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Direct interactions between molecular chaperones heat-shock protein (Hsp) 70 and Hsp40: yeast Hsp70 Ssa1 binds the extreme C-terminal region of yeast Hsp40 Sis1.

ABSTRACT: Heat-shock protein 40 (Hsp40) enables Hsp70 to play critical roles in a number of cellular processes, such as protein folding, assembly, degradation and translocation in vivo. Hsp40 recognizes and binds non-native polypeptides and delivers them to Hsp70. Then Hsp40 stimulates the ATPase activity of Hsp70 to fold the polypeptides. By using yeast Hsp40 Sis1 and yeast Hsp70 Ssa1 as our model proteins, we found that the Sis1 peptide-binding fragment interacts directly with the full-length Ssa1 in vitro. Further studies showed that the C-terminal lid domain of Ssa1 could interact with Sis1 peptide-binding domain physically in vitro. The Sis1 peptide-binding fragment forms a stable complex with the Ssa1 C-terminal lid domain in solution. The interactions between these two proteins appear to be charge-charge interactions because high-ionic-strength buffer can dissociate the complex. Further mapping studies showed that the Sis1 peptide-binding fragment binds the extreme C-terminal 15 amino acid residues of Ssa1. A flexible glycine-rich region is followed by these 15 residues in the Ssa1 primary sequence. Atomic force microscopy of the Sis1-Ssa1 complex showed that only one end of the Ssa1 lid domain binds the Sis1 peptide-binding-fragment dimer at the upper level of the huge groove within the Sis1 dimer. Based on the data, we propose an "anchoring and docking" model to illustrate the mechanisms by which Hsp40 interacts with Hsp70 and delivers the non-native polypeptide to Hsp70.

SUBMITTER: Qian X

PROVIDER: S-EPMC1222294 | biostudies-other | 2002 Jan

REPOSITORIES: biostudies-other

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Single methyl group determines prion propagation and protein degradation activities of yeast heat shock protein (Hsp)-70 chaperones Ssa1p and Ssa2p.

Project description:Organisms encode multiple homologous heat shock protein (Hsp)-70s, which are essential protein chaperones that play the major role in cellular protein "quality control." Although Hsp70s are functionally redundant and highly homologous, many possess distinct functions. A regulatory motif underlying such distinctions, however, is unknown. The 98% identical cytoplasmic Hsp70s Ssa1p and Ssa2p function differently with regard to propagation of yeast [URE3] prions and in the vacuolar-mediated degradation of gluconeogenesis enzymes, such as FBPase. Here, we show that the Hsp70 nucleotide binding domain (NBD) regulates these functional specificities. We find little difference in ATPase, protein refolding, and amyloid inhibiting activities of purified Ssa1p and Ssa2p, but show that interchanging NBD residue alanine 83 (Ssa1p) and glycine 83 (Ssa2p) switched functions of Ssa1p and Ssa2p in [URE3] propagation and FBPase degradation. Disrupting the degradation pathway did not affect prion propagation, however, indicating these are two distinct processes where Ssa1/2p chaperones function differently. Our results suggest that the primary evolutionary pressure for Hsp70 functional distinctions is not to specify interactions of Hsp70 with substrate, but to specify the regulation of this activity. Our data suggest a rationale for maintaining multiple Hsp70s and suggest that subtle differences among Hsp70s evolved to provide functional specificity without affecting overall enzymatic activity.

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Prion-like nuclear aggregation of TDP-43 during heat shock is regulated by HSP40/70 chaperones.

