Cytosolic Hsc70 Research Articles

Targeting chaperone-mediated autophagy in neurodegenerative diseases: mechanisms and therapeutic potential.

The pathological hallmarks of various neurodegenerative diseases including Parkinson's disease and Alzheimer's disease prominently feature the accumulation of misfolded proteins and neuroinflammation. Chaperone-mediated autophagy (CMA) has emerged as a distinct autophagic process that coordinates the lysosomal degradation of specific proteins bearing the pentapeptide motif Lys-Phe-Glu-Arg-Gln (KFERQ), a recognition target for the cytosolic chaperone HSC70. Beyond its role in protein quality control, recent research underscores the intimate interplay between CMA and immune regulation in neurodegeneration. In this review, we illuminate the molecular mechanisms and regulatory pathways governing CMA. We further discuss the potential roles of CMA in maintaining neuronal proteostasis and modulating neuroinflammation mediated by glial cells. Finally, we summarize the recent advancements in CMA modulators, emphasizing the significance of activating CMA for the therapeutic intervention in neurodegenerative diseases.

Activation of Disulfide Redox Switch in REDD1 Promotes Oxidative Stress Under Hyperglycemic Conditions.

The stress response protein regulated in development and DNA damage response 1 (REDD1) has been implicated in visual deficits in patients with diabetes. The aim here was to investigate the mechanism responsible for the increase in retinal REDD1 protein content that is observed with diabetes. We found that REDD1 protein expression was increased in the retina of streptozotocin-induced diabetic mice in the absence of a change in REDD1 mRNA abundance or ribosome association. Oral antioxidant supplementation reduced retinal oxidative stress and suppressed REDD1 protein expression in the retina of diabetic mice. In human retinal Müller cell cultures, hyperglycemic conditions increased oxidative stress, enhanced REDD1 expression, and inhibited REDD1 degradation independently of the proteasome. Hyperglycemic conditions promoted a redox-sensitive cross-strand disulfide bond in REDD1 at C150/C157 that was required for reduced REDD1 degradation. Discrete molecular dynamics simulations of REDD1 structure revealed allosteric regulation of a degron upon formation of the disulfide bond that disrupted lysosomal proteolysis of REDD1. REDD1 acetylation at K129 was required for REDD1 recognition by the cytosolic chaperone HSC70 and degradation by chaperone-mediated autophagy. Disruption of REDD1 allostery upon C150/C157 disulfide bond formation prevented the suppressive effect of hyperglycemic conditions on REDD1 degradation and reduced oxidative stress in cells exposed to hyperglycemic conditions. The results reveal redox regulation of REDD1 and demonstrate the role of a REDD1 disulfide switch in development of oxidative stress.

The Role of Chaperone-Mediated Autophagy in Hepatitis C Virus-Induced Pathogenesis.

Lysosome incorporate and degrade proteins in a process known as autophagy. There are three types of autophagy; macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Although autophagy is considered a nonselective degradation process, CMA is known as a selective degradation pathway. All proteins internalized in the lysosome via CMA contain a pentapeptide KFERQ-motif, also known as a CMA-targeting motif, which is necessary for selectivity. CMA directly delivers a substrate protein into the lysosome lumen using the cytosolic chaperone HSC70 and the lysosomal receptor LAMP-2A for degradation. Hepatitis C virus (HCV) NS5A protein interacts with hepatocyte-nuclear factor 1α (HNF-1α) together with HSC70 and promotes the lysosomal degradation of HNF-1α via CMA, resulting in HCV-induced pathogenesis. HCV NS5A promotes recruitment of HSC70 to the substrate protein HNF-1α. HCV NS5A plays a crucial role in HCV-induced CMA. Further investigations of HCV NS5A-interacting proteins containing CMA-targeting motifs may help to elucidate HCV-induced pathogenesis.

A plant reovirus hijacks the DNAJB12-Hsc70 chaperone complex to promote viral spread in its planthopper vector.

