FRANCISCO RAFAEL MARTINS LAURINDO
Projetos de Pesquisa
Unidades Organizacionais
Instituto do Coração, Hospital das Clínicas, Faculdade de Medicina
LIM/64, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/64, Hospital das Clínicas, Faculdade de Medicina - Líder
9 resultados
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- Protein disulfide isomerase plasma levels in healthy humans reveal proteomic signatures involved in contrasting endothelial phenotypes(2019) OLIVEIRA, Percillia Victoria Santos de; GARCIA-ROSA, Sheila; SACHETTO, Ana Teresa Azevedo; MORETTI, Ana Iochabel Soares; DEBBAS, Victor; BESSA, Tiphany Coralie De; SILVA, Nathalia Tenguan; PEREIRA, Alexandre da Costa; MARTINS-DE-SOUZA, Daniel; SANTORO, Marcelo Larami; LAURINDO, Francisco Rafael MartinsRedox-related plasma proteins are candidate reporters of protein signatures associated with endothelial structure/function. Thiol-proteins from protein disulfide isomerase (PDI) family are unexplored in this context. Here, we investigate the occurrence and physiological significance of a circulating pool of PDI in healthy humans. We validated an assay for detecting PDI in plasma of healthy individuals. Our results indicate high inter-individual (median = 330 pg/mL) but low intra-individual variability over time and repeated measurements. Remarkably, plasma PDI levels could discriminate between distinct plasma proteome signatures, with PDI-rich ( > median) plasma differentially expressing proteins related to cell differentiation, protein processing, housekeeping functions and others, while PDI-poor plasma differentially displayed proteins associated with coagulation, inflammatory responses and immunoactivation. Platelet function was similar among individuals with PDI-rich vs. PDI-poor plasma. Remarkably, such protein signatures closely correlated with endothelial function and phenotype, since cultured endothelial cells incubated with PDI-poor or PDI-rich plasma recapitulated gene expression and secretome patterns in line with their corresponding plasma signatures. Furthermore, such signatures translated into functional responses, with PDI-poor plasma promoting impairment of endothelial adhesion to fibronectin and a disturbed pattern of wound-associated migration and recovery area. Patients with cardiovascular events had lower PDI levels vs. healthy individuals. This is the first study describing PDI levels as reporters of specific plasma proteome signatures directly promoting contrasting endothelial phenotypes and functional responses.
- Protein Disulfide Isomerase-A1 (PDIA1) Remodels Endoplasmic Reticulum -Plasma Membrane Contact Sites: Possible Role of Nogo-B Protein Regulation(2022) BESSA, Tiphany De; OLIVEIRA, Percillia; DEBBAS, Victor; WOSNIAK JR., Joao; SANTOS, Celio; SHAH, Ajay; LAURINDO, Francisco R.
- Evidence for a protective role of Protein Disulfide Isomerase-A1 against aortic dissection(2023) PORTO, Fernando Garcez; TANAKA, Leonardo Yuji; BESSA, Tiphany Coralie de; OLIVEIRA, Percillia Victoria Santos; SOUZA, Julia Martins Felipe de; KAJIHARA, Daniela; FERNANDES, Carolina Goncalves; SANTOS, Patricia Nolasco; LAURINDO, Francisco Rafael MartinsBackground and aims: Redox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection. Methods: Initially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days. Results: Transgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mor-tality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%-50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echo-cardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation. Conclusions: Our data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially un-covers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling.
- Protein Disulfide Isomerase-A1 (PDIA1) Governs the Morphology of Endoplasmic Reticulum-Plasma Membrane Contact Sites(2022) BESSA, Tiphany De; OLIVEIRA, Percillia; WOSNIAK JR., Joao; DEBBAS, Victor; SANTOS, Celio; SHAH, Ajay; LAURINDO, Francisco R.
- Endoplasmic reticulum - plasma membrane contact sites as a potential hub for Protein Disulfide Isomerase-A1 (PDIA1) / NADPH oxidase cross-talk(2023) BESSA, Tiphany De; OLIVEIRA, Percillia; DEBBAS, Victor; WOSNIAK JR., Joao; SANTOS, Celio; SHAH, Ajay; LAURINDO, Francisco R.
- DNAJB12 and DNJB14 are non-redundant Hsp40 redox chaperones involved in endoplasmic reticulum protein reflux(2024) PURIFICACA, Aline Dias da; DEBBAS, Victor; TANAKA, Leonardo Yuji; GABRIEL, Gabriele Veronica de Mello; WOSNIAK JUNIOR, Joao; BESSA, Tiphany Coralie De; GARCIA-ROSA, Sheila; LAURINDO, Francisco Rafael Martins; OLIVEIRA, Percillia Victoria SantosBackground: The endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux.Methods: We investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/ regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI).Results: We show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells.Conclusions and general significance: Our findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux.
