scholarly journals The Cotranslational Maturation of the Type I Membrane Glycoprotein Tyrosinase: The Heat Shock Protein 70 System Hands Off to the Lectin-based Chaperone System

2005 ◽  
Vol 16 (8) ◽  
pp. 3740-3752 ◽  
Author(s):  
Ning Wang ◽  
Robert Daniels ◽  
Daniel N. Hebert
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Disulfide Bonds ◽  
Stop Codon ◽  
Signal Sequence ◽  
Membrane Glycoprotein ◽  
Type I ◽  
Nascent Chain ◽  
Chaperone Binding

The maturation of eukaryotic secretory cargo initiates cotranslationally and cotranslocationally as the polypeptide chain emerges into the endoplasmic reticulum lumen. Here, we characterized the cotranslational maturation pathway for the human type I membrane glycoprotein tyrosinase. To recapitulate the cotranslational events, including glycosylation, signal sequence cleavage, chaperone binding, and oxidation, abbreviated transcripts lacking a stop codon were in vitro translated in the presence of semipermeabilized melanocyte membranes. This created a series of ribosome/translocon-arrested chains of increasing lengths, simulating intermediates in the cotranslational folding process. Initially, nascent chains were found to associate with the heat shock protein (Hsp) 70 family member BiP. As the nascent chains elongated and additional glycans were transferred, BiP binding rapidly decreased and the lectin-based chaperone system was recruited in its place. The lectin chaperone calnexin bound to the nascent chain after the addition of two glycans, and calreticulin association followed upon the addition of a third. The glycan-specific oxidoreductase ERp57 was cross-linked to tyrosinase when calnexin and calreticulin were associated. This timing coincided with the formation of disulfide bonds within tyrosinase and the cleavage of its signal sequence. Therefore, tyrosinase maturation initiates cotranslationally with the Hsp70 system and is handed off to the lectin chaperone system that first uses calnexin before calreticulin. Interestingly, divergence in the maturation pathways of wild-type and mutant albino tyrosinase can already be observed for translocon-arrested nascent chains.

scholarly journals Two distinct classes of co-chaperones compete for the EEVD motif in heat shock protein 70 (Hsp70) to tune its activity

2021 ◽  
Author(s):  
Oleta T Johnson ◽  
Cory M Nadel ◽  
Emma C Carroll ◽  
Taylor Arhar ◽  
Jason E Gestwicki
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Heat Shock Protein 70 ◽  
Protein Protein Interactions ◽  
Hsp70 Family ◽  
C Terminus ◽  
Class B ◽  
Chaperone Binding

Chaperones of the heat shock protein 70 (Hsp70) family engage in protein-protein interactions (PPIs) with many co-chaperones. One hotspot for co-chaperone binding is the EEVD motif that is found at the extreme C-terminus of cytoplasmic Hsp70s. This motif is known to bind tetratricopeptide repeat (TPR) domain co-chaperones, such as the E3 ubiquitin ligase CHIP, and Class B J-domain proteins (JDPs), such as DnaJB4. Although complexes between Hsp70-CHIP and Hsp70-DnaJB4 are both important for chaperone functions, the molecular determinants that dictate the competition between these co-chaperones are not clear. Using a collection of EEVD-derived peptides, we find that DnaJB4 binds to the IEEVD motif of Hsp70s, but not the related MEEVD motif of cytoplasmic Hsp90s. Then, we explored which residues are critical for binding to CHIP and DnaJB4, revealing that they rely on some shared features of the IEEVD motif, such as the C-terminal carboxylate. However, they also had unique preferences, especially at the isoleucine position. Finally, we observed a functionally important role for competition between CHIP and DnaJB4 in vitro, as DnaJB4 can limit the ubiquitination activity of the Hsp70-CHIP complex, while CHIP suppresses the chaperone activities of Hsp70-DnaJB4. Together, these results suggest that the EEVD motif has evolved to support diverse PPIs, such that competition between co-chaperones could help guide whether Hsp70-bound proteins are folded or degraded.

