A comparison of Hsp90α and Hsp90β interactions with cochaperones and substrates

2008 ◽  
Vol 86 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Aliakbar Taherian ◽  
Patrick H. Krone ◽  
Nick Ovsenek
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Xenopus Oocytes ◽  
Stress Conditions ◽  
Differentially Expressed ◽  
Substrate Interactions ◽  
Functional Activities ◽  
Functional Differences ◽  
Hsp90 Chaperone ◽  
The Individual

Hsp90 chaperone complexes function in assembly, folding, and activation of numerous substrates. The 2 vertebrate homologues encoded by the genes hsp90a and hsp90b are differentially expressed in embryonic and adult tissues and during stress; however, it is not known whether they possess identical functional activities in chaperone complexes. This question was addressed by examining potential differences between the Hsp90 isoforms with respect to both cochaperone and substrate interactions. Epitope-tagged proteins were expressed in mammalian cells or Xenopus oocytes and subjected to immunoprecipitation with an array of cochaperones. Both isoforms were shown to participate equally in multichaperone complexes, and no significant differences in cochaperone distribution were observed. The substrates Raf-1, HSF1, Cdc37, and MEK1 interacted with both Hsp90α and Hsp90β, and the relative patterns of these interactions were not affected by heat shock. The substrate kinases c-Src, CKIIB, A-raf, and Erk interacted with both isoforms; however, significantly more Hsp90α was recovered after heat shock. The data demonstrate that Hsp90α and Hsp90β exhibit similar interactions with cochaperones, but significantly different behaviors with respect to substrate interactions under stress conditions. These results reveal both functional similarities and key functional differences in the individual members of this protein family.

scholarly journals Human N-acetylglucosaminyltransferase II substrate recognition uses a modular architecture that includes a convergent exosite

2018 ◽  
Vol 115 (18) ◽  
pp. 4637-4642 ◽  
Author(s):  
Renuka Kadirvelraj ◽  
Jeong-Yeh Yang ◽  
Justin H. Sanders ◽  
Lin Liu ◽  
Annapoorani Ramiah ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biosynthetic Pathway ◽  
Secretory Pathway ◽  
Substrate Recognition ◽  
Structural Basis ◽  
Modular Architecture ◽  
Complex Type ◽  
Substrate Interactions ◽  
Enzyme Substrate ◽  
The Individual

Asn-linked oligosaccharides are extensively modified during transit through the secretory pathway, first by trimming of the nascent glycan chains and subsequently by initiating and extending multiple oligosaccharide branches from the trimannosyl glycan core. Trimming and branching pathway steps are highly ordered and hierarchal based on the precise substrate specificities of the individual biosynthetic enzymes. A key committed step in the synthesis of complex-type glycans is catalyzed by N-acetylglucosaminyltransferase II (MGAT2), an enzyme that generates the second GlcNAcβ1,2- branch from the trimannosyl glycan core using UDP-GlcNAc as the sugar donor. We determined the structure of human MGAT2 as a Mn2+-UDP donor analog complex and as a GlcNAcMan3GlcNAc2-Asn acceptor complex to reveal the structural basis for substrate recognition and catalysis. The enzyme exhibits a GT-A Rossmann-like fold that employs conserved divalent cation-dependent substrate interactions with the UDP-GlcNAc donor. MGAT2 interactions with the extended glycan acceptor are distinct from other related glycosyltransferases. These interactions are composed of a catalytic subsite that binds the Man-α1,6- monosaccharide acceptor and a distal exosite pocket that binds the GlcNAc-β1,2Man-α1,3Manβ- substrate “recognition arm.” Recognition arm interactions are similar to the enzyme–substrate interactions for Golgi α-mannosidase II, a glycoside hydrolase that acts just before MGAT2 in the Asn-linked glycan biosynthetic pathway. These data suggest that substrate binding by MGAT2 employs both conserved and convergent catalytic subsite modules to provide substrate selectivity and catalysis. More broadly, the MGAT2 active-site architecture demonstrates how glycosyltransferases create complementary modular templates for regiospecific extension of glycan structures in mammalian cells.