- Proteins Cross-talking with Nox Complexes: The Social Life of Noxes(2023) BESSA, T. C. de; LAURINDO, F. R. M.Nox NADPH Oxidases exhibit a basic organization comprising a catalytic transmembrane subunit closely regulated by canonical regulatory subunits, discussed in other chapters of this book. However, many additional proteins regulate the expression, assembly, structure, activity and subcellular traffic of Nox subunits. As such, they gravitate around Nox complexes and physically associate with at least one among the regulatory or catalytic subunits. Given that such associated proteins, in turn, exert canonical effects distinct from Nox regulation, they connect Nox function to physiological cell programs, mediating cross-talk to and from Noxes. This chapter provides a systematic overview of proteins for which the physical interaction with Noxes has been validated by “wet-lab” experiments. Such proteins support both stimulatory or inhibitory effects towards several aspects of Nox regulation and can be roughly classified as: (a) kinase-related organizers; (b) general organizers; (c) chaperone-like organizers; (d) RhoGTPase and/or cytoskeleton-related organizers; (e) scaffold proteins. In addition, we provide an overview of the Nox interactome “in silico”, indicating that Noxes cross-talk with their environment preferentially via interactive protein hubs associated with their regulatory, rather than catalytic subunits. Characterizing the roles of Nox-associated proteins is essential to provide an integrative understanding of Noxes within multiple cellular physiological contexts. © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023.
- Impaired vascular smooth muscle cell force-generating capacity and phenotypic deregulation in Marfan Syndrome mice(2020) NOLASCO, Patricia; FERNANDES, Carolina Goncalves; RIBEIRO-SILVA, Joao Carlos; OLIVEIRA, Percillia V. S.; SACRINI, Mariana; BRITO, Isis Vasconcelos de; BESSA, Tiphany Coralie De; PEREIRA, Lygia V.; TANAKA, Leonardo Y.; ALENCAR, Adriano; LAURINDO, Francisco Rafael MartinsMechanisms whereby fibrillin-1 mutations determine thoracic aorta aneurysms/dissections (TAAD) in Marfan Syndrome (MFS) are unclear. Most aortic aneurysms evolve from mechanosignaling deregulation, converging to impaired vascular smooth muscle cell (VSMC) force-generating capacity accompanied by synthetic phenotype switch. However, little is known on VSMC mechanoresponses in MFS pathophysiology. Here, we investigated traction force-generating capacity in aortic VSMC cultured from 3-month old mg Delta(lpn) MFS mice, together with morpho-functional and proteomic data. Cultured MFS-VSMC depicted marked phenotype changes vs. wild-type (WT) VSMC, with overexpressed cell proliferation markers but either lower (calponin-1) or higher (SM alphaactin and SM22) differentiation marker expression. In parallel, the increased cell area and its complex non-fusiform shape suggested possible transition towards a mesenchymal-like phenotype, confirmed through several markers (e.g. N-cadherin, Slug). MFS-VSMC proteomic profile diverged from that of WT-VSMC particularly regarding lower expression of actin cytoskeleton-regulatory proteins. Accordingly, MFS-VSMC displayed lower traction force-generating capacity and impaired contractile moment at physiological substrate stiffness, and markedly attenuated traction force responses to enhanced substrate rigidity. Such impaired mechanoresponses correlated with decreased number, altered morphology and delocalization of focal adhesions, as well as dis-organized actin stress fiber network vs. WT-VSMC. In VSMC cultured from 6-month-old mice, phenotype changes were attenuated and both WT-VSMC and MFS-VSMC generated less traction force, presumably involving VSMC aging, but without evident senescence. In summary, MFS-VSMC display impaired force-generating capacity accompanying a mesenchymal-like phenotype switch connected to impaired cytoskeleton/focal adhesion organization. Thus, MFS-associated TAAD involves mechanoresponse impairment common to other TAAD types, but through distinct mechanisms.
- Subverted regulation of Nox1 NADPH oxidase-dependent oxidant generation by protein disulfide isomerase A1 in colon carcinoma cells with overactivated KRas(2019) BESSA, Tiphany Coralie De; PAGANO, Alessandra; MORETTI, Ana Iochabel Soares; OLIVEIRA, Percillia Victoria Santos; MENDONCA, Samir Andrade; KOVACIC, Herve; LAURINDO, Francisco Rafael MartinsProtein disulfide isomerases including PDIA1 are implicated in cancer progression, but underlying mechanisms are unclear. PDIA1 is known to support vascular Nox1 NADPH oxidase expression/activation. Since deregulated reactive oxygen species (ROS) production underlies tumor growth, we proposed that PDIA1 is an upstream regulator of tumor-associated ROS. We focused on colorectal cancer (CRC) with distinct KRas activation levels. Analysis of RNAseq databanks and direct validation indicated enhanced PDIA1 expression in CRC with constitutive high (HCT116) vs. moderate (HKE3) and basal (Caco2) Ras activity. PDIA1 supported Nox1-dependent superoxide production in CRC; however, we first reported a dual effect correlated with Ras-level activity: in Caco2 and HKE3 cells, loss-of-function experiments indicate that PDIA1 sustains Nox1-dependent superoxide production, while in HCT116 cells PDIA1 restricted superoxide production, a behavior associated with increased Rac1 expression/activity. Transfection of Rac1G12V active mutant into HKE3 cells induced PDIA1 to become restrictive of Nox1-dependent superoxide, while in HCT116 cells treated with Rac1 inhibitor, PDIA1 became supportive of superoxide. PDIA1 silencing promoted diminished cell proliferation and migration in HKE3, not detectable in HCT116 cells. Screening of cell signaling routes affected by PDIA1 silencing highlighted GSK3 beta and Stat3. Also, E-cadherin expression after PDIA1 silencing was decreased in HCT116, consistent with PDIA1 support of epithelial-mesenchymal transition. Thus, Ras overactivation switches the pattern of PDIA1-dependent Rac1/Nox1 regulation, so that Ras-induced PDIA1 bypass can directly activate Rac1. PDIA1 may be a crucial regulator of redox-dependent adaptive processes related to cancer progression.