scholarly journals Structure-based mutagenesis studies of the peptide substrate binding fragment of type I heat-shock protein 40

2005 ◽  
Vol 386 (3) ◽  
pp. 453-460 ◽  
Author(s):  
Jingzhi LI ◽  
Bingdong SHA
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Molecular Chaperone ◽  
Critical Role ◽  
In Vitro Assays ◽  
Major Type ◽  
Type I ◽  
Peptide Substrate ◽  
Hydrophobic Pocket

Ydj1 is the major type I Hsp40 (heat-shock protein 40) family member in yeast. Ydj1 can pair with yeast Hsp70 Ssa1 to facilitate protein translocation and protein folding. Ydj1 itself can also function as a molecular chaperone to bind the non-native polypeptides and suppress protein aggregations in vitro. The crystal structure of Ydj1 complexed with its peptide substrate GWLYEIS reveals that a hydrophobic pocket located on Ydj1 domain I may play a major role in mediating the interactions between Ydj1 and the peptide substrate. To understand the mechanism by which Ydj1 interacts with non-native polypeptide, we have mutated the residues forming the hydrophobic pocket, based on the structural information. We have also constructed deletion mutations of the zinc-finger motifs within Ydj1. We have examined the functional consequences of these Ydj1 mutants by in vivo and in vitro assays. The results indicated that the hydrophobic pocket located on Ydj1 plays a critical role in its molecular chaperone activity by mediating interactions with the non-native polypeptides.

scholarly journals Heat Shock Protein 70 Improves In Vitro Embryo Yield and Quality from Heat Stressed Bovine Oocytes

Animals ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 1794
Author(s):  
Konstantina Stamperna ◽  
Themistoklis Giannoulis ◽  
Eleni Dovolou ◽  
Maria Kalemkeridou ◽  
Ioannis Nanas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Cumulus Cells ◽  
Intramolecular Interactions ◽  
Developmental Competence ◽  
Embryo Yield ◽  
Bovine Oocytes

Heat shock protein 70 (HSP70) is a chaperon that stabilizes unfolded or partially folded proteins, preventing inappropriate inter- and intramolecular interactions. Here, we examined the developmental competence of in vitro matured oocytes exposed to heat stress with or without HSP70. Bovine oocytes were matured for 24 h at 39 °C without (group C39) or with HSP70 (group H39) and at 41 °C for the first 6 h, followed by 16 h at 39 °C with (group H41) or without HSP70 (group C41). After insemination, zygotes were cultured for 9 days at 39 °C. Cleavage and embryo yield were assessed 48 h post insemination and on days 7, 8, 9, respectively. Gene expression was assessed by RT-PCR in oocytes, cumulus cells and blastocysts. In C41, blastocysts formation rate was lower than in C39 and on day 9 it was lower than in H41. In oocytes, HSP70 enhanced the expression of three HSP genes regardless of incubation temperature. HSP70 at 39 °C led to tight coordination of gene expression in oocytes and blastocysts, but not in cumulus cells. Our results imply that HSP70, by preventing apoptosis, supporting signal transduction, and increasing antioxidant protection of the embryo, protects heat stressed maturing bovine oocyte and restores its developmental competence.

scholarly journals Stimulation of the Human Heat Shock Protein 70 Promoter in Vitro by Simian Virus 40 Large T Antigen

1989 ◽  
Vol 264 (27) ◽  
pp. 16160-16164
Author(s):  
I C Taylor ◽  
W Solomon ◽  
B M Weiner ◽  
E Paucha ◽  
M Bradley ◽  
...  
Keyword(s):  
Heat Shock ◽  
Heat Shock Protein ◽  
Heat Shock Protein 70 ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Large T Antigen ◽  
Human Heat Shock Protein ◽  
Stimulation Of
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