hrvatski

Uloga proteasoma u degradaciji proteina oštećenih dimom cigareta

Mehanizam kojim pušenje cigareta vodi k razvoju kronične opstrukcijske plućne bolesti još uvijek je nejasan. Dim cigareta pridonosi oksidativnom oštećenju proteina u plućima. Jedan od glavnih načina zaštite stanica od oštećenih proteina je ubikvitin-proteasomalni put. Razgradnja proteina ovim putem uključuje konjugaciju više molekula ubikvitina i razgradnju tako označenog proteina putem proteasoma na peptide koji se dalje proteazama i aminopeptidazama brzo hidroliziraju na aminokiseline. Dokazano je da dim cigareta dovodi do porasta sadržaja slobodnih aminokiselina unutar A549 plućnih epitelnih stanica. Postavlja se pitanje je li proteasom uključen u razgradnju proteina oštećenih dimom cigareta. Izlaganjem A549 stanica dimu cigareta uz Epoximicin (inhibitor aktivnosti proteasoma) metodom LC-MS pokazali smo da proteasom je uključen u porast sadržaja slobodnih aminokiselina (p<0, 001). Western blot analizom smo pokazali da dim cigareta uzrokuje povećanje ubikvitin-proteinskih konjugata. Obilježavanjem aktivnih mjesta proteasoma fluorescentnom bojom MV151 dokazali smo da se aktivna mjesta proteasoma uglavnom ne mijenjaju pod utjecajem dima cigareta. Upotrebom fluorogenih supstrata pokazali smo da dim cigareta mijenja sve tri vrste aktivnosti proteasoma. Ukratko, naši rezultati pokazuju da dim cigareta inducira oštećenje proteina koji se potom ubikvitiniraju i obilježavaju za razgradnju putem proteasoma. Proteasom je uključen u uklanjanje proteina oštećenih dimom cigareta. Iako aktivna mjesta proteasoma ostaju očuvana, dim cigareta značajno mijenja aktivnost proteasoma što može pridonijeti agregaciji oštećenih proteina i povećanoj smrti stanica. The mechanism by which cigarette smoking leads to development of chronic obstructive pulmonary disease is still unclear. Cigarette smoke contributes to oxidative damage of proteins inside the lung. One of the main systems to protect cells from damaged proteins is ubiquitin-proteasome pathway. Degradation of proteins via this pathway involves conjugation of multiple ubiquitin moieties and degradation of the tagged protein by proteasome into peptides which are rapidly hydrolyzed into amino acids by proteases and aminopeptidases. It was shown that cigarette smoke causes an increase of free amino acids inside the A549 airway epithelial cells. The question is weather the proteasome is involved in degradation of proteins damaged by cigarette smoke. We have exposed A549 cells to cigarette smoke and Epoxomicin (inhibitor of proteasomal activity) and we have shown, using LC-MS method, that proteasome is involved in the increase of free amino acids (p<0, 001). Western blot analysis has shown that cigarette smoke causes an increase of ubiquitin-protein conjugates. Proteasomal active sites were labelled by using the fluorescent probe MV151 and in general were not disturbed by cigarette smoke. Using the fluorogenic substrates we have shown that cigarette smoke changes all three kinds of proteasomal activities. Briefly, our data suggest that cigarette smoke induces damage to proteins which are subsequently ubiquitinated and tagged for proteasomal degradation. Proteasome is involved in elimination of proteins damaged by cigarette smoke. Although proteasomal active sites remain intact, cigarette smoke significantly disturbs proteasomal activity which may contribute to the aggregation of damaged proteins and enhanced cell death.

pušenje ; kronična opstrukcijska plućna bolest ; ubikvitin ; proteasom

nije evidentirano

engleski

Cigarette smoke - induced protein damage: role of the proteasome

Mehanizam kojim pušenje cigareta vodi k razvoju kronične opstrukcijske plućne bolesti još uvijek je nejasan. Dim cigareta pridonosi oksidativnom oštećenju proteina u plućima. Jedan od glavnih načina zaštite stanica od oštećenih proteina je ubikvitin-proteasomalni put. Razgradnja proteina ovim putem uključuje konjugaciju više molekula ubikvitina i razgradnju tako označenog proteina putem proteasoma na peptide koji se dalje proteazama i aminopeptidazama brzo hidroliziraju na aminokiseline. Dokazano je da dim cigareta dovodi do porasta sadržaja slobodnih aminokiselina unutar A549 plućnih epitelnih stanica. Postavlja se pitanje je li proteasom uključen u razgradnju proteina oštećenih dimom cigareta. Izlaganjem A549 stanica dimu cigareta uz Epoximicin (inhibitor aktivnosti proteasoma) metodom LC-MS pokazali smo da proteasom je uključen u porast sadržaja slobodnih aminokiselina (p<0, 001). Western blot analizom smo pokazali da dim cigareta uzrokuje povećanje ubikvitin-proteinskih konjugata. Obilježavanjem aktivnih mjesta proteasoma fluorescentnom bojom MV151 dokazali smo da se aktivna mjesta proteasoma uglavnom ne mijenjaju pod utjecajem dima cigareta. Upotrebom fluorogenih supstrata pokazali smo da dim cigareta mijenja sve tri vrste aktivnosti proteasoma. Ukratko, naši rezultati pokazuju da dim cigareta inducira oštećenje proteina koji se potom ubikvitiniraju i obilježavaju za razgradnju putem proteasoma. Proteasom je uključen u uklanjanje proteina oštećenih dimom cigareta. Iako aktivna mjesta proteasoma ostaju očuvana, dim cigareta značajno mijenja aktivnost proteasoma što može pridonijeti agregaciji oštećenih proteina i povećanoj smrti stanica. The mechanism by which cigarette smoking leads to development of chronic obstructive pulmonary disease is still unclear. Cigarette smoke contributes to oxidative damage of proteins inside the lung. One of the main systems to protect cells from damaged proteins is ubiquitin-proteasome pathway. Degradation of proteins via this pathway involves conjugation of multiple ubiquitin moieties and degradation of the tagged protein by proteasome into peptides which are rapidly hydrolyzed into amino acids by proteases and aminopeptidases. It was shown that cigarette smoke causes an increase of free amino acids inside the A549 airway epithelial cells. The question is weather the proteasome is involved in degradation of proteins damaged by cigarette smoke. We have exposed A549 cells to cigarette smoke and Epoxomicin (inhibitor of proteasomal activity) and we have shown, using LC-MS method, that proteasome is involved in the increase of free amino acids (p<0, 001). Western blot analysis has shown that cigarette smoke causes an increase of ubiquitin-protein conjugates. Proteasomal active sites were labelled by using the fluorescent probe MV151 and in general were not disturbed by cigarette smoke. Using the fluorogenic substrates we have shown that cigarette smoke changes all three kinds of proteasomal activities. Briefly, our data suggest that cigarette smoke induces damage to proteins which are subsequently ubiquitinated and tagged for proteasomal degradation. Proteasome is involved in elimination of proteins damaged by cigarette smoke. Although proteasomal active sites remain intact, cigarette smoke significantly disturbs proteasomal activity which may contribute to the aggregation of damaged proteins and enhanced cell death.

cigarette smoking ; chronic obstructive pulmonary disease ; ubiqutin ; proteasome

nije evidentirano