Many viruses usurp the functions of endoplasmic reticulum (ER) for virus‐encoded membrane proteins proper functional folding or assembly to promote virus spread. Southern rice black‐streaked dwarf virus (SRBSDV), a plant reovirus, exploits virus‐containing tubules composed of nonstructural membrane protein P7‐1 to spread in its planthopper vector Sogatella furcifera. Here, we report that two factors of the ER‐associated degradation (ERAD) machinery, the ER chaperone DNAJB12 and its cytosolic co‐chaperone Hsc70, are activated by SRBSDV to facilitate ER‐to‐cytosol export of P7‐1 tubules in S. furcifera. Both P7‐1 of SRBSDV and Hsc70 directly bind to the J‐domain of DNAJB12. DNAJB12 overexpression induces ER retention of P7‐1, but Hsc70 overexpression promotes the transport of P7‐1 from the ER to the cytosol to initiate tubule assembly. Thus, P7‐1 is initially retained in the ER by interaction with DNAJB12 and then delivered to Hsc70. Furthermore, the inhibitors of the ATPase activity of Hsc70 reduce P7‐1 tubule assembly, suggesting that the proper folding and assembly of P7‐1 tubules is dependent on the ATPase activity of Hsc70. The DNAJB12–Hsc70 chaperone complex is recruited to P7‐1 tubules in virus‐infected midgut epithelial cells in S. furcifera. The knockdown of DNAJB12 or Hsc70 strongly inhibits P7‐1 tubule assembly in vivo, finally suppressing effective viral spread in S. furcifera. Taken together, our results indicate that the DNAJB12–Hsc70 chaperone complex in the ERAD machinery facilitates the ER‐to‐cytosol transport of P7‐1 for proper assembly of tubules, enabling viral spread in insect vectors in a manner dependent on ATPase activity of Hsc70.

HSC70 expression is reduced in lymphomonocytes of sporadic ALS patients and contributes to TDP-43 accumulation

Aim: The demonstration that chaperone-mediated autophagy (CMA) contributes to the degradation of TDP-43, the main constituent of cytoplasmic inclusions typically found in motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS), has pointed out a possible involvement of CMA in aggregate formation. To explore this possibility, in this study, we verified the presence of a possible systemic CMA alteration in sALS patients and its effect on TDP-43 expression. Materials and methods: Gene and protein expression of the cytosolic chaperone HSC70 and the lysosome receptor LAMP2A, the two pivotal mediators of CMA, was assessed in peripheral blood mononuclear cells (PBMCs) derived from 30 sALS patients and 30 healthy controls. The expression of TDP-43 and co-chaperones BAG1 and BAG3 was also analyzed. Results: We found reduced HSC70 expression in patient cells, with no change in LAMP2A, and increased insoluble TDP-43 protein levels, with an aberrant intracellular localization. We also observed an unbalanced expression of co-chaperones BAG1 and BAG3. HSC70 down-regulation was confirmed in immortalized lymphoblastoid cell lines derived from sporadic and TARDBP mutant ALS patients. Lastly, we demonstrated that HSC70 silencing directly increases TDP-43 protein levels in human neuroblastoma cells. Discussion: Our results do not support the existence of a systemic CMA alteration in sALS patients but indicate a direct involvement of HSC70 alterations in ALS pathogenesis.

Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones.

Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases.

Macronutrient deprivation modulates antigen trafficking and immune recognition through HSC70 accessibility.

B lymphocytes exploit macroautophagy to capture cytoplasmic and nuclear proteins within autophagosomes. Fusion of autophagosomes with lysosomes and endosomes facilitates content proteolysis, with the resulting peptides selectively binding MHC class II (MHC II) molecules, which are displayed for recognition by T lymphocytes. Nutrient deprivation or stress amplified this pathway, favoring increased MHC II presentation of cytoplasmic Ags targeted to autophagosomes. By contrast, this stress diminished MHC II presentation of membrane Ags including the BCR and cytoplasmic proteins that use the chaperone-mediated autophagy pathway. Whereas intracellular protease activity increased with nutrient stress, endocytic trafficking and proteolytic turnover of the BCR was impaired. Addition of macronutrients such as high molecular mass proteins restored endocytosis and Ag presentation, evidence of tightly regulated membrane trafficking dependent on macronutrient status. Altering cellular levels of the cytosolic chaperone HSC70 was sufficient to overcome the inhibitory effects of nutritional stress on BCR trafficking and Ag presentation. Together, these results reveal a key role for macronutrient sensing in regulating immune recognition and the importance of HSC70 in modulating membrane trafficking pathways during cellular stress.

