In this Opinion article, we aim to address how cells adapt to stress and the repercussions chronic stress has on cellular function. We consider acute and chronic stress-induced changes at the cellular level, with a focus on a regulator of cellular stress, the chaperome, which is a protein assembly that encompasses molecular chaperones, co-chaperones and other co-factors. We discuss how the chaperome takes on distinct functions under conditions of stress that are executed in ways that differ from the one-on-one cyclic, dynamic functions exhibited by distinct molecular chaperones. We argue that through the formation of multimeric stable chaperome complexes, a state of chaperome hyperconnectivity, or networking, is gained. The role of these chaperome networks is to act as multimolecular scaffolds, a particularly important function in cancer, where they increase the efficacy and functional diversity of several cellular processes. We predict that these concepts will change how we develop and implement drugs targeting the chaperome to treat cancer.

Significance: Understanding isoform-and context-specific subcellular Nox reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase compartmentalization allows relevant functional inferences. This review addresses the interplay between Nox NADPH oxidases and the endoplasmic reticulum (ER), an increasingly evident player in redox pathophysiology given its role in redox protein folding and stress responses. Recent Advances: Catalytic/regulatory transmembrane subunits are synthesized in the ER and their processing includes folding, N-glycosylation, heme insertion, p22phox heterodimerization, as shown for phagocyte Nox2. Dual oxidase (Duox) maturation also involves the regulation by ER-resident Duoxa2. The ER is the activation site for some isoforms, typically Nox4, but potentially other isoforms. Such location influences redox/Nox-mediated calcium signaling regulation via ER targets, such as sarcoendoplasmic reticulum calcium ATPase (SERCA). Growing evidence suggests that Noxes are integral signaling elements of the unfolded protein response during ER stress, with Nox4 playing a dual prosurvival/proapoptotic role in this setting, whereas Nox2 enhances proapoptotic signaling. ER chaperones such as protein disulfide isomerase (PDI) closely interact with Noxes. PDI supports growth factor-dependent Nox1 activation and mRNA expression, as well as migration in smooth muscle cells, and PDI overexpression induces acute spontaneous Nox activation. Critical Issues: Mechanisms of PDI effects include possible support of complex formation and RhoGTPase activation. In phagocytes, PDI supports phagocytosis, Nox activation, and redox-dependent interactions with p47phox. Together, the results implicate PDI as possible Nox organizer. Future Directions: We propose that convergence between Noxes and ER may have evolutive roots given ER-related functional contexts, which paved Nox evolution, namely calcium signaling and pathogen killing. Overall, the interplay between Noxes and the ER may provide relevant insights in Nox-related (patho)physiology. Antioxid. Redox Signal. 20, 2755Signal. 20, -2775

Highlights d N-glycosylation transforms a chaperone, GRP94, from a folder into a scaffolding protein d These changes are pathologic in nature as they remodel proteome-wide connectivity d The N-glycosylated GRP94 variant is a small and distinct fraction of the GRP94 pool d Proteome dysfunctions mediated by the N-glycosylated GRP94 variant are actionable

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V ascular remodeling involves changes for the whole-vessel circumference and crucially determines lumen caliber 1 in physio(patho)logical situations, such as shear-stress responses, restenosis postangioplasty, 1-3 or native atherosclerosis. 1,4 Moreover, cellular mechanisms governing conductance vessel remodeling are likely shared by small-vessel remodeling, for example, in hypertension.5 Despite such relevance, mechanisms of vessel remodeling are not well known. The identification of endothelium 6 and nitric oxide 7 dependency of shear-induced remodeling raised possible mediator roles of redox processes, as indeed supported by many subsequent studies. 3,8 We showed previously that superoxide dismutase underactivity favors constrictive remodeling after injury (AI), whereas exogenous replenishment of SOD3(ecSOD) rescued bioactive nitric oxide from inducible NO synthase and normalized vessel caliber by counteracting constrictive remodeling rather than neointimal growth. 3 The randomized clinical trial Multivitamins and Probucol Study showed that the antioxidant probucol significantly prevented restenosis postballoon angioplasty, essentially by preventing constrictive remodeling rather than neointima formation. 9Such redox-responsiveness is in line with the importance of redox pathways in cytoskeletal dynamics 10 and extracellular matrix organization, 11 which are both crucially involved in vessel remodeling. However, molecular determinants of such redox signaling pathways remain unclear.Abstract-Whole-vessel remodeling critically determines lumen caliber in vascular (patho)physiology, and it is reportedly redox-dependent. We hypothesized that the cell-surface pool of the endoplasmic reticulum redox chaperone protein disulfide isomerase-A1 (peri/epicellular=pecPDI), which is known to support thrombosis, also regulates disease-associated vascular architecture. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling and plaque stability. In a rabbit iliac artery overdistension model, there was unusually high PDI upregulation (≈25-fold versus basal, 14 days postinjury), involving both intracellular and pecPDI. PecPDI neutralization with distinct anti-PDI antibodies did not enhance endoplasmic reticulum stress or apoptosis. In vivo pecPDI neutralization with PDI antibodycontaining perivascular gel from days 12 to 14 post injury promoted 25% decrease in the maximally dilated arteriographic vascular caliber. There was corresponding whole-vessel circumference loss using optical coherence tomography without change in neointima, which indicates constrictive remodeling. This was accompanied by decreased hydrogen peroxide generation. Constrictive remodeling was corroborated by marked changes in collagen organization, that is, switching from circumferential to radial fiber orientation and to a more rigid fiber type. The cytoskeleton architecture was also disrupted; there was a loss of stress fiber coherent organization and a switch from thin to medium thickness actin fibers, all leading to...

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Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.

Streptococcus sanguinis is a pioneer commensal species of dental biofilms, abundant in different oral sites and commonly associated with opportunist cardiovascular infections. In this study, we addressed intra-species functional diversity to better understand the S. sanguinis commensal and pathogenic lifestyles. Multiple phenotypes were screened in nine strains isolated from dental biofilms or from the bloodstream to identify conserved and strain-specific functions involved in biofilm formation and/or persistence in oral and cardiovascular tissues. Strain phenotypes of biofilm maturation were independent of biofilm initiation phenotypes, and significantly influenced by human saliva and by aggregation mediated by sucrose-derived exopolysaccharides (EPS). The production of H2O2 was conserved in most strains, and consistent with variations in extracellular DNA (eDNA) production observed in few strains. The diversity in complement C3b deposition correlated with the rates of opsonophagocytosis by human PMN and was influenced by culture medium and sucrose-derived EPS in a strain-specific fashion. Differences in C3b deposition correlated with strain binding to recognition proteins of the classical pathway, C1q and serum amyloid protein (SAP). Importantly, differences in strain invasiveness into primary human coronary artery endothelial cells (HCAEC) were significantly associated with C3b binding, and in a lesser extent, with binding to host glycoproteins (such as fibrinogen, plasminogen, fibronectin, and collagen). Thus, by identifying conserved and strain-specific phenotypes involved in host persistence and systemic virulence, this study indicates potential new functions involved in systemic virulence and highlights the need of including a wider panel of strains in molecular studies to understand S. sanguinis biology.