TY - CONF
T1 - Human HSP10 variants downregulation after cigarette smoke extract exposure in lung cells
AU - Zummo, Giovanni
AU - Lo Iacono, Melania
AU - Farina, Felicia
AU - La Rocca, Giampiero
AU - Campanella, Claudia
AU - Cappello, Francesco
AU - Anzalone, Rita
AU - Corrao, Simona
AU - Loria, Tiziana
PY - 2009
Y1 - 2009
N2 - The impact of cigarette-smoke stress (a form of oxidative stress) on human lung fibroblasts and epithelial cells, particularly its effect on Hsp10 expression, has not been characterized despite the fact that a role for mitochondrial chaperonins in the development of lung diseases, ranging from chronic obstructive pulmonary disease to bronchial carcinogenesis, has been suggested (1). We studied the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of Hsp10 in human lung fibroblasts (HFL-1 line) and epithelial cells (16HBE line). Proteomics was carried out using 2D-IPG, silver stain, western blotting, and mass-spectrometry; mRNA was measured by RT-PCR. Database searches and chaperonomics were used to identify the proteins and genes of interest. Hsp10 levels changed significantly following CSE cell exposure as compared with unstressed cells. In unstressed cells, three isoelectric variants of Hsp10 were found, which have not yet been reported for other systems. After CSE exposure, only the most basic variant was still expressed; the other two were no longer detectable. In order to investigate the nature of the three variants found in unstressed cells, e.g., posttranslational modifications of a single product from the canonical human hsp10 gene, or different products from different genes with variations in primary structure, we performed mass spectrometric analysis. Spots were excised from matched Coomassie-stained gels, trypsin digested, and analyzed by nano-RP-HPLC-ESI-MS/MS to determine the fragments’ amino-acid sequences. The sequencing results and database searches showed that the most basic variant was human Hsp10. Partial sequencing of the other two variants indicated they are also human Hsp10. While these determinations continue to complete sequencing coverage, the data thus far indicate that: a) the three variants have the same amino acid sequence; b) they are, most likely, the product of the same hsp10 canonical gene; and c) post-translational modifications are responsible for the observed protein variants. Current experiments are underway to determine the biochemical nature of such modifications, and the possible regulatory pathways driven by CSE that lead to changes in Hsp10 variant levels.
AB - The impact of cigarette-smoke stress (a form of oxidative stress) on human lung fibroblasts and epithelial cells, particularly its effect on Hsp10 expression, has not been characterized despite the fact that a role for mitochondrial chaperonins in the development of lung diseases, ranging from chronic obstructive pulmonary disease to bronchial carcinogenesis, has been suggested (1). We studied the effects of non-lethal doses of cigarette smoke extract (CSE) on the expression of Hsp10 in human lung fibroblasts (HFL-1 line) and epithelial cells (16HBE line). Proteomics was carried out using 2D-IPG, silver stain, western blotting, and mass-spectrometry; mRNA was measured by RT-PCR. Database searches and chaperonomics were used to identify the proteins and genes of interest. Hsp10 levels changed significantly following CSE cell exposure as compared with unstressed cells. In unstressed cells, three isoelectric variants of Hsp10 were found, which have not yet been reported for other systems. After CSE exposure, only the most basic variant was still expressed; the other two were no longer detectable. In order to investigate the nature of the three variants found in unstressed cells, e.g., posttranslational modifications of a single product from the canonical human hsp10 gene, or different products from different genes with variations in primary structure, we performed mass spectrometric analysis. Spots were excised from matched Coomassie-stained gels, trypsin digested, and analyzed by nano-RP-HPLC-ESI-MS/MS to determine the fragments’ amino-acid sequences. The sequencing results and database searches showed that the most basic variant was human Hsp10. Partial sequencing of the other two variants indicated they are also human Hsp10. While these determinations continue to complete sequencing coverage, the data thus far indicate that: a) the three variants have the same amino acid sequence; b) they are, most likely, the product of the same hsp10 canonical gene; and c) post-translational modifications are responsible for the observed protein variants. Current experiments are underway to determine the biochemical nature of such modifications, and the possible regulatory pathways driven by CSE that lead to changes in Hsp10 variant levels.
KW - 2D-electrophoresis
KW - Hsp10
KW - IPG
KW - bronchial epithelial cells
KW - chaperonins
KW - cigarette smoke
KW - isoelectric variants
KW - lung fibroblasts
KW - oxidative stress
KW - 2D-electrophoresis
KW - Hsp10
KW - IPG
KW - bronchial epithelial cells
KW - chaperonins
KW - cigarette smoke
KW - isoelectric variants
KW - lung fibroblasts
KW - oxidative stress
UR - http://hdl.handle.net/10447/40979
M3 - Other
SP - 51
EP - 51
ER -