scholarly journals Quantification of Hsp90 availability reveals differential coupling to the heat shock response

2018 ◽  
Vol 217 (11) ◽  
pp. 3809-3816 ◽  
Author(s):  
Brian D. Alford ◽  
Onn Brandman
Keyword(s):  
Heat Shock ◽  
Heat Shock Response ◽  
Protein Quality ◽  
Multiple Stressors ◽  
Stress Conditions ◽  
Proteotoxic Stress ◽  
Shock Response ◽  
Hsp90 Chaperone ◽  
Differential Coupling ◽  
Expression Program

The heat shock response (HSR) is a protective gene expression program that is activated by conditions that cause proteotoxic stress. While it has been suggested that the availability of free chaperones regulates the HSR, chaperone availability and the HSR have never been precisely quantified in tandem under stress conditions. Thus, how the availability of chaperones changes in stress conditions and the extent to which these changes drive the HSR are unknown. In this study, we quantified Hsp90 chaperone availability and the HSR under multiple stressors. We show that Hsp90-dependent and -independent pathways both regulate the HSR, and the contribution of each pathway varies greatly depending on the stressor. Moreover, stressors that regulate the HSR independently of Hsp90 availability do so through the Hsp70 chaperone. Thus, the HSR responds to diverse defects in protein quality by monitoring the state of multiple chaperone systems independently.

Tissue specificity of expression of heat shock gene ATHSP70-10 in Arabidopsis thaliana seedlings under normal and stress conditions

2020 ◽  
Vol 2020 (3) ◽  
pp. 37-47
Author(s):  
L.Ye. Kozeko ◽  
◽  
E.L. Kordyum ◽  
Keyword(s):  
Arabidopsis Thaliana ◽  
Heat Shock ◽  
Water Deficit ◽  
Tissue Specificity ◽  
Abiotic Factors ◽  
Root Apex ◽  
Stress Conditions ◽  
Heat Shock Gene ◽  
Organ Specificity ◽  
Pcr Analysis

Mitochondrial heat shock proteins of HSP70 family support protein homeostasis in mitochondria under normal and stress conditions. They provide folding and complex assembly of proteins encoded by mitochondrial genome, as well as import of cytosolic proteins to mitochondria, their folding and protection against aggregation. There are reports about organ-specificity of mitochondrial HSP70 synthesis in plants. However, tissue specificity of their functioning remains incompletely characterized. This problem was studied for mitochondrial AtHSP70-10 in Arabidopsis thaliana seedlings using a transgenic line with uidA signal gene under normal conditions, as well as high temperature and water deficit. Under normal conditions, histochemical GUS-staining revealed the expression of AtHSP70-10 in cotyledon and leaf hydathodes, stipules, central cylinder in root differentiation and mature zones, as well as weak staining in root apex and root-shoot junction zone. RT-PCR analysis of wild-type seedlings exposed to 37°C showed rapid upregulation of AtHSP70-10, which reached the highest level within 2 h. In addition, the gradual development of water deficit for 5 days caused an increase in transcription of this gene, which became more pronounced after 3 days and reached a maximum after 5 days of dehydration. Histochemical analysis showed complete preservation of tissue localization of AtHSP70-10 expression under both abiotic factors. The data obtained indicate the specific functioning of mitochondrial chaperone AtHSP70-10 in certain plant cellular structures.

