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Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin.

Corrao S, Anzalone R, Lo Iacono M, Corsello T, Di Stefano A, D'Anna SE, Balbi B, Carone M, Sala A, Corona D, Timperio AM, Zolla L, Farina F, de Macario EC, Macario AJ, Cappello F, La Rocca G - Open Biol (2014)

Bottom Line: 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 distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD).Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) 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.

View Article: PubMed Central - PubMed

Affiliation: Istituto Euro-Mediterraneo di Scienza e Tecnologia (IEMEST), Palermo, Italy Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, Università degli Studi di Palermo, Palermo, Italy.

ABSTRACT
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 distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) 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.

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2D SDS-PAGE and mass spectrometry analyses. 2D SDS-PAGE of Hsp60 (a,b) and Hsp10 (c,d) before (a,c) and after (b,d) treatment of the cells with CSE 5% for 24 h. Both Hsp10 and Hsp60 showed changes in Mr and pI after treatment. (e) Proteomic and mass spectrometry analyses confirmed the identity of the three spots in (c) as isoforms of Hsp10.
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RSOB140125F5: 2D SDS-PAGE and mass spectrometry analyses. 2D SDS-PAGE of Hsp60 (a,b) and Hsp10 (c,d) before (a,c) and after (b,d) treatment of the cells with CSE 5% for 24 h. Both Hsp10 and Hsp60 showed changes in Mr and pI after treatment. (e) Proteomic and mass spectrometry analyses confirmed the identity of the three spots in (c) as isoforms of Hsp10.

Mentions: In order to determine if qualitative variations in the Hsp10 and Hsp60 molecules did occur after CSE exposure, 2D SDS-PAGE analyses were performed on 100 µg of total protein extracts. To perform this set of experiments we used two sources of materials for protein extraction, NT cells as control and cells exposed to 5% CSE for 24 h. The choice of this CSE concentration was based on 1D western blot results: we used the highest concentration that did not determine changes in the Hsp10 and Hsp60 levels. Western blots and resulting spots were analysed using gel matching, with reference maps in our laboratory database, as explained in Material and Methods. Figure 5 shows the results of 2D-IPG western blot of the Hsp60 isoforms in the HFL-1 cell line under control condition and after 24 h of treatment with 5% of CSE. Control cells showed four Hsp60 isoforms with the same relative molecular weight (Mr = 57 500) but distinct pIs, 5.09, 5.14, 5.18 and 5.24 (figure 5a). After 5% CSE exposure, HFL-1 showed only the absence of the Hsp60 5.09 isoform (figure 5b). More differences were instead found when analysing the results of Hsp10 isoforms. Control conditions (figure 5c) showed three different isoforms of Hsp10: one, the more highly expressed and basic isoform (likely to be the typical canonical Hsp10), had an apparent Mr of 10 800 and pI of 8.7 (relative to the most expressed isoform); the other two isoforms had a Mr of 10 600, with pI 8.3, and Mr 11 000, with pI 7.5. Considerable modifications were visible following CSE treatment. On the one hand, the canonical isoform showed Mr and pI shifts (Mr = 11 500; pI = 8.9), on the other hand, the two acid forms became undetectable after treatment (figure 5d). Very similar results were obtained for 16HBE cells (data not shown). Hence, CSE exposure in bronchial cell lines induced qualitative modifications in the two chaperonins, particularly in Hsp10.Figure 5.


Hsp10 nuclear localization and changes in lung cells response to cigarette smoke suggest novel roles for this chaperonin.

