR's membrane spanning domains. Interestingly, efficient folding of NBD2 was also found to be dependent upon calnexin binding to CFTR. Furthermore, we identified folding defects caused by deletion of F508 that occurred before and after the calnexin-dependent association of MSD1 and MSD2. Early folding defects are evident upon translation of the NBD1 and R-domain and are sensed by the RMA-1 ubiquitin ligase complex.

Heat shock protein (Hsp)40s play an essential role in protein metabolism by regulating the polypeptide binding and release cycle of Hsp70. The Hsp40 family is large, and specialized family members direct Hsp70 to perform highly specific tasks. Type I and Type II Hsp40s, such as yeast Ydj1 and Sis1, are homodimers that dictate functions of cytosolic Hsp70, but how they do so is unclear. Type I Hsp40s contain a conserved, centrally located cysteine-rich domain that is replaced by a glycine- and methionine-rich region in Type II Hsp40s, but the mechanism by which these unique domains influence Hsp40 structure and function is unknown. This is the case because high-resolution structures of full-length forms of these Hsp40s have not been solved. To fill this void, we built low-resolution models of the quaternary structure of Ydj1 and Sis1 with information obtained from biophysical measurements of protein shape, small-angle X-ray scattering, and ab initio protein modeling. Low-resolution models were also calculated for the chimeric Hsp40s YSY and SYS, in which the central domains of Ydj1 and Sis1 were exchanged. Similar to their human homologs, Ydj1 and Sis1 each has a unique shape with major structural differences apparently being the orientation of the J domains relative to the long axis of the dimers. Central domain swapping in YSY and SYS correlates with the switched ability of YSY and SYS to perform unique functions of Sis1 and Ydj1, respectively. Models for the mechanism by which the conserved cysteine-rich domain and glycine- and methionine-rich region confer structural and functional specificity to Type I and Type II Hsp40s are discussed.

Molecular chaperones such as heat shock protein 70 (Hsp70) are crucial for protein folding. Crystal structures of Hsp70 in a complex with the nucleotide exchange factor (NEF) Hsp110 reported in this issue of Cell (Polier et al., 2008) and in Molecular Cell (Schuermann et al., 2008) provide new insights into how NEF action specifies Hsp70 cellular function.

Protein conformational diseases are associated with the aberrant accumulation of amyloid protein aggregates, but whether amyloid formation is cytotoxic or protective is unclear. To address this issue, we investigated a normally benign amyloid formed by the yeast prion [RNQ(+)]. Surprisingly, modest overexpression of Rnq1 protein was deadly, but only when preexisting Rnq1 was in the [RNQ(+)] prion conformation. Molecular chaperones protect against protein aggregation diseases and are generally believed to do so by solubilizing their substrates. The Hsp40 chaperone, Sis1, suppressed Rnq1 proteotoxicity, but instead of blocking Rnq1 protein aggregation, it stimulated conversion of soluble Rnq1 to [RNQ(+)] amyloid. Furthermore, interference with Sis1-mediated [RNQ(+)] amyloid formation exacerbated Rnq1 toxicity. These and other data establish that even subtle changes in the folding homeostasis of an amyloidogenic protein can create a severe proteotoxic gain-of-function phenotype and that chaperone-mediated amyloid assembly can be cytoprotective. The possible relevance of these findings to other phenomena, including prion-driven neurodegenerative diseases and heterokaryon incompatibility in fungi, is discussed.

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin-proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRDeltaF508, by specifically promoting ubiquitylation of CFTRDeltaF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRDeltaF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRDeltaF508 and catalyzes further polyubiquitylation of CFTRDeltaF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRDeltaF508.

McKusick-Kaufman syndrome (MKKS) is a recessively inherited human genetic disease characterized by several developmental anomalies. Mutations in the MKKS gene also cause Bardet-Biedl syndrome (BBS), a genetically heterogeneous disorder with pleiotropic symptoms. However, little is known about how MKKS mutations lead to disease. Here, we show that disease-causing mutants of MKKS are rapidly degraded via the ubiquitin-proteasome pathway in a manner dependent on HSC70 interacting protein (CHIP), a chaperone-dependent ubiquitin ligase. Although wild-type MKKS quickly shuttles between the centrosome and cytosol in living cells, the rapidly degraded mutants often fail to localize to the centrosome. Inhibition of proteasome functions causes MKKS mutants to form insoluble structures at the centrosome. CHIP and partner chaperones, including heat-shock protein (HSP)70/heat-shock cognate 70 and HSP90, strongly recognize MKKS mutants. Modest knockdown of CHIP by RNA interference moderately inhibited the degradation of MKKS mutants. These results indicate that the MKKS mutants have an abnormal conformation and that chaperone-dependent degradation mediated by CHIP is a key feature of MKKS/BBS diseases.