Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation (original) (raw)
Related papers
Journal of Biosciences, 1996
The α-crystallin-related stress protein HSP27, which promotes cellular resistance to different types of stress, is constitutively expressed during the growth of several primate tissue culture cells. Here, we report an analysis of the cellular localization of this protein in CV-1 monkey cells. Following cell lysis and fractionation in the absence of detergent about 2 5 % of the cellular content of HSP27 was recovered in the particulate fractions while the remaining of this protein was in the soluble cytoplasmic fraction. This association of HSP27 with particulate fractions was no more observed when cells were lysed in the presence of non-ionic detergent or when cells were pretreated with drugs, such as monensin and colcemid, that disrupt cytoskeletal architecture. Immunofluorescence analysis revealed that HSP27 is concentrated in a polarized perinuclear zone of CV-1 cells from where microtubules radiate. The particular locale of HSP27 was investigated in cells exposed to drugs or treatments, such as monensin, colcemid, cold stess and serum starvation, that disrupt the cellular architecture of microtubules. A correlation was observed between HSP27 cellular locale and microtubules integrity. Our results suggest a possible interaction of a fraction of HSP27 with cytoplasmic organelles or structures, different from the Golgi apparatus, whose distribution depends upon the organization of microtubules.
Heat shock resistance conferred by expression of the human HSP27 gene in rodent cells
Journal of Cell Biology, 1989
Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenou...
The Journal of Cell Biology
Heat shock induces in cells the synthesis of specific proteins called heat shock proteins (HSPs) and a transient state of thermotolerance. The putative role of one of the HSPs, HSP27, as a protective molecule during thermal stress has been directly assessed by measuring the resistance to hyperthermia of Chinese hamster and mouse cells transfected with the human HSP27 gene contained in plasmid pHS2711. One- and two-dimensional gel electrophoresis of [3H]leucine- and [32P]orthophosphate-labeled proteins, coupled with immunological analysis using Ha27Ab and Hu27Ab, two rabbit antisera that specifically recognize the hamster and the human HSP27 protein respectively, were used to monitor expression and inducibility of the transfected and endogenous proteins. The human HSP27 gene cloned in pHS2711 is constitutively expressed in rodent cells, resulting in accumulation of the human HSP27 and all phosphorylated derivatives. No modification of the basal or heat-induced expression of endogenou...
Journal of Pharmaceutical Research International
HSP27, also known as HSPB1, was first discovered with a molecular weight 27kDa belonging to the four member gene family. Elevated levels of HSP27 are seen when different unfavorable conditions prevail such as increase in temperature and oxidative stress or exposure to heavy metals or organic solvents. They possess ATP-independent chaperone like activity which helps in maintaining protein homeostasis. It can also form large oligomers (300-600 kDa) containing different numbers of subunits. It is composed of total 205 amino acids. HSP27 undergoes post-translational modifications i.e. phosphorylation thereby converting large oligomers into dimers. It can act as an anti-apoptotic and antioxidant molecule during oxidative stress. The elevated form of HSP27 is also seen in some cancer belongs to breast, ovary, prostate, brain, colorectal, hepatocellular carcinoma, lung, liver, and cervical regions. Keeping in view of molecular roles of HSP27 signaling in various pathways, we have proposed...
Journal of cell …, 1993
We have previously reported that a novel 40 kDa protein is induced by heat shock and several environmental stresses in mammalian and avian cells and that the N-terminal amino acid sequence of this 40 kDa protein has homology with the bacterial DnaJ heat-shock protein. We have purified this protein (40 kDa heat-shock protein, hsp40) from HeLa cells by modified two-dimensional gel electrophoresis and generated a polyclonal antibody against hsp40. This antibody was highly specific for human hsp40 and cross-reacted weakly with rat and Chinese hamster hsp40. Indirect immunofluorescence revealed that the hsp40 in HeLa cells accumulates in the nucleus, especially in the nucleolus, during heat shock and returns to the cytoplasm during the recovery period. The kinetics of the accumulation in the nucleoli and subsequent return to the cytoplasm of hsp40 was similar to that of hsp70. In addition, hsp40 was co-localized with hsc70(p73) in heat-shocked HeLa cells as demonstrated by double immunofluorescence staining. These results suggest that hsp40 (a DnaJ homologue) and hsp70 (a DnaK homologue) may act in concert to repair (refold) denatured proteins and protein aggregates in the nuclei and nucleoli of heat-shocked HeLa cells.
