We have previously observed that subunits of the chaperonin required for actin production (type-II chaperonin containing T-complex polypeptide 1 [CCT]) localize at sites of microfilament assembly. In this article we extend this observation by showing that substantially substoichiometric CCT reduces the initial rate of pyrene-labeled actin polymerization in vitro where eubacterial chaperonin GroEL had no such effect. CCT subunits bound selectively to F-actin in cosedimentation assays, and CCT reduced elongation rates from both purified actin filament “seeds” and the short and stabilized, minus-end blocked filaments in erythrocyte membrane cytoskeletons. These observations suggest CCT might remain involved in biogenesis of the actin cytoskeleton, by acting at filament ( ) ends, beyond its already well-established role in producing new actin monomers.

The surprisingly efficient uptake of peptide-loaded heat shock proteins (Hsps) by antigen-presenting cells (APCs) has been recently associated with a specific receptor-ligand–based mechanism, and the identity of at least 1 receptor has been determined. In this study, we tested how the domain composition of the stress protein affected its surface association and internalization by APCs, and this was facilitated by the availability of the 70-kDa human heat shock protein (Hsp70) and its various deletion mutants. We show that both these processes strictly depend on the presence of all 3 domains of Hsp70. We propose that the previously described interdomain interactions as a determinant of a favorable conformational status might also govern a sterical adaptation of Hsps to components of the internalization machinery.

Synthesis of heat shock proteins (Hsps) in response to elevated temperatures and other denaturing agents is a common feature of prokaryotic and eukaryotic cells. The heat-induced expression of Hsp70 family members in the gills and mantle of Ostrea edulis, a highly valued fisheries resource inhabiting primarily estuarine environments, has been studied. O edulis is exposed to a variety of natural and anthropogenic stresses in the environment. Two isoforms of about 72 kDa and 77 kDa were constitutively present in unstressed organisms, reflecting the housekeeping function performed by these proteins under normal circumstances. Their expression in animals undergoing thermal stress was highly variable, and on the average, little change occurred under different experimental conditions. A third isoform of about 69 kDa was induced in both tissues after exposure to ≥32°C; its synthesis was detected within 4 hours of poststress recovery at 15°C, reaching the maximum expression after 24 hours in the gills and after 48 hours in the mantle and declining thereafter. Hsp69 expression was low at 38°C, a temperature lethal for about 50% of the individuals tested. Densitometric analysis of Western blots revealed that Hsp69 was mostly responsible for the significant heat-induced overexpression of Hsp70s in O edulis. Comparison with heat shock responses in tissues of Crassostrea gigas indicated a similar pattern of Hsp70 expression. In this organism, however, Hsp69 was induced after exposure to ≥38°C. We conclude that tissue expression of Hsp69 in O edulis, and possibly other bivalves, is an early sign of thermal stress; determining whether these changes also correlate with other major environmental stresses is the goal of ongoing studies.

Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By “Far-Western” and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95–195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (ΔSGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex.

Inflammation of the human bronchial epithelium, as observed in asthmatics, is characterized by the selective death of the columnar epithelial cells, which desquamate from the basal cells. Tissue repair initiates from basal cells that resist inflammation. Here, we have evaluated the extent of apoptosis as well as the Hsp27 level of expression in epithelial cells from bronchial biopsy samples taken from normal and asthmatic subjects. Hsp27 is a chaperone whose expression protects against oxidative stress. We report that in asthmatic subjects the basal epithelium cells express a high level of Hsp27 but no apoptotic morphology. In contrast, apoptotic columnar cells are devoid of Hsp27 expression. Moreover, we observed a decreased resistance to hydrogen peroxide–induced apoptosis in human bronchial epithelial 16–HBE cells when they were genetically modified to express reduced levels of Hsp27.

A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the protein's distribution within living cells.

To assess the ability of the heat-inducible molecular chaperone heat-shock protein 70 (Hsp70) to mitigate a specific developmental lesion, we administered the antimicrotubule drugs vinblastine (VB) and colchicine (COL) to larvae of Drosophila engineered to express differing levels of Hsp70 after heat pretreatment (HP). VB and COL decreased survival during metamorphosis, disrupted development of the adult eye and other structures as well as their precursor imaginal disks, and induced chromosome nondisjunction in the wing imaginal disk as indicated by the somatic mutation and recombination test (SMART) assay. Hsp70-inducing HP reduced many of these effects. For the traits viability, adult eye morphology, eye imaginal disk morphology, cell death in the eye imaginal disks, and single and total mutant clone formation in the SMART assay, HP reduced the impact of VB to a greater extent in Drosophila with 6 hsp70 transgenes than in a sister strain from which the transgenes had been excised. Because the extra-copy strain has higher levels of Hsp70 than does the excision strain but is otherwise almost identical in genetic background to the excision strain, these outcomes are attributable to Hsp70. The hsp70 copy number had a variable interaction with HP and COL administration.

Mortalin/mthsp70/PBP74/Grp75 (called mortalin hereafter), a member of the Hsp70 family of chaperones, was shown to have different subcellular localizations in normal and immortal cells. It has been assigned to multiple subcellular sites and implicated in multiple functions ranging from stress response, intracellular trafficking, antigen processing, control of cell proliferation, differentiation, and tumorigenesis. The present article compiles and reviews information on the multiple sites and functions of mortalin in different organisms. The relevance of its differential distributions and functions in normal and immortal cell phenotypes is discussed.