Streptococcus pneumonia 감염으로 변화한 사람 폐 상피세포 단백질의 프로테오믹 분석

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia throughout the world. The bacteria invade through lung tissue and cause sepsis, shock, and serious sequelae, including rheumatic fever and acute glomerulonephritis. However, the molecular mechanism associated with pneumonia’s penetration of lung tissue and invasion of the blood stream are still unclear. We attempted to investigate the host cell response at protein levels to S. pneumoniae D39 invasion using human lung cancer epithelial cells, A549. Streptococcus pneumoniae D39 began to change the morphology of A549 cells to become round with filopodia at 2 hours post-infection. A549 cell proteins obtained at each infection time point were separated by SDS-PAGE and analyzed using MALDI-TOF. We identified several endoplasmic reticulum (ER) resident proteins such as Grp94 and Grp78 and mitochondrial proteins such as ATP synthase and Hsp60 that increased after S. pneumoniae D39 infection. Cytosolic Hsc70 and Hsp90 were, however, identified to decrease. These proteins were also confirmed by Western blot analysis. The identified ER resident proteins were known to be induced during ER stress signaling. These/ data, therefore, suggest that S. pneumoniae D39 infection may induce ER stress.

The Cytosolic Chaperone Hsc70 Promotes Traffic to the Cell Surface of Intracellular Retained Melanocortin-4 Receptor Mutants

Inherited modifications in protein structure frequently cause a loss-of-function by interfering with protein synthesis, transport, or stability. For the obesity-linked melanocortin-4 receptor (MC4R) and other G protein-coupled receptors, many mutants are intracellular retained. The biogenesis and trafficking of G protein-coupled receptors are regulated by multiple factors, including molecular chaperone networks. Here, we have investigated the ability of the cytosolic cognate 70-kDa heat-shock protein (Hsc70) chaperone system to modulate cell surface expression of MC4R. Clinically occurring MC4R mutants S58C, P78L, and D90N were demonstrated to have reduced trafficking to the plasma membrane and to be retained at the endoplasmic reticulum (ER). Analyses by fluorescence recovery after photobleaching revealed that the mobility of MC4R mutant protein at the ER was reduced, implying protein misfolding. In cells expressing MC4R, overexpression of Hsc70 resulted in increased levels of wild-type and mutant receptors at the cell surface. MC4R and Hsc70 coimmunoprecipitated, and fluorescence recovery after photobleaching analyses showed that increasing cellular levels of Hsc70 promoted the mobility of ER retained MC4R. Moreover, expression of HSJ1b, a cochaperone that enhances degradation of Hsc70 clients, reduced cellular levels of MC4R. Hsp70 and Hsp90 chaperone systems collaborate in the cellular processing of clients. For MC4R, inhibition of endogenous Hsp90 by geldanamycin reduced receptor levels. By contrast, expression of the Hsp90 cochaperone Aha1 (activator of Hsp90 ATPase) increased cellular levels of MC4R. Finally, we demonstrate that signaling of intracellular retained MC4R mutants is increased in cells overexpressing Hsc70. These data indicate that cytosolic chaperone systems can facilitate rescue of intracellular retained MC4R by improving folding. They also support proteostasis networks as a potential target for MC4R-linked obesity.

The endoplasmic reticulum–associated Hsp40 DNAJB12 and Hsc70 cooperate to facilitate RMA1 E3–dependent degradation of nascent CFTRΔF508

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.

Heat shock protein 70 (hsp70) as an emerging drug target.

Heat shock protein 70 (Hsp70) is a molecular chaperone that is expressed in response to stress. In this role, Hsp70 binds to its protein substrates and stabilize them against denaturation or aggregation until conditions improve.1 In addition to its functions during a stress response, Hsp70 has multiple responsibilities during normal growth; it assists in the folding of newly synthesized proteins,2, 3 the subcellular transport of proteins and vesicles,4 the formation and dissociation of complexes,5 and the degradation of unwanted proteins.6, 7 Thus, this chaperone broadly shapes protein homeostasis by controlling protein quality control and turnover during both normal and stress conditions.8 Consistent with these diverse activities, genetic and biochemical studies have implicated it in a range of diseases, including cancer, neurodegeneration, allograft rejection and infection. This review provides a brief review of Hsp70 structure and function and then explores some of the emerging opportunities (and challenges) for drug discovery. Hsp70 is Highly Conserved Members of the Hsp70 family are ubiquitously expressed and highly conserved; for example, the major Hsp70 from Escherichia coli, termed DnaK, is approximately 50% identical to human Hsp70s.9 Eukaryotes often express multiple Hsp70 family members with major isoforms found in all the cellular compartments: Hsp72 (HSPA1A) and heat shock cognate 70 (Hsc70/HSPA8) in the cytosol and nucleus, BiP (Grp78/HSPA5) in the endoplasmic reticulum and mtHsp70 (Grp75/mortalin/HSPA9) in mitochondria. Some of the functions of the cytosolic isoforms, Hsc70 and Hsp72, are thought to be redundant, but the transcription of Hsp72 is highly responsive to stress and Hsc70 is constitutively expressed. In the ER and mitochondria, the Hsp70 family members are thought to fulfill specific functions and have unique substrates, with BiP playing key roles in the folding and quality control of ER proteins and mtHsp70 being involved in the import and export of proteins from the mitochondria. For the purposes of this review, we will often use Hsp70 as a generic term to encompass the shared properties of the family members.