A manganese oxide nanozyme prevents the oxidative damage of biomolecules without affecting the endogenous antioxidant system

Nanoscale ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 3855-3863 ◽  
Author(s):  
Namrata Singh ◽  
Mohammed Azharuddin Savanur ◽  
Shubhi Srivastava ◽  
Patrick D'Silva ◽  
Govindasamy Mugesh
Keyword(s):  
Oxidative Stress ◽  
Oxidative Damage ◽  
Manganese Oxide ◽  
Antioxidant System ◽  
Mammalian Cells ◽  
Redox State ◽  
Stress Conditions ◽  
System P ◽  
Endogenous Antioxidant ◽  
Antioxidant Machinery

Multi-enzyme mimetic Mn3O4 nanoflowers (Mp) modulate the redox state of mammalian cells without altering the cellular antioxidant machinery under oxidative stress conditions.

scholarly journals Changes in Membrane Fluid State and Heat Shock Response Cause Attenuation of Virulence

2010 ◽  
Vol 192 (7) ◽  
pp. 1999-2005 ◽  
Author(s):  
Amalia Porta ◽  
Annamaria Eletto ◽  
Zsolt Török ◽  
Silvia Franceschelli ◽  
Attila Glatz ◽  
...  
Keyword(s):  
Heat Shock ◽  
Genetic Modification ◽  
Heat Shock Response ◽  
Mammalian Cells ◽  
Histoplasma Capsulatum ◽  
Genetic Manipulation ◽  
Microbial Pathogens ◽  
Fluid State ◽  
Shock Response ◽  
Serovar Typhimurium

ABSTRACT So far attenuation of pathogens has been mainly obtained by chemical or heat treatment of microbial pathogens. Recently, live attenuated strains have been produced by genetic modification. We have previously demonstrated that in several prokaryotes as well as in yeasts and mammalian cells the heat shock response is controlled by the membrane physical state (MPS). We have also shown that in Salmonella enterica serovar Typhimurium LT2 (Salmonella Typhimurium) overexpression of a Δ12-desaturase gene alters the MPS, inducing a sharp impairment of transcription of major heat shock genes and failure of the pathogen to grow inside macrophage (MΦ) (A. Porta et al., J. Bacteriol. 192:1988-1998, 2010). Here, we show that overexpression of a homologous Δ9-desaturase sequence in the highly virulent G217B strain of the human fungal pathogen Histoplasma capsulatum causes loss of its ability to survive and persist within murine MΦ along with the impairment of the heat shock response. When the attenuated strain of H. capsulatum was injected in a mouse model of infection, it did not cause disease. Further, treated mice were protected when challenged with the virulent fungal parental strain. Attenuation of virulence in MΦ of two evolutionarily distant pathogens was obtained by genetic modification of the MPS, suggesting that this is a new method that may be used to produce attenuation or loss of virulence in both other intracellular prokaryotic and eukaryotic pathogens. This new procedure to generate attenuated forms of pathogens may be used eventually to produce a novel class of vaccines based on the genetic manipulation of a pathogen's membrane fluid state and stress response.

The CRZ-1 Transcription Factor Regulates Hsp 80 Involves in the Acquisition of Thermotolerance, and NCA-2 for Calcium Stress Tolerance in Neurospora Crassa

2021 ◽  
Author(s):  
Avishek Roy ◽  
Ranjan Tamuli
Keyword(s):  
Transcription Factor ◽  
Heat Shock ◽  
Neurospora Crassa ◽  
Stress Condition ◽  
Stress Conditions ◽  
Start Codon ◽  
Expression Levels ◽  
Heat Shock Stress ◽  
Shock Proteins ◽  
Shock Stress

