Roles moleculaires des proteines de choc thermique dans le fonctionnement des organismes a des temperatures normales et suite a des chocs thermiques; differents genes impliques

The heat-shock proteins

In the cell, as in vitro, the final conformation of a protein is determined by its amino-acid sequence. But whereas some isolated proteins can be denatured and refolded in vitro in the absence of other macromolecular cellular components, folding and assembly of polypeptides in vivo involves other proteins, many of which belong to families that have been highly conserved during evolution.

Protein folding in the cell.

HSP70; CHARACTERISTICS AND FUNCTION S 582 HSP70 Diversity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 582 Cytop lasmic HSP70 Genes and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . .. 583 Cytoplasmic HSP70 Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 586 HSP70 in the Endoplasmic Reticulum . . . . .. . . ..... . . .... . . . . . . . . ... . 588 Mitochondrial and Chloroplast HSP70s . . . .. . .. . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588 H SP60 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590 Mitochondrial HSP60 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590 Chloroplast Chaperonin 60 . . . . . . . .... . . . . . .. . . . ....... ........ . ......... . ....... . . . . .. . . . ...... 591 LMW HSPs . . . . . . . . " . . ... .... ,... ....... . . . ........ . ...... . ...... 593 The LMW HSP Gene Supeifamily . . . . . . . . .. . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . 593 LMW HSP Structure . . . . . . . . . . . . . . . . . . . . . . . . . , .... , , 597 P lastid LMW HSPs . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . , ..... .... , 599 Endomembrane LMW HSPs . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 601 LMW HSP Function . .. . . . .. . . . . . . .... . . . . . .... . . . .. . . . . . . . . . . . . . . . . . . . ... . . . . . . . .... . . . . . . . . . . . . 602 OTHER H SPs . . 604 HSP90 and HSPlJO. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 604 Ubiquitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ... ,......... 605 Additional Heat-Regulated Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 606 RELEV ANCE OF HSPS TO PLANT G ROWTH AND DEVELOPMENT 607 Expression of HSPs in the Natural Environment . . .. . . . . . . . ..... . . . . . .. . . . . . . . . . . ....... .. . 607 HSP Expression During Development 608 Role of HSPs in Thermotolerance . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . .. . . . . . . . . . 609 SUMMARY AND FUTURE PROSPECTS....... ....... ..... . .... .. . .. ....... ..... 611

The Roles of Heat Shock Proteins in Plants

The cellular-stress response can mediate cellular protection through expression of heat-shock protein (Hsp) 70, which can interfere with the process of apoptotic cell death. Stress-induced apoptosis proceeds through a defined biochemical process that involves cytochrome c, Apaf-1 and caspase proteases. Here we show, using a cell-free system, that Hsp70 prevents cytochrome c/dATP-mediated caspase activation, but allows the formation of Apaf-1 oligomers. Hsp70 binds to Apaf-1 but not to procaspase-9, and prevents recruitment of caspases to the apoptosome complex. Hsp70 therefore suppresses apoptosis by directly associating with Apaf-1 and blocking the assembly of a functional apoptosome.

Heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the Apaf-1 apoptosome.

Heat shock proteins.

Expression of the major heat shock and stress-induced protein, HSP70, is under complex regulatory control in human cells. In addition to being induced by physiological stress such as heat shock or transition metals, the HSP70 gene is induced by serum stimulation and immortalizing products of the adenovirus E1A 13S and polyoma large tumor antigen genes. Here we show that expression of the human HSP70 gene is tightly regulated during the cell cycle. Using selective mitotic detachment, a noninductive method to obtain synchronous populations of HeLa cells, we show that levels of HSP70 mRNA rapidly increase 10- to 15-fold upon entry into S phase and decline by late S and G2. A transient increase in HSP70 synthesis is detected during early S phase. The subcellular localization of HSP70 varies throughout the cell cycle; the protein is diffusely distributed in the nucleus and cytoplasm in G1, localized in the nucleus in S, and again diffusely distributed in G2 cells. We suggest that the temporal pattern of HSP70 expression during S phase, the nuclear localization, and activation by trans-acting immortalizing proteins indicate a role for HSP70 in the nucleus of replicating cells.

