Dissulfeto isomerase protéica (PDIA1 ou PDI) é uma chaperona e ditiol-dissulfeto oxido-redutase residente do reticulo endoplasmático (RE). PDI é essencial à regulação da proteostase por ter função no enovelamento oxidativo de proteínas e na via de degradação associada ao RE (ERAD). Além disso, PDI interage fisicamente e regula a atividade de NADPH oxidases, e fora da célula é um regulador redox essencial à atividade de proteínas extracelulares. Este pool epi/pericelular da PDI (pecPDI) regula função de proteínas de membrana/secretadas, como integrinas, glicoproteínas gp120 do virus HIV e outras, com múltiplas funções que incluem: trombose, ativação plaquetária, adesão celular, infecção viral e remodelamento vascular. A rota de externalização da PDI permanece obscura, e seu conhecimento pode indicar mecanismos dos efeitos (fisio)patológicos da PDI. A secreção da PDI pela rota RE-Golgi foi sugerida em células endoteliais infectadas pelo vírus da dengue, células pancreáticas e tireoideanas. No entanto, uma varredura sistemática das possíveis rotas de externalização da PDI não foi previamente realizada. Neste estudo, mostramos que células endoteliais (EC) externalizam constitutivamente, por rotas distintas, dois pools de PDI, de superfície celular e solúvel, enquanto na EC não estimulada PDI não foi detectada significativamente em micropartículas. PDI externalizada corresponde a ca.1,4% do pool total de PDI celular. Tanto a PDI de superfície celular como a solúvel foram majoritariamente secretadas pela via de secreção não-convencional do tipo IV independente de GRASP. Contudo, a via de secreção clássica também contribui para externalização basal da PDI de superfície celular, mas não da solúvel basal ou estimulada por PMA, ATP e trombina indicando que todas envolvem escape do Golgi. Além disso, a externalização constitutiva da PDI de superfície em célula muscular lisa vascular também ocorre por via independente de Golgi. Externalização da PDI não foi detectavelmente mediada pela secreção não-convencional do tipo I, II, III, lisossomos secretórios, endossoma de reciclagem e transporte ativo (dependente de ATP) em EC. Considerando que chaperonas são vias essenciais de resposta a estresses, investigamos o efeito de estresse do RE e choque térmico na pecPDI. Estresse do RE não altera a PDI de superfície celular, mas aumenta PDI solúvel. Ambos os pools de PDI não foram alterados por choque térmico, embora a recuperação desse estresse diminua a secreção de PDI. Estes dados sugerem que a liberação de PDI é um processo regulado, dependente da natureza do estresse. Bloqueio da síntese de proteínas com cicloheximida não altera pecPDI, indicando que PDI recém-sintetizada não é preferencialmente externalizada e que o tráfego da PDI independe de outras proteínas recém-sintetizadas. Um aspecto importante do estudo foi indicar uma resiliência da pecPDI à modulação individual de distintas vias secretoras, consistente com uma estrita auto-regulação e possibilidade de vias sinérgicas e complementares. Estes resultados indicam que a externalização da PDI de superfície e PDI secretada possam ser externalizadas por mecanismos independentes. Estes processos compõem um processo regulado estritamente, consistente com papel homeostático da pecPDI / Protein disulfide isomerase (PDIA1 or PDI) is dithiol-disulfide oxireductase chaperone resident in the endoplasmic reticulum (ER). PDI is essential for proteostasis, due to its support of oxidative protein folding and ER-associated protein degradation (ERAD). In addition, PDI associates with NADPH oxidase(s) and regulate its activity, while outside of the cell, PDI redox-dependently modulates extracellular proteins. This epi/pericellular PDI (pecPDI) pool is known to regulate membrane/secreted proteins such as integrins, HIV glycoprotein gp120 and others, with functions that involve thrombosis, platelet function, cell adhesion, viral infection and vascular remodeling. PDI externalization route remains enigmatic and its elucidation can help understand some (patho)physiological PDI effects. An ER-Golgi route for PDI secretion has been as described on dengue virus-infected endothelial cells pancreatic and thyroid) cells. However, none of these papers addressed PDI secretion routes in a systematic fashion. Here, we show that endothelial cells (EC) constitutively externalize, through different routes, two PDI pools, a cell-surface and a secreted one, while in nonstimulated ECs PDI was not significantly detected in