Novel in vitro and in vivo data on the cellular localization of Hsp10 in smokers affected by COPD and in lung-derived cell lines exposed to cigarette smoke extract as stressor

Cigarette smoke is a potent stressor for the respiratory system, contributing to pathogenesis, for instance in chronic obstructive pulmonary disease (COPD), but its effects on the expression, function, and cellular localization of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and characterized the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial (16HBE). We applied various in vitro methods: immunohistochemistry (IHC), subcellular fractionation analyses (SFA), Western blotting (WB), immunocytochemistry (ICC), and transmission electron microscopy (TEM) immunogold, and used bioinformatics and databases searches to gather structural in silico data for interpreting and complementing the in vitro results. IHC showed that in smokers and non-smokers COPD patients Hsp10 was localized in both, the cytoplasm and the nucleus of epithelial and lamina propria cells, while Hsp60 was present only in the cytosol. ICC, SFA, and WB on both CSE-exposed cell lines confirmed the presence of nuclear Hsp10, with an increasing trend in parallel to CSE concentration. TEM immunogold further confirmed Hsp10 in the nucleus, in addition to its presence in the cytoplasm and mitochondria, on both cell lines. Bioinformatics and in silico structural analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals in its primary sequence. Our data suggest a link between exposure to exogenous oxidative stress and cell response, involving Hsp10, which would play roles different from its canonical functions. It is known that Hsp10 can display an array of functions depending on its location: cytoplasm, mitochondria, or extracellular. Here, we show for the first time the presence of Hsp10 in the nucleus of epithelial and stromal human-lung cell lines, paralleling the observations in vivo in COPD patients, and indicating that intranuclear Hsp10 levels are affected by oxidative stress due to an exogenous stressor like cigarette-smoke. The questions now are by what mechanism Hsp10 becomes a resident of the nucleus and what are its functions there.

Nuclear localization and new isoforms detection give new insights on Hsp10 functions in normal and cigarette smoke-stressed lung cells

Heat-shock protein (Hsp)10 is the co-chaperone for Hsp60 inside mitochondria, but it also resides outside the organelle. Variations in its levels and intracellular dis- tribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS) is a potent stressor for the respiratory system, but its effects on the expression, function, and cellular locali- zation of mitochondrial chaperonins are still largely unknown. We studied in vivo (airways biopsies) the localization of Hsp10 and Hsp60 in patients (smokers and non-smokers) affected by mild-moderate COPD, and charac- terized the effects of non-lethal doses of CS extract (CSE) on the expression of these molecules in two human cell lines: lung fibroblasts (HFL-1) and bronchial epithelial cells (16HBE). We applied various in vitro methods: IHC, subcellular fractionation analyses (SFA), western blotting (WB), ICC, transmission electron microscopy (TEM) immunogold, chromati protein extracts (CPE), as well as 2D-gel based proteomics analyses. Bioinformatics was used to gather structural in silico data. IHC showed that Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers. ICC, SFA, and WB showed that 16HBE and HFL-1 cells featured nuclear Hsp10, before and after CSE exposure; TEM immunogold further confirmed this observation. Proteomics data showed that CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts. Bioinformatics analyses indicated that Hsp10 can localize in extramitochondrial sites, such as the nucleus, even if Hsp10 lacks known DNA-binding motifs or nuclear import signals. Hsp10 nuclear levels increased after CSE stimulation in HFL-1, indicating cytosol to nucleus migration, and although Hsp10 did not bind DNA, it bound a DNA-associated protein as suggested by CPE/gel retardation experiments. Data reported here indicate that in human cells of the respiratory mucosa there are at least three different intracellular locales for Hsp10: mitochondrial, nuclear, and cyto- solic. Further experiments are en route for the definition of the mechanisms underlying the transfer of Hsp10 to the nucleus and other cellular/extracellular compartments. This work was supported by grants from University of Palermo (FFR 2012) to GLR

Clinical anatomic, immunomorphologic and molecular anatomic data suggest interplay of thyroidal molecules, autoantibodies and Hsp60 in Hashimoto’s disease

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM)

Chaperonin of Group I: Oligomeric spectrum and biochemical and biological implications

Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM), is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still incompletely understood. Chaperonin I in eukaryotes occurs in various locations, not just the mitochondrion, which is its canonical place of residence and function. Eukaryotic Chaperonin I, namely Hsp60 (designated HSP60 or HSPD1 in humans) has, indeed, been found in the cytosol; the plasma-cell membrane; on the outer surface of cells; in the intercellular space; in biological liquids such as lymph, blood, and cerebrospinal fluid; and in secretions, for instance saliva and urine. Hsp60 has also been found in cell-derived vesicles such as exosomes. The functions of Hsp60 in all these non-canonical locales are still poorly characterized and one of the questions not yet answered is in what form, i.e., monomer or oligomer, is the chaperonin present in these non-canonical locations. In view of the steady increase in interest on chaperonopathies over the last several years, we have studied human HSP60 to determine its role in various diseases, its locations in cells and tissues and migrations in the body, and its post-translational modifications that might have an impact on its location and function. We also carried out experiments to characterize the oligomeric status of extramitochondrial of HSP60 in solution. Here, we provide an overview of our results, focusing on the oligomeric equilibrium and stability of the various forms of HSP60 in comparison with GroEL. We also discuss post-translational modifications associated with anti-cancer drugs to indicate the potential of Hsp60 in Medicine, as a biomarker and etiopathogenic factor