Structural Analysis of the Ribosome-associated Complex (RAC) Reveals an Unusual Hsp70/Hsp40 Interaction

Yeast Zuotin and Ssz are members of the conserved Hsp40 and Hsp70 chaperone families, respectively, but compared with canonical homologs, they atypically form a stable heterodimer termed ribosome-associated complex (RAC). RAC acts as co-chaperone for another Hsp70 to assist de novo protein folding. In this study, we identified the molecular basis for the unusual Hsp70/Hsp40 pairing using amide hydrogen exchange (HX) coupled with mass spectrometry and mutational analysis. Association of Ssz with Zuotin strongly decreased the conformational dynamics mainly in the C-terminal domain of Ssz, whereas Zuotin acquired strong conformational stabilization in its N-terminal segment. Deletion of the highly flexible N terminus of Zuotin abolished stable association with Ssz in vitro and caused a phenotype resembling the loss of Ssz function in vivo. Thus, the C-terminal domain of Ssz, the N-terminal extension of Zuotin, and their mutual stabilization are the major structural determinants for RAC assembly. We furthermore found dynamic changes in the J-domain of Zuotin upon complex formation that might be crucial for RAC co-chaperone function. Taken together, we present a novel mechanism for converting Zuotin and Ssz chaperones into a functionally active dimer.

Transgenic Expression of Hsc70 in Pancreatic Islets Enhances Autoimmune Diabetes in Response to β Cell Damage

Inflammation following tissue damage promotes lymphocyte recruitment, tissue remodeling, and wound healing while maintaining self tolerance. Endogenous signals associated with tissue damage and cell death have been proposed to initiate and instruct immune responses following injury. In this study, we have examined the effects of elevated levels of a candidate endogenous danger signal, heat shock cognate protein 70 (hsc70), on stimulation of inflammation and autoimmunity following cell damage. We find that damage to pancreatic beta cells expressing additional cytosolic hsc70 leads to an increased incidence of diabetes in a transgenic mouse model. Steady-state levels of activated APC and T cell populations in the draining lymph node were enhanced, which further increased following streptozotocin-induced beta cell death. In addition, proinflammatory serum cytokines, and lymphocyte recruitment were increased in hsc70 transgenic mice. Islet Ag-specific T cells underwent a greater extent of proliferation in the lymph nodes of mice expressing hsc70 following beta cell damage, suggesting elevated Ag presentation following release of Ag in the presence of hsc70. These findings suggest that an elevated content of hsc70 in cells undergoing necrotic or apoptotic cell death can increase the extent of sterile inflammation and increase the susceptibility to autoimmunity.

Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34(+) erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61alpha), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.

Interaction of human heat shock protein 70 with tumor-associated peptides

Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immunological approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity (K(d) = 0.58 microm at 25 degrees C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase (K(d) = 32 microm) and melanoma antigen recognized by T cells (MART-1) (K(d) = 2.4 microm). Adding a peptide sequence (pep70) with high Hsp70 binding affinity (K(d) = 0.04 microm) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation.

Mitochondrial carrier protein biogenesis: role of the chaperones Hsc70 and Hsp90.

Metabolite carrier proteins of the mitochondrial inner membrane share homology in their transmembrane domains, which also carries their targeting information. In addition, some carriers have cleavable presequences which are not essential for targeting, but have some other function before import. The cytosolic chaperones Hsc70 (heat-shock cognate 70) and Hsp90 (heat-shock protein 90) complex with carrier precursors and interact specifically with the Tom (translocase of the mitochondrial outer membrane) 70 import receptor to promote import. We analysed how the presequences of the PiC (phosphate carrier) and CIC (citrate carrier) relate to the mechanisms of chaperone-mediated import. Deletion of the PiC presequence reduced the efficiency of import but, notably, not by causing aggregation. Instead, binding of the protein to Hsc70 was reduced, as well as the dependence on Hsc70 for import. Hsp90 binding and function in import was not greatly affected, but it could not entirely compensate for the lack of Hsc70 interaction. Deletion of the presequence from CIC was shown to cause its aggregation, but had little effect on the contribution to import of either Hsc70 or Hsp90. The presequence of PiC, but not that of CIC, conferred Hsc70 binding to dihydrofolate reductase fusion proteins. In comparison, OGC (oxoglutarate carrier) lacks a presequence and was more soluble, though it is still dependent on both Hsc70 and Hsp90. We propose that carrier presequences evolved to improve targeting competence by different mechanisms, depending on physical properties of the precursors in the cytosolic targeting environment.

Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS

Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

Post-translational integration of tail-anchored proteins is facilitated by defined molecular chaperones

Tail-anchored (TA) proteins provide an ideal model for studying post-translational integration at the endoplasmic reticulum (ER) of eukaryotes. There are multiple pathways for delivering TA proteins from the cytosol to the ER membrane yet, whereas an ATP-dependent route predominates, none of the cytosolic components involved had been identified. In this study we have directly addressed this issue and identify novel interactions between a model TA protein and the two cytosolic chaperones Hsp40 and Hsc70. To investigate their function, we have reconstituted the membrane integration of TA proteins using purified components. Remarkably, we find that a combination of Hsc70 and Hsp40 can completely substitute for the ATP-dependent factors present in cytosol. On the basis of this in vitro analysis, we conclude that this chaperone pair can efficiently facilitate the ATP-dependent integration of TA proteins.

In vitro evidence of Hsc70 functioning as a molecular chaperone during cold stress

Hsp70 molecular chaperones have been shown to play an important role in helping cells to cope with adverse environments, especially in response to high temperatures. The molecular chaperone function of Hsc70 at low temperature was investigated. A cold-inducible spinach cytosolic Hsc70 was subcloned into a protein expression vector and the recombinant protein was expressed in bacterial cells. Recombinant Hsc70 bound a permanently unfolded substrate: α-carboxymethylated lactalbumin (CMLA) in the presence of 3 mM ATP and MgCl 2 at low temperature (4 and –4 °C). Radiolabeling with 35S-Met and 35S-Cys and immunoprecipitation with cytosolic Hsc70 monoclonal antibodies showed that there were several proteins co-immunoprecipitated at low temperature (4 and –4 °C) but not at room temperature. Enhanced co-purification of sHsp17.7 with Hsc70 at low temperature was observed and suggests that co-chaperone interactions can contribute to molecular chaperone function during cold stress. These results suggest that the molecular chaperone Hsc70 may have a functional role in plants during low temperature stress.

Tid1 Isoforms Are Mitochondrial DnaJ-like Chaperones with Unique Carboxyl Termini That Determine Cytosolic Fate

Tid1 is a human homolog of bacterial DnaJ and the Drosophila tumor suppressor Tid56 that has two alternatively spliced isoforms, Tid1-long and -short (Tid1-L and -S), which differ only at their carboxyl termini. Although Tid1 proteins localize overwhelmingly to mitochondria, published data demonstrate principally nonmitochondrial protein interactions and activities. This study was undertaken to determine whether Tid1 proteins function as mitochondrial DnaJ-like chaperones and to resolve the paradox of how proteins targeted primarily to mitochondria function in nonmitochondrial pathways. Here we demonstrate that Tid1 isoforms exhibit a conserved mitochondrial DnaJ-like function substituting for the yeast mitochondrial DnaJ-like protein Mdj1p. Like Mdj1p, Tid1 localizes to human mitochondrial nucleoids, which are large protein complexes bound to mitochondrial DNA. Unlike other DnaJs, Tid1-L and -S form heterocomplexes; both unassembled and complexed Tid1 are observed in human cells. Results demonstrate that Tid1-L has a longer residency time in the cytosol prior to mitochondrial import as compared with Tid1-S; Tid1-L is also significantly more stable in the cytosol than Tid1-S, which is rapidly degraded. The longer cytosolic residency time and the half-life of Tid1-L are explained by its interaction with cytosolic Hsc70 and potential protein substrates such as the STAT1 and STAT3 transcription factors. We show that the unique carboxyl terminus of Tid1-L is required for interaction with Hsc70 and STAT1 and -3. We propose that the association of Tid1 with chaperones and/or protein substrates in the cytosol provides a mechanism for the alternate fates and functions of Tid1 in mitochondrial and nonmitochondrial pathways.