Cell Proliferation, 1997
We have used human mammary cells of the MCF-7 strain, which constitutively express high levels of the small heat shock protein HSP27 and we have compared the changes in the phosphorylation status of this protein together with changes in cell growth and/or morphology induced by the action of one of the following agents: (1) TPA (12-O-tetradecanoylphorbol-13-acetate), known as a differentiation inducer in MCF-7 cells; (2) OH-TAM (hydroxytamoxifen), which exerts a cytostatic and cytotoxic action; or (3) TNFa (tumour necrosis factor), which induces apoptotic cell death in this cell line. Our data show that TPA and TNF stimulate an immediate and massive phosphorylation of HSP27, whereas OH-TAM affect the phosphorylation status of the protein only after a 3 day delay. In the case of TPA, high levels of HSP27 phosphorylation were maintained for at least 4 days, along with growth inhibition and acquisition by the cells of a secretory phenotype. TPA and OH-TAM exerted similar immediated effects on cell growth, despite the different time course of their action on HSP27 phosphorylation. This excludes the possibility that the latter is a necessary consequence of, or an absolute requisite to, growth inhibition. With OH-TAM and TNF the increase in HSP27 phosphorylation was concomitant with the appearance of apoptosis, not observed with TPA. This indicates that increased phosphorylation of HSP27 is not specifically associated with the triggering or the execution of apoptosis in these cells. Altogether, our data support the concept that phosphorylated HSP27 is involved (and might then be rate limiting in some instances) in the execution of vital cell programmes (including resistance to stress, proliferation and differentiation), as well as in that of cell death. This is consistent with its role in actin polymerization and its position downstream of the p38iRK-type MAPkinase, itself a point of convergence for diverse signal transduction pathways.
Decreased stress inducibility of the HSP68 protein in a rat hepatoma variant clone
European Journal of Biochemistry, 1992
Analysis of the stress response of closely related rat hepatoma clones revealed that the major inducible heat-shock protein 68 (HSP68) was only slightly inducible upon stress in the glucocorticoidresistant, dedifferentiated clone-2 cells, but strongly activated in the differentiated, glucocorticoidsensitive Faza 967 cells from which clone 2 was derived. The decreased inducibility of HSP68 in clone-2 cells was not the consequence of altered kinetics of protein synthesis recovery, was not correlated with the deficient inducibility of other major heat-shock proteins and had no effect on the heat sensitivity of the cells. This deficiency was observed after treatment with mild and strong heat and various chemicals. The results of nuclear run-on experiments suggested that the impairment of HSP68 mRNA induction most likely occurs at the transcriptional level and is probably specific for the corresponding gene. In Faza 967 and clone-2 cells, stress activated comparable levels of heat-shockfactor binding to the heat-shock element, and the expression of a reporter gene under the control of murine HSP70.1 promoter was strongly stimulated in both cells. Therefore, our results raise the possibility that the deficient stress inducibility of HSP68 is due to some specific regulation of the endogeneous HSP68 gene, rather than to a deficiency of the heat-shock factor or mutation of the corresponding gene, In response to heat shock and exposure to various chemicals, the transcription of a small set of genes, the heat-shock genes, is specifically activated in most cells. This activation results in the vigorous synthesis of several heat-shock proteins (HSP) (for a review, see [l]). The major HSP display essential functions in unstressed cells, related to protein folding and unfolding, such as chaperoning unfolded protein precursors, assisting translocation across membranes, multiprotein assembly and protein degradation. During heat shock and other stresses, several proteins are denatured and HSPs are thought to prevent protein aggregation, to help recovery from denaturation and to assist in the degradation of denatured proteins (for reviews see [l-31). The synthesis of HSP is regulated at both the transcriptional and post-transcriptional levels (for reviews see [4, 51). Repeats of the GAA-TTC-DNA motif constitute the heatshock elements (HSE) of eukaryotic heat-shock genes and confer the stress inducibility of transcription [l, 61. Stress promotes the HSE binding of the heat-shock transcription factor(s) (HSF) [7]. Although the HSF plays an essential role in transcriptional regulation of the stress genes, the binding of HSF to the HSE does not appear to be sufficient for transcriptional activation [8, 91.