Solution NMR investigation on the structure and function of the isolated J-domain from Sis1: Evidence of transient inter-domain interactions in the full-length protein

2019 ◽  
Vol 669 ◽  
pp. 71-79 ◽  
Author(s):  
Glaucia M.S. Pinheiro ◽  
Gisele C. Amorim ◽  
Anwar Iqbal ◽  
Fabio C.L. Almeida ◽  
C.H.I. Ramos
Structure ◽  
2020 ◽  
Vol 28 (7) ◽  
pp. 830-846.e9
Author(s):  
Corey D. Seacrist ◽  
Georg Kuenze ◽  
Reece M. Hoffmann ◽  
Brandon E. Moeller ◽  
John E. Burke ◽  
...  

2020 ◽  
Author(s):  
Bryan D. Ryder ◽  
Irina Matlahov ◽  
Sofia Bali ◽  
Jaime Vaquer-Alicea ◽  
Patrick C.A. van der Wel ◽  
...  

AbstractThe Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed new light on the domain interactions of the Hsp40 DnaJB8, and how they regulate recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a new model for DnaJB8-Hsp70 regulation, whereby the JD-CTD interaction of DnaJB8 acts as a reversible autoinhibitory switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Bryan D. Ryder ◽  
Irina Matlahov ◽  
Sofia Bali ◽  
Jaime Vaquer-Alicea ◽  
Patrick C. A. van der Wel ◽  
...  

AbstractThe Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.


Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1060
Author(s):  
Alexander S. Goryashchenko ◽  
Andrey A. Mozhaev ◽  
Oxana V. Serova ◽  
Tatiana N. Erokhina ◽  
Alexander N. Orsa ◽  
...  

To study the structure and function of the pH-regulated receptor tyrosine kinase insulin receptor-related receptor (IRR), а member of the insulin receptor family, we obtained six mouse monoclonal antibodies against the recombinant IRR ectodomain. These antibodies were characterized in experiments with exogenously expressed full-length IRR by Western blotting, immunoprecipitation, and immunocytochemistry analyses. Utilizing a previously obtained set of IRR/IR chimeras with swapped small structural domains and point amino acid substitutions, we mapped the binding sites of the obtained antibodies in IRR. Five of them showed specific binding to different IRR domains in the extracellular region, while one failed to react with the full-length receptor. Unexpectedly, we found that 4D5 antibody can activate IRR at neutral pH, and 4C2 antibody can inhibit activation of IRR by alkali. Our study is the first description of the instruments of protein nature that can regulate activity of the orphan receptor IRR and confirms that alkali-induced activation is an intrinsic property of this receptor tyrosine kinase.


2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Wenping Hu ◽  
Xinlong Dong ◽  
Zhilong Tian ◽  
Zhuangbiao Zhang ◽  
Jishun Tang ◽  
...  

Abstract Background JUNO and IZUMO1 are the first receptor-ligand protein pairs discovered to be essential for sperm-oocyte fusion; their interaction is indispensable for fertilization. Methods PCR was used to clone the full-length DNA sequence of the Juno gene in sheep. The single nucleotide polymorphism (SNP) loci of Juno were genotyped by Sequenom MassARRAY®. PCR combined with rapid amplification of cDNA Ends were used to clone the full-length cDNA sequence of Juno and Izumo1. Reverse transcriptase-PCR (RT-PCR) and real time-quantitative-PCR (RT-qPCR) were used to analyze the genes’ expression in tissues of sheep, and single cell RNA-seq was used to analyze the genes’ expression in oocytes, granulosa cells and follicular theca of polytocous and monotocous Small Tail Han ewes. Bioinformatics was used to analyze advanced structure and phylogeny of JUNO and IZUMO1 proteins. Results The full-length DNA sequence of the Juno gene in sheep was cloned and nine SNPs were screened. We found a significant association between the g.848253 C > A locus of Juno and litter size of Small Tail Han sheep (P < 0.05). The full-length cDNA sequence of Juno and Izumo1 genes from Small Tail Han sheep were obtained. We found a new segment of the Izumo1 CDS consisting of 35 bp, and we confirmed the Izumo1 gene has 9 exons, not 8. RT-qPCR showed that Juno and Izumo1 genes were highly expressed in ovarian and testicular tissues, respectively (P < 0.01). Single cell RNA-seq showed Juno was specifically expressed in oocytes, but not in granulosa cells or follicular theca, while Izumo1 displayed little to no expression in all three cell types. There was no difference in expression of the Juno gene in oocyte and ovarian tissue in sheep with different litter sizes, indicating expression of Juno is not related to litter size traits. Bioinformatic analysis revealed the g.848253 C > A locus of Juno results in a nonconservative missense point mutation leading to a change from Phe to Leu at position 219 in the amino acid sequence. Conclusions For the first time, this study systematically analyzed the expression, structure and function of Juno and Izumo1 genes and their encoded proteins in Small Tail Han sheep, providing the basis for future studies of the regulatory mechanisms of Juno and Izumo1 genes.


PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17624 ◽  
Author(s):  
Debanu Das ◽  
Mireille Hervé ◽  
Julie Feuerhelm ◽  
Carol L. Farr ◽  
Hsiu-Ju Chiu ◽  
...  

2006 ◽  
Vol 26 (8) ◽  
pp. 3256-3265 ◽  
Author(s):  
Jessica J. Connelly ◽  
Peihua Yuan ◽  
Hao-Chi Hsu ◽  
Zhizhong Li ◽  
Rui-Ming Xu ◽  
...  

ABSTRACT Previous work has shown that the N terminus of the Saccharomyces cerevisiae Sir3 protein is crucial for the function of Sir3 in transcriptional silencing. Here, we show that overexpression of N-terminal fragments of Sir3 in strains lacking the full-length protein can lead to some silencing of HML and HMR. Sir3 contains a BAH (bromo-adjacent homology) domain at its N terminus. Overexpression of this domain alone can lead to silencing as long as Sir1 is overexpressed and Sir2 and Sir4 are present. Overexpression of the closely related Orc1 BAH domain can also silence in the absence of any Sir3 protein. A previously characterized hypermorphic sir3 mutation, D205N, greatly improves silencing by the Sir3 BAH domain and allows it to bind to DNA and oligonucleosomes in vitro. A previously uncharacterized region in the Sir1 N terminus is required for silencing by both the Sir3 and Orc1 BAH domains. The structure of the Sir3 BAH domain has been determined. In the crystal, the molecule multimerizes in the form of a left-handed superhelix. This superhelix may be relevant to the function of the BAH domain of Sir3 in silencing.


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