Corrao S, Anzalone R, Lo Iacono M, Corsello T, Di Stefano A, D'Anna SE, Balbi B, Carone M, Sala A, Corona D, Timperio AM, Zolla L, Farina F, de Macario EC, Macario AJ, Cappello F, La Rocca G - Open Biol (2014)

2D SDS-PAGE and mass spectrometry analyses. 2D SDS-PAGE of Hsp60 (a,b) and Hsp10 (c,d) before (a,c) and after (b,d) treatment of the cells with CSE 5% for 24 h. Both Hsp10 and Hsp60 showed changes in Mr and pI after treatment. (e) Proteomic and mass spectrometry analyses confirmed the identity of the three spots in (c) as isoforms of Hsp10.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4221893&req=5

RSOB140125F5: 2D SDS-PAGE and mass spectrometry analyses. 2D SDS-PAGE of Hsp60 (a,b) and Hsp10 (c,d) before (a,c) and after (b,d) treatment of the cells with CSE 5% for 24 h. Both Hsp10 and Hsp60 showed changes in Mr and pI after treatment. (e) Proteomic and mass spectrometry analyses confirmed the identity of the three spots in (c) as isoforms of Hsp10.
Mentions: In order to determine if qualitative variations in the Hsp10 and Hsp60 molecules did occur after CSE exposure, 2D SDS-PAGE analyses were performed on 100 µg of total protein extracts. To perform this set of experiments we used two sources of materials for protein extraction, NT cells as control and cells exposed to 5% CSE for 24 h. The choice of this CSE concentration was based on 1D western blot results: we used the highest concentration that did not determine changes in the Hsp10 and Hsp60 levels. Western blots and resulting spots were analysed using gel matching, with reference maps in our laboratory database, as explained in Material and Methods. Figure 5 shows the results of 2D-IPG western blot of the Hsp60 isoforms in the HFL-1 cell line under control condition and after 24 h of treatment with 5% of CSE. Control cells showed four Hsp60 isoforms with the same relative molecular weight (Mr = 57 500) but distinct pIs, 5.09, 5.14, 5.18 and 5.24 (figure 5a). After 5% CSE exposure, HFL-1 showed only the absence of the Hsp60 5.09 isoform (figure 5b). More differences were instead found when analysing the results of Hsp10 isoforms. Control conditions (figure 5c) showed three different isoforms of Hsp10: one, the more highly expressed and basic isoform (likely to be the typical canonical Hsp10), had an apparent Mr of 10 800 and pI of 8.7 (relative to the most expressed isoform); the other two isoforms had a Mr of 10 600, with pI 8.3, and Mr 11 000, with pI 7.5. Considerable modifications were visible following CSE treatment. On the one hand, the canonical isoform showed Mr and pI shifts (Mr = 11 500; pI = 8.9), on the other hand, the two acid forms became undetectable after treatment (figure 5d). Very similar results were obtained for 16HBE cells (data not shown). Hence, CSE exposure in bronchial cell lines induced qualitative modifications in the two chaperonins, particularly in Hsp10.Figure 5.

Bottom Line: 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 distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD).Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) 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.

View Article: PubMed Central - PubMed

Affiliation: Istituto Euro-Mediterraneo di Scienza e Tecnologia (IEMEST), Palermo, Italy Dipartimento di Biomedicina Sperimentale e Neuroscienze Cliniche, Università degli Studi di Palermo, Palermo, Italy.

ABSTRACT
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 distribution have been documented in pathological conditions, e.g. cancer and chronic obstructive pulmonary disease (COPD). Here, we show that Hsp10 in COPD undergoes changes at the molecular and subcellular levels in bronchial cells from human specimens and derived cell lines, intact or subjected to stress induced by cigarette smoke extract (CSE). Noteworthy findings are: (i) Hsp10 occurred in nuclei of epithelial and lamina propria cells of bronchial mucosa from non-smokers and smokers; (ii) human bronchial epithelial (16HBE) and lung fibroblast (HFL-1) cells, in vitro, showed Hsp10 in the nucleus, before and after CSE exposure; (iii) CSE stimulation did not increase the levels of Hsp10 but did elicit qualitative changes as indicated by molecular weight and isoelectric point shifts; and (iv) 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.

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Related in: MedlinePlus