Nitric oxide (NO) is present in soil and air, and is produced by bacteria, animals and plants. Superoxide (O2-) arises in all organisms inhabiting aerobic environments. Thus, many organisms are likely to encounter peroxynitrite (OONO-), a product of NO and O2- that forms at near diffusion-limited rates, and rapidly decomposes upon protonation through isomerization to nitrate (NO3-; ref. 1) while generating hydroxyl radical (*OH) and nitrogen dioxide radical (*NO2) (refs 2, 3), both more reactive than peroxynitrite's precursors. The oxidative, inflammatory, mutagenic and cytotoxic potential (ref. 4) of peroxynitrite contrasts with the antioxidant, anti-inflammatory and tissue-protective properties ascribed to NO itself. Thus, the ability of cells to cope with peroxynitrite is central in determining the biological consequences of NO production. We considered whether cells might be equipped with enzymes to detoxify peroxynitrite. Peroxiredoxins have been identified in most genomes sequenced, but their functions are only partly understood. Here we show that the peroxiredoxin alkylhydroperoxide reductase subunit C (AhpC) from Salmonella typhimurium catalytically detoxifies peroxynitrite to nitrite fast enough to forestall the oxidation of bystander molecules such as DNA. Results are similar with peroxiredoxins from Mycobacterium tuberculosis and Helicobacter pylori. Thus, peroxynitrite reductase activity may be widespread among bacterial genera.

Peroxynitrite reductase activity of bacterial peroxiredoxins

The rapid yet transient transcriptional activation of heat shock genes is mediated by the reversible conversion of HSF1 from an inert negatively regulated monomer to a transcriptionally active DNA-binding trimer. During attenuation of the heat shock response, transcription of heat shock genes returns to basal levels and HSF1 reverts to an inert monomer. These events coincide with elevated levels of Hsp70 and other heat shock proteins (molecular chaperones). Here, we show that the molecular chaperone Hsp70 and the cochaperone Hdj1 interact directly with the transactivation domain of HSF1 and repress heat shock gene transcription. Overexpression of either chaperone represses the transcriptional activity of a transfected GAL4-HSF1 activation domain fusion protein and endogenous HSF1. As neither the activation of HSF1 DNA binding nor inducible phosphorylation of HSF1 was affected, the primary autoregulatory role of Hsp70 is to negatively regulate HSF1 transcriptional activity. These results reveal that the repression of heat shock gene transcription, which occurs during attenuation, is due to the association of Hsp70 with the HSF1 transactivation domain, thus providing a plausible explanation for the role of molecular chaperones in at least one key step in the autoregulation of the heat shock response.

/pdf/molecular-chaperones-as-hsf1-specific-transcriptional-4bfy45dnjo.pdf

Molecular chaperones as HSF1-specific transcriptional repressors.

Phosphorylation of heat shock protein 27 (HSP27) can modulate actinfilament dynamics in response to growth factors. During heat shock, HSP27 is phosphorylated at the same sites and by the same protein kinase as during mitogenic stimulation. This suggests that the same function of the protein may be activated during growth factor stimulation and the stress response. To determine the role of HSP27 phosphorylation in the heat shock response, several stable Chinese hamster cell lines that constitutively express various levels of the wild-type HSP27 (HU27 cells)oranonphosphorylatableformofhumanHSP27(HU27pm3cells)weredeveloped.IncontrasttoHU27cells, whichshowedincreasedsurvivalafterheatshock,HU27pm3cellsshowedonlyslightlyenhancedsurvival.Evidence is presented that stabilization of microfilaments is a major target of the protective function of HSP27. In the HU27pm3 cells, the microfilaments were thermosensitized compared with those in the control cells, whereas wild-typeHSP27causedanincreasedstabilityofthesestructuresinHU27cells.HU27butnotHU27pm3cellswere highly resistant to cytochalasin D treatment compared with control cells. Moreover, in cells treated with cytochalasin D, wild-type HSP27 but not the phosphorylated form of HSP27 accelerated the reappearance of actin filaments. The mutations in human HSP27 had no effect on heat shock-induced change in solubility and cellular localizationoftheprotein,indicatingthatphosphorylationwasnotinvolvedintheseprocesses.However,induction ofHSP27phosphorylationbystressingagentsormitogenscausedareductioninthemultimericsizeofthewild-type protein, an effect which was not observed with the mutant protein. We propose that early during stress, phosphorylation-induced conformational changes in the HSP27 oligomers regulate the activity of the protein at the level of microfilamentdynamics,resultinginbothenhancedstabilityandacceleratedrecoveryofthefilaments.Thelevelof protectionprovidedbyHSP27duringheatshockmaythusrepresentthecontributionofbettermaintenanceofactin filament integrity to overall cell survival.

Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27.

