RESUMO
Alterations in protein expression within the initiation phase of acute pancreatitis (AP) might play an important role in the development of this disease, lysosomes being involved in its pathophysiology. The use of pancreatic subcellular fractions in proteomic analysis, simplifies protein maps and helps in the identification of new protein changes and biomarkers characterizing tissue damage. The present study aims to determine the differentially expressed acidic proteins in the pancreatic soluble and lysosomal+mitochondrial (L+M) fractions from rats during the early phase of the experimental model of cerulein (Cer)-induced AP. Subcellular pancreatic extracts from diseased and control rats were analyzed by 2-DE (3-5.6 pH range) and MALDI-TOF/TOF MS. Comparative analysis afforded the conclusive identification of 13 (soluble fraction) and 7 (L+M fraction) proteins or protein fragments occuring in different amounts between diseased and control pancreas, some of them being newly described in AP. In the soluble fraction, we detected changes related to inflammation and apoptosis (α1-inhibitor-3, α-1 antitrypsin, α-1 macroglobulin, haptoglobin, STRAP), oxidative stress and stress response (peroxiredoxin-2, thioredoxin-like 1, GRP94/TRA1, heat shock cognate 71kDa protein), digestive proteases (elastase 3B), serine protease inhibition (serpins B6 and A3L) and translation processes (EF 1-δ). In the L+M fraction, we detected changes mainly related to energy generation or cellular metabolism (ATP synthase ß subunit, chymotrypsinogen B, triacylglycerol lipase), cell redox homeostasis (iodothyronine 5´monodeiodinase) and digestive proteases (carboxypeptidase B1). The data should provide valuable information for unraveling the early pathophysiologic mechanisms of Cer-induced AP.
Assuntos
Ceruletídeo , Lisossomos/química , Mitocôndrias/química , Pâncreas/química , Pancreatite/metabolismo , Proteoma/análise , Doença Aguda , Animais , Apoptose , Biomarcadores/análise , Eletroforese em Gel Bidimensional , Concentração de Íons de Hidrogênio , Lisossomos/metabolismo , Masculino , Mitocôndrias/metabolismo , Pâncreas/metabolismo , Pâncreas/patologia , Pancreatite/induzido quimicamente , Pancreatite/patologia , Proteômica , Ratos , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações Subcelulares/químicaRESUMO
Paxillin is a scaffold protein that participates in focal adhesion signaling in mammalian cells. Fission yeast paxillin ortholog, Pxl1, is required for contractile actomyosin ring (CAR) integrity and collaborates with the ß-glucan synthase Bgs1 in septum formation. We show here that Pxl1's main function is to recruit calcineurin (CN) phosphatase to the actomyosin ring; and thus the absence of either Pxl1 or calcineurin causes similar cytokinesis defects. In turn, CN participates in the dephosphorylation of the Cdc15 F-BAR protein, which recruits and concentrates Pxl1 at the CAR. Our findings suggest the existence of a positive feedback loop between Pxl1 and CN and establish that Pxl1 is a crucial component of the CN signaling pathway during cytokinesis.
Assuntos
Calcineurina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Citocinese/fisiologia , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Glucosiltransferases/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiologia , Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Calcineurina/genética , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Proteínas de Ligação ao GTP/genética , Glucosiltransferases/genética , Proteólise , Proteínas de Schizosaccharomyces pombe/genética , beta-Glucanas/metabolismoRESUMO
In the fission yeast Schizosaccharomyces pombe the cell integrity pathway (CIP) orchestrates multiple biological processes like cell wall maintenance and ionic homeostasis by fine tuning activation of MAPK Pmk1 in response to various environmental conditions. The small GTPase Rho2 positively regulates the CIP through protein kinase C ortholog Pck2. However, Pmk1 retains some function in mutants lacking either Rho2 or Pck2, suggesting the existence of additional upstream regulatory elements to modulate its activity depending on the nature of the environmental stimulus. The essential GTPase Rho1 is a candidate to control the activity of the CIP by acting upstream of Pck2, whereas Pck1, a second PKC ortholog, appears to negatively regulate Pmk1 activity. However, the exact regulatory nature of these two proteins within the CIP has remained elusive. By exhaustive characterization of strains expressing a hypomorphic Rho1 allele (rho1-596) in different genetic backgrounds we show that both Rho1 and Pck1 are positive upstream regulatory members of the CIP in addition to Rho2 and Pck2. In this new model Rho1 and Rho2 control Pmk1 basal activity during vegetative growth mainly through Pck2. Notably, whereas Rho2-Pck2 elicit Pmk1 activation in response to most environmental stimuli, Rho1 drives Pmk1 activation through either Pck2 or Pck1 exclusively in response to cell wall damage. Our study reveals the intricate and complex functional architecture of the upstream elements participating in this signaling pathway as compared to similar routes from other simple eukaryotic organisms.
Assuntos
Sistema de Sinalização das MAP Quinases/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Proteínas rho de Ligação ao GTP/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteína Quinase C/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Rho1 GTPase is the main activator of cell wall glucan biosynthesis and regulates actin cytoskeleton in fungi, including Schizosaccharomyces pombe. We have obtained a fission yeast thermosensitive mutant strain carrying the rho1-596 allele, which displays reduced Rho1 GTPase activity. This strain has severe cell wall defects and a thermosensitive growth, which is partially suppressed by osmotic stabilization. In a global screening for rho1-596 multicopy suppresors the pmp1+ gene was identified. Pmp1 is a dual specificity phosphatase that negatively regulates the Pmk1 mitogen-activated protein kinase (MAPK) cell integrity pathway. Accordingly, elimination of Pmk1 MAPK partially rescued rho1-596 thermosensitivity, corroborating the unexpected antagonistic functional relationship of these genes. We found that rho1-596 cells displayed increased basal activation of the cell integrity MAPK pathway and therefore were hypersensitive to MgCl2 and FK506. Moreover, the absence of calcineurin was lethal for rho1-596. We found a higher level of calcineurin activity in rho1-596 than in wild-type cells, and overexpression of constitutively active calcineurin partially rescued rho1-596 thermosensitivity. All together our results suggest that loss of Rho1 function causes an increase in the cell integrity MAPK activity, which is detrimental to the cells and turns calcineurin activity essential.