Expression of human HSP70 during the synthetic phase of the cell cycle

The human HSP70 gene was modified in vitro using oligonucleotide-directed mutagenesis to add sequences encoding a peptide from the testis-specific form of human lactate dehydrogenase (LDH) to the carboxy terminus of HSP70. The peptide-tagged HSP70 can be distinguished from the endogenous HSP70 protein using an LDH peptide-specific antiserum in indirect immunofluorescence assays of cells transiently transfected with an expression vector containing the tagged HSP70 gene regulated by the human HSP70 promoter. A series of deletion mutants within the HSP70 protein coding region were generated. Using double-label indirect immunofluorescence with the LDH peptide-specific antiserum and HSP70-specific mAbs, we compared the intracellular distribution of the deletion mutants to that of endogenous HSP70. We have determined that sequences in the carboxy terminus of HSP70 are necessary for proper nucleolar localization after heat shock. In contrast, sequences in the amino terminus of HSP70 are responsible for the ATP-binding ability of the protein. Mutants that were unable to bind ATP, however, still displayed nucleolar association, indicating that ATP binding is apparently not required for interaction with substrate. Additional support that HSP70 appears to be composed of at least two domains follows from the results of trypsin digestions of wild type and mutant HSP70. Protease digestion of the mutant HSP70 proteins identified a region of HSP70 that, when deleted, affected HSP70 conformation.

https://rupress.org/jcb/article-pdf/109/5/1947/1058683/1947.pdf

Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding.

In asynchronous populations of HeLa cells maintained at control or heat shock temperatures, HSP70 levels and its subcellular distribution exhibit substantial heterogeneity as demonstrated by indirect immunofluorescence with HSP70-specific monoclonal antibodies. Of particular interest is a subpopulation of cells in which the characteristic nuclear accumulation and nucleolar association of HSP70 is not detected after heat shock treatment. This apparent variation in the heat shock response is not observed when synchronized cells are examined. In this study, we demonstrate that three monoclonal antibodies to HSP70, in particular, do not detect nucleolar-localized HSP70 in heat-shocked G2 cells. This is not due to an inability of G2 cells to respond to heat shock as measured by increased HSP70 mRNA and protein synthesis, or due to a lack of accumulation of HSP70 after heat shock in G2. Rather the epitopes recognized by the various antibodies appear to be inaccessible, perhaps due to the association of HSP70 with other proteins. Non-denaturing immunoprecipitations with these HSP70-specific antibodies suggest that HSP70 may interact with other cellular proteins in a cell cycle-dependent manner.

/pdf/cell-cycle-dependent-association-of-hsp70-with-specific-3xkuvzcsof.pdf

Cell cycle-dependent association of HSP70 with specific cellular proteins.

I. VERTEBRATE HEAT SHOCK GENES The eukaryotic genome encodes a large multigene family of hsp70-related proteins. In human cells, there are at least five distinct protein members of the hsp70 gene family (Fig. 1) that exhibit constitutive and inducible regulation and share biochemical and antigenic properties. The genes that encode many of these hsp70-related proteins have been cloned, and comparison of the corresponding nucleotide sequences has revealed a high degree of evolutionary conservation among members of the hsp70 family within a single species and between species (Moran et al. 1982; Lowe et al. 1983; Hunt and Morimoto 1985; Mues et al. 1986). For example, Escherichia coli DnaK and human hsp70 are 50% identical at the amino acid level (Bardwell and Craig 1984; Hunt and Morimoto 1985) whereas grp78, p72, and hsp70 are approximately 75–85% related. Under normal conditions of cell growth, different members of the hsp70 family are localized to particular cellular compartments and in response to physiological stress are redistributed to different intracellular locales. This feature is best represented by hsp70 and p72 which are typically found in the cytoplasm of cells in the G 1 phase of the cell cycle and, following heat shock or other forms of stress, redistribute from the cytoplasm to the nucleus and nucleolus. Although the function of the hsp70-related proteins has not been firmly established, recent biochemical evidence suggests multiple roles for the hsp70-related proteins in the formation, maintenance, and disaggregation of complex multimeric structures and in protein folding (Chirico et al. 1988;...

Kim L. Milarski

Papers

Expression of human HSP70 during the synthetic phase of the cell cycle

Mutational analysis of the human HSP70 protein: distinct domains for nucleolar localization and adenosine triphosphate binding.

Cell cycle-dependent association of HSP70 with specific cellular proteins.

14 Expression and Function of Vertebrate hsp70 Genes