Induction of Chinese hamster HSP27 gene expression in mouse cells confers resistance to heat shock : HSP27 stabilization of the microfilament organization

The 25-kD inhibitor of actin polymerization (25-kD IAP), isolated from turkey smooth muscle (Miron, T., M. Wilchek, and B. Geiger, 1988. Eur. J. Biochem. 178:543-553), is shown here to be a low molecular mass heat shock protein (HSP). Direct sequence analysis of the purified protein, as well as cloning and sequencing of the respective cDNA, disclosed a high degree of homology (67% identity, 80% similarity) to the human 27-kD HSP. Southern blot of chicken genomic DNA disclosed one band, suggesting the presence of a single gene, and Northern blot analysis revealed abundant transcript of approximately 1 kb in gizzard and heart tissues and lower amounts in total 18-d chick embryo RNA and in cultured fibroblasts. Exposure of the latter cells to 45 degrees C resulted in over 15-fold increase in the apparent level of the 25-kD IAP protein, confirming that its expression is regulated by heat shock. Immunofluorescent microscopic localization indicated that after heat treatment, the levels of the 25-kD IAP were markedly increased and the protein was apparently associated with cytoplasmic granules. Heat shock also had a transient, yet prominent, effect on the microfilament system in cultured fibroblasts: stress fibers disintegrated within 10-15 min after incubation at 45 degrees C, yet upon further incubation at the elevated temperature, conspicuous actin bundles were apparently reformed.

https://rupress.org/jcb/article-pdf/114/2/255/1061578/255.pdf

A 25-kD inhibitor of actin polymerization is a low molecular mass heat shock protein.

We earlier discovered a novel 40-kDa protein (hsp40) induced by heat shock and other stresses in mammalian and avian cells. In this report, we purified the hsp40 in HeLa cells, using modified two-dimensional gel electrophoresis, and determined the amino terminal amino acid sequence of this protein. The hsp40 is homologous to DnaJ, an Escherichia coli heat-shock protein, as well as to DnaJ-homologous proteins in yeast such as SCJ1, Sec63/Np11, YDJ1 and SIS1. Indirect immunofluorescence staining using an anti-hsp40 polyclonal antibody demonstrated that hsp40 was localized faintly throughout the cell in non-heat-shocked cells and was accumulated in nuclei and nucleoli in heat-shocked cells. The intracellular localization of hsp40 was very similar to that of hsp70, suggesting that these two hsps colocalize in heat-shocked HeLa cells.

Intracellular Localization and Partial Amino Acid Sequence of a Stress-Inducible 40-kDa Protein in HeLa Cells

When murine mastocytoma cells (FMA 1) were heat shocked (42 degrees C for 4 h), nine heat shock proteins (HSPs) were detected by two-dimensional gel electrophoresis. Their apparent molecular weights were 100, 85, 69, 68, 32, 30, and 23 kDa (3 of 23 kDa). The structural homology of 4, 69, 68, 32, and 30 kDa, was demonstrated by two-dimensional tryptic peptide mapping. The 69- and 68-kDa HSPs were purified and rabbit antisera against these HSPs were prepared. A small fraction (less than 10%) of the 69- and 68-kDa HSPs were copurified with the microtubules and were present in the Triton X-100/KCl cytoskeletal fraction as shown by immunoblotting with the antiserum and by peptide mapping. Our results are consistent with the hypothesis of a cytoskeletal role for HSPs.

Possible cytoskeletal association of 69,000- and 68,000-dalton heat shock proteins and structural relations among heat shock proteins in murine mastocytoma cells.

There are many discrepancies in the molecular weight of terminal deoxynucleotidyl transferase (TdT) reported, e.g. 32- and 79-kilodalton(k) forms1–7, and also in the enzyme structure, i.e. two-sub-unit form (the α and β subunits)1–3 or single polypeptide form4–7. However, it has not been established yet whether these differences reflect species- or tissue-specificities of the enzyme, or are due simply to the proteolytic degradation during purification process. In order to clarify this issue, we have developed a new, rapid purification method, immunoadsorbent column chromatography, which is suitable to purify TdT’s from limited amounts of tissues such as rodent thymus8. Furthermore, the enzyme proteins from various species of mammals were compared by two-dimensional peptide mapping.

Purification of Terminal Deoxynucleotidyl Transferase of 60,000 Dalton from Mammalian Thymus and Thymona by Immunoadsorbent Column and Comparison of Peptide Structures

Kazushi Tanabe

Papers

Intracellular Localization and Partial Amino Acid Sequence of a Stress-Inducible 40-kDa Protein in HeLa Cells

Possible cytoskeletal association of 69,000- and 68,000-dalton heat shock proteins and structural relations among heat shock proteins in murine mastocytoma cells.

Purification of Terminal Deoxynucleotidyl Transferase of 60,000 Dalton from Mammalian Thymus and Thymona by Immunoadsorbent Column and Comparison of Peptide Structures