Abstract Heat shock proteins (Hsps) are molecular chaperones and required for survival of organisms under heat stress conditions. In this study, we studied Hsp80, a member of the Hsp90 family, in Neurospora crassa. The expression of hsp80 was severely reduced in the N. crassa calcineurin B subunit RIP-mutant (cnb-1RIP) strains under the heat shock conditions. Furthermore, the expression levels of cnb-1, hsp60, hsp80, and the calcineurin-regulated transcription factor crz-1 were increased, but expression levels were reduced in the presence of the calcineurin inhibitor FK506 under the heat shock stress in the N. crassa wild type. Therefore, the calcineurin-crz-1 signaling pathway transcriptionally regulates hsp60 and hsp80 under the heat shock stress condition in N. crassa. In addition, the transcript levels of trm-9 and nca-2, a Ca2+ sensor and a Ca2+ ATPase, respectively, were increased under the heat shock stress condition. Moreover, the expression of the hsp80, but not the hsp60, was reduced in the Δtrm-9, Δnca-2, and the Δtrm-9 Δnca-2 double mutants. These results suggested that hsp80, trm-9, and nca-2 play a role in coping the heat shock stress in N. crassa. We found that CRZ-1 binds to 5ʹ-CCTTCACA-3ʹ and 5ʹ-AGCGGAGC-3ʹ 8 bp nucleotide sequences, located about 1075 bp and 679 bp upstream of the ATG start codon, respectively, of hsp80. We also found that CRZ-1 binds to an 8 bp nucleotide sequence 5ʹ-ACCGCGCC-3ʹ, located 234 bp upstream of the ATG start codon of nca-2 under Ca2+ stress condition. Thus, cnb-1, hsp60, hsp80, and crz-1 are involved in the heat shock stress response in N. crassa. Moreover, CRZ-1 upregulates the expressions of hsp80 and nca-2 under the heat shock stress and Ca2+ stress conditions, respectively, in N. crassa.

Topological requirements for homologous recombination among DNA molecules transfected into mammalian cells

1985 ◽  
Vol 5 (8) ◽  
pp. 2080-2089
Author(s):  
C T Wake ◽  
F Vernaleone ◽  
J H Wilson
Keyword(s):  
Homologous Recombination ◽  
Mammalian Cells ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Animal Cells ◽  
Linear Molecules ◽  
Sv40 Genome ◽  
Foreign Dna ◽  
The Individual ◽  
Sv40 Dna

Cultured animal cells rearrange foreign DNA very efficiently by homologous recombination. The individual steps that constitute the mechanism(s) of homologous recombination in transfected DNA are as yet undefined. In this study, we examined the topological requirements by using the genome of simian virus 40 (SV40) as a probe. By assaying homologous recombination between defective SV40 genomes after transfection into CV1 monkey cells, we showed that linear molecules are preferred substrates for homologous exchanges, exchanges are distributed around the SV40 genome, and the frequency of exchange is not diminished significantly by the presence of short stretches of non-SV40 DNA at the ends. These observations are considered in relation to current models of homologous recombination in mammalian cells, and a new model is proposed. The function of somatic cell recombination is discussed.

scholarly journals DjA1 maintains Golgi integrity via interaction with GRASP65

2019 ◽  
Vol 30 (4) ◽  
pp. 478-490 ◽  
Author(s):  
Jie Li ◽  
Danming Tang ◽  
Stephen C. Ireland ◽  
Yanzhuang Wang
Keyword(s):  
Heat Shock ◽  
Membrane Fusion ◽  
Structure Formation ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Golgi Membrane ◽  
Biochemical Methods ◽  
Golgi Fragmentation ◽  
Golgi Structure

In mammalian cells, the Golgi reassembly stacking protein of 65 kDa (GRASP65) has been implicated in both Golgi stacking and ribbon linking by forming trans-oligomers. To better understand its function and regulation, we used biochemical methods to identify the DnaJ homolog subfamily A member 1 (DjA1) as a novel GRASP65-binding protein. In cells, depletion of DjA1 resulted in Golgi fragmentation, short and improperly aligned cisternae, and delayed Golgi reassembly after nocodazole washout. In vitro, immunodepletion of DjA1 from interphase cytosol reduced its activity to enhance GRASP65 oligomerization and Golgi membrane fusion, while adding purified DjA1 enhanced GRASP65 oligomerization. DjA1 is a cochaperone of Heat shock cognate 71-kDa protein (Hsc70), but the activity of DjA1 in Golgi structure formation is independent of its cochaperone activity or Hsc70, rather, through DjA1-GRASP65 interaction to promote GRASP65 oligomerization. Thus, DjA1 interacts with GRASP65 to enhance Golgi structure formation through the promotion of GRASP65 trans-oligomerization.

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