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1.
Cell Mol Life Sci ; 79(3): 185, 2022 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-35279766

RESUMO

Golgi membrane proteins such as glycosyltransferases and other glycan-modifying enzymes are key to glycosylation of proteins and lipids. Secretion of soluble Golgi enzymes that are released from their membrane anchor by endoprotease activity is a wide-spread yet largely unexplored phenomenon. The intramembrane protease SPPL3 can specifically cleave select Golgi enzymes, enabling their secretion and concomitantly altering global cellular glycosylation, yet the entire range of Golgi enzymes cleaved by SPPL3 under physiological conditions remains to be defined. Here, we established isogenic SPPL3-deficient HEK293 and HeLa cell lines and applied N-terminomics to identify substrates cleaved by SPPL3 and released into cell culture supernatants. With high confidence, our study identifies more than 20 substrates of SPPL3, including entirely novel substrates. Notably, our N-terminome analyses provide a comprehensive list of SPPL3 cleavage sites demonstrating that SPPL3-mediated shedding of Golgi enzymes occurs through intramembrane proteolysis. Through the use of chimeric glycosyltransferase constructs we show that transmembrane domains can determine cleavage by SPPL3. Using our cleavage site data, we surveyed public proteome data and found that SPPL3 cleavage products are present in human blood. We also generated HEK293 knock-in cells expressing the active site mutant D271A from the endogenous SPPL3 locus. Immunoblot analyses revealed that secretion of select novel substrates such as the key mucin-type O-glycosylation enzyme GALNT2 is dependent on endogenous SPPL3 protease activity. In sum, our study expands the spectrum of known physiological substrates of SPPL3 corroborating its significant role in Golgi enzyme turnover and secretion as well as in the regulation of global glycosylation pathways.


Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Complexo de Golgi/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Proteólise , Proteoma/análise , Ácido Aspártico Endopeptidases/deficiência , Ácido Aspártico Endopeptidases/genética , Domínio Catalítico/genética , Edição de Genes , Células HEK293 , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , N-Acetilgalactosaminiltransferases/genética , Proteômica/métodos , RNA Guia de Cinetoplastídeos/metabolismo , Especificidade por Substrato , Polipeptídeo N-Acetilgalactosaminiltransferase
2.
Am J Physiol Cell Physiol ; 315(5): C636-C642, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30088793

RESUMO

Bisphenol A (BPA) is a synthetic, organic compound frequently present in consumer plastics, including plastic-lined cans, water bottles, toys, and teeth sutures. Previous studies have shown that BPA can produce adverse health effects that include defects in reproductive function and altered prenatal/childhood development. However, little is known regarding the effects of BPA on immune function. In this study, we assessed the effect of BPA on human neutrophils, a critical component of the innate immune system's defense against pathogens. We found that BPA induces a concentration-dependent increase in reactive oxygen species (ROS) generation by neutrophils, which is inhibited by the estrogen receptor-ß antagonist PHTPP. Furthermore, incubation with the membrane-permeable calcium chelator BAPTA-AM and/or removal of extracellular calcium inhibited BPA-induced ROS production, indicating that the process is calcium dependent. Transwell chemotaxis assays revealed that BPA exposure reduces the chemotactic capacity of neutrophils in a gradient of the bacterial cell wall component f-Met-Leu-Phe, a potent chemoattractant. Exposure to BPA also inhibits the ability of neutrophils to kill methicillin-resistant Staphylococcus aureus, a leading human pathogen. Our findings reveal that BPA alters the in vitro function of neutrophils, including ROS production, chemotaxis, and bacterial killing, and raises the possibility of altered innate immunity in vivo, especially in those with compromised immune function and who can be exposed to BPA in a wide variety of products.


Assuntos
Compostos Benzidrílicos/imunologia , Imunidade Inata/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/imunologia , Neutrófilos/imunologia , Fenóis/imunologia , Compostos Benzidrílicos/toxicidade , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/imunologia , Humanos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/patogenicidade , Neutrófilos/efeitos dos fármacos , Fenóis/toxicidade , Espécies Reativas de Oxigênio/imunologia
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