Project description:TDP-43 aggregation in the cytoplasm or nucleus is a key feature of the pathology of amyotrophic lateral sclerosis and frontotemporal dementia and is observed in numerous other neurodegenerative diseases, including Alzheimer's disease. Despite this fact, the inciting events leading to TDP-43 aggregation remain unclear. We observed that endogenous TDP-43 undergoes reversible aggregation in the nucleus after the heat shock and that this behavior is mediated by the C-terminal prion domain. Substitution of the prion domain from TIA-1 or an authentic yeast prion domain from RNQ1 into TDP-43 can completely recapitulate heat shock-induced aggregation. TDP-43 is constitutively bound to members of the Hsp40/Hsp70 family, and we found that heat shock-induced TDP-43 aggregation is mediated by the availability of these chaperones interacting with the inherently disordered C-terminal prion domain. Finally, we observed that the aggregation of TDP-43 during heat shock led to decreased binding to hnRNPA1, and a change in TDP-43 RNA-binding partners suggesting that TDP-43 aggregation alters its function in response to misfolded protein stress. These findings indicate that TDP-43 shares properties with physiologic prions from yeast, in that self-aggregation is mediated by a Q/N-rich disordered domain, is modulated by chaperone proteins and leads to altered function of the protein. Furthermore, they indicate that TDP-43 aggregation is regulated by chaperone availability, explaining the recurrent observation of TDP-43 aggregates in degenerative diseases of both the brain and muscle where protein homeostasis is disrupted.

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Heat shock protein (Hsp) 70 is an activator of the Hsp104 motor.

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Project description:Cadmium (Cd) is an environmental carcinogenic pollutant known to inactivate several proteins involved in DNA repair systems while at the same time creating an oxidative stress that can result in additional DNA lesions. The testis and the lung are the target organs for cadmium carcinogenesis. Increased production of oxidants in vivo can cause damage to intracellular macromolecules such as DNA, proteins and lipids, which in turn lead to oxidative injury. So, this investigation aimed to evaluate the protective role of L-Carnitine through up regulation of HSPs against DNA damage induced by cadmium chloride. The current study was carried out on forty adult male rats, each with average weight 220-250g., were divided into 4 equal groups. 1(st) group was received saline solution (0.5 ml/100 g body weight) and kept as control. 2(nd) group was received 500mg / kg body weight L-Carnitine intraperitoneally (IP). 3(rd) group was administered 1.2 mg cadmium chloride IP. 4(th) group was received both cadmium chloride and L-Carnitine simultaneously. The comet assay parameters showed significantly increased HSP70 and DNA damage in testis cells after 10 and 56 days in the third group. Meanwhile, HSP70 showed significantly decreased levels after 10 days and 56 days in the fourth group after L-Carnitine treatment simultaneously with cadmium chloride. The results of the present study demonstrate a damaging effect of cadmium chloride on DNA of the testis cells (with low stress response). This damaging effect increases the synthesis of HSP70 that upregulated by L-Carnitine treatment and showed ameliorative effect of the cells for recovery.

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Project description:Hsp70 is a molecular chaperone that binds to "client" proteins and protects them from protein degradation. Hsp70 is essential for the survival of many cancer cells, but it is not yet clear which of its clients are involved. Using structurally distinct chemical inhibitors, we found that many of the well-known clients of the related chaperone, Hsp90, are not strikingly responsive to Hsp70 inhibition. Rather, Hsp70 appeared to be important for the stability of the RIP1 (RIPK1) regulators: cIAP1/2 (BIRC1 and BIRC3), XIAP, and cFLIPS/L (CFLAR). These results suggest that Hsp70 limits apoptosis and necroptosis pathways downstream of RIP1. Consistent with this model, MDA-MB-231 breast cancer cells treated with Hsp70 inhibitors underwent apoptosis, while cotreatment with z-VAD.fmk switched the cell death pathway to necroptosis. In addition, cell death in response to Hsp70 inhibitors was strongly suppressed by RIP1 knockdown or inhibitors. Thus, these data indicate that Hsp70 plays a previously unrecognized and important role in suppressing RIP1 activity.Implications: These findings clarify the role of Hsp70 in prosurvival signaling and suggest IAPs as potential new biomarkers for Hsp70 inhibition. Mol Cancer Res; 16(1); 58-68. ©2017 AACR.

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