microparticles. Externalized PDI corresponds to < 2% of total cellular PDI pool. Both cell-surface and soluble PDI were predominantly externalized through unconventional type IV GRASP-independent pathway(s). However, the classical secretory pathway also contributes to basal cell-surface, but not soluble, PDI externalization, as PMA, ATP or thrombin-stimulated secretion also involve Golgi bypass. Furthermore, constitutive cell-surface PDI externalization in vascular smooth muscle cells also occurs in a Golgi-independent way. PDI externalization was not detectably mediated by non-conventional type I, II and III secretion routes, secretory lysosomes, recycling endosomes and ATP dependent active transport in EC. Since chaperones are essential for cellular stress response, we assessed the effects of ER stress and heat-shock on pecPDI. ER stress did not affect cell-surface PDI but increased the soluble pool. Both PDI pools were unaltered by heat shock, while stress recovery decreased PDI secretion. These data suggest that PDI release is finely tuned and dependent on the type of stress. Blockade of protein synthesis with cycloheximide did not change pecPDI levels, suggesting that newly-synthesized PDI is not preferentially externalized and that PDI traffic does not require newly-synthesized proteins. An important aspect of the study was the evidence for pecPDI resilience to individual modulation of distinct secretion routes, consistent with strict auto-regulation and possible synergic or complementary pathways. Overall, our data suggest that cell-surface and secreted PDI pool externalization are regulated through independent mechanisms, which in both cases involve Type IV non-conventional routes, with some minor contribution of Golgi-dependent secretory pathway. These patterns compose a strictly regulated process, consistent with an important homeostatic role for pecPDI

Thesis (Ph. D.)--University of Missouri--Columbia, 2001. / "May 2001." Typescript. Vita. Includes bibliographical references (leaves 176-195). Also available on the Internet.

Wong Cheuk Ying. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 101-124). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgement --- p.vi / List of Abbreviations --- p.vii / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter Chapter 2. --- Methods --- p.4 / Chapter 2.1 --- Measurement of cAMP and cGMP in VSMCs --- p.4 / Chapter 2.1.1 --- Cell culture --- p.4 / Chapter 2.1.2 --- Enzyme-immunoassay colorimetric measurement for cAMP and cGMP --- p.5 / Chapter 2.1.3 --- Statistical analysis --- p.6 / Chapter 2.2 --- Measurement of apoptosis in VSMCs and bone marrow-derived stem cells --- p.6 / Chapter 2.2.1 --- Cell culture --- p.6 / Chapter 2.2.2 --- Hoechst33258 --- p.7 / Chapter 2.2.3 --- Cell Death ELISA plus --- p.7 / Chapter 2.2.4 --- Protein extraction and Western blot analysis of PKG expression --- p.8 / Chapter 2.2.5 --- Statistical analysis --- p.9 / Chapter 2.3 --- Measurement of cell proliferation in VSMCs and bone marrow-derived stem cells --- p.9 / Chapter 2.3.1 --- Cell culture --- p.9 / Chapter 2.3.2 --- Cell count --- p.10 / Chapter 2.3.3 --- MTT assay --- p.11 / Chapter 2.3.4 --- BrdU-(5`Bromo-2-deoxyuridine) ELISA colorimetric assay --- p.11 / Chapter 2.3.5 --- Statistical analysis --- p.12 / Chapter Chapter 3. --- Effects of hyperglycemia on CGRP-induced cAMP response in VSMCs / Chapter 3.1 --- Introduction --- p.13 / Chapter 3.2 --- Results --- p.18 / Chapter 3.3 --- Discussion --- p.22 / Chapter Chapter 4. --- Role of cGMP and protein kinase G in regulation of apoptosis in VSMCs / Chapter 4.1 --- Introduction --- p.26 / Chapter 4.2 --- Results --- p.30 / Chapter 4.3 --- Discussion --- p.44 / Chapter Chapter 5. --- Role of protein kinase G in regulation of proliferation in VSMCs / Chapter 5.1 --- Introduction --- p.55 / Chapter 5.2 --- Results --- p.58 / Chapter 5.3 --- Discussion --- p.67 / Chapter Chapter 6. --- Effects of aging and eNOS- and iNOS-gene deletion (using eNOS- and iNOS-knockout mice) on apoptosis of VSMCs / Chapter 6.1 --- Introduction --- p.73 / Chapter 6.2 --- Results --- p.76 / Chapter 6.3 --- Discussion --- p.79 / Chapter Chapter 7. --- Role of protein kinase G in regulation of apoptosis and proliferation of bone marrow stromal stem cells / Chapter 7.1 --- Introduction --- p.81 / Chapter 7.2 --- Results --- p.84 / Chapter 7.3 --- Discussion --- p.92 / Chapter Chapter 8. --- Overall discussion --- p.95 / Chapter Chapter 9. --- References --- p.101