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Abstract Caspase-2 is a unique and conserved cysteine protease that is involved in several cellular processes, including different forms of cell death, maintenance of genomic stability, and the response to reactive oxygen species. Despite advances in caspase-2 research in recent years, the mechanisms underlying its activation remain largely unclear. Although caspase-2 is activated in the PIDDosome complex, its processing could occur even in the absence of PIDD1 and/or RAIDD, suggesting the existence of an alternative platform for caspase-2 activation. Here, we show that caspase-2 undergoes ubiquitination and interacts with scaffolding protein p62/sequestosome-1 (SQSTM1) under normal conditions and in response to DNA damage. p62 promotes proteasomal but not autophagic caspase-2 degradation as well as its dimerization and activation that triggers the caspase cascade and, subsequently, cell death. Inhibition of p62 expression attenuates cisplatin-induced caspase-2 processing and apoptosis. Notably, the ZZ domain of p62 is critical for caspase-2 binding, whereas the UBA domain is seemingly required to stabilize the p62–caspase-2 complex. Thus, we have uncovered the dual role of p62 in regulating caspase-2 activity: it can foster the degradation of caspase-2 in the proteasome or facilitate its activation by acting as a scaffold platform.

Chronic stress can trigger several pathologies including mood disorders for which no clear diagnostic molecular markers have been established yet. Attractive biomarker sources are extracellular vesicles (EVs). Evs are released by cells in health and disease and contain genetic material, proteins and lipids characteristic of the cell state. Here we show that Evs recovered from the blood of animals exposed to a repeated interrupted stress protocol (RIS) have a different protein profile compared to those obtained from control animals. Proteomic analysis indicated that proteins differentially present in bulk serum Evs from stressed animals were implicated in metabolic and inflammatory pathways and several of them were previously related to psychiatric disorders. Interestingly, these serum Evs carry brain-enriched proteins including the stress-responsive neuronal protein M6a. Then, we used an in-utero electroporation strategy to selectively overexpress M6a-GFP in brain neurons and found that M6a-GFP could also be detected in bulk serum Evs suggesting a neuronal origin. Finally, to determine if these Evs could have functional consequences, we administered Evs from control and RIS animals intranasally to naïve mice. Animals receiving stress EVs showed changes in behavior and brain M6a levels similar to those observed in physically stressed animals. Such changes could therefore be attributed, or at least in part, to EV protein transfer. Altogether these findings show that EVs may participate in stress signaling and propose proteins carried by EVs as a valuable source of biomarkers for stress-induced diseases.

Abstract Background Emergency department (ED) attendances are contributing to rising costs of the National Health Service (NHS) in England. Critically assessing the impact of new services to reduce emergency department use can be difficult as new services may create additional access points, unlocking latent demand. The study evaluated an Acute Visiting Scheme (AVS) in a primary care context. We asked if AVS reduces overall ED demand and whether or not it changed utilisation patterns for frequent attenders. Method The study used a pre post single cohort design. The impact of AVS on all-cause ED attendances was hypothesised as a substitution effect, where AVS duty doctor visits would replace emergency department visits. Primary outcome was frequency of ED attendances. End points were reduction of frequency of service use and increase of intervals between attendances by frequent attenders. Results ED attendances for AVS users rose by 47.6%. If AVS use was included, there was a more than fourfold increase of total service utilisation, amounting to 438.3%. It shows that AVS unlocked significant latent demand. However, there was some reduction in the frequency of ED attendances for some patients and an increase in time intervals between ED attendances for others. Conclusion The study demonstrates that careful analysis of patient utilisation can detect a differential impact of AVS on the use of ED. As the new service created additional access points for patients and hence introduces an element of choice, the new service is likely to unlock latent demand. This study illustrates that AVS may be most useful if targeted at specific patient groups who are most likely to benefit from the new service.

BackgroundThe DHTKD1-encoded 2-oxoadipate dehydrogenase (OADH) oxidizes 2-oxoadipate—a common intermediate of the lysine and tryptophan catabolism. The mostly low and cell-specific flux through these pathways, and similar activities of OADH and ubiquitously expressed 2-oxoglutarate dehydrogenase (OGDH), agree with often asymptomatic phenotypes of heterozygous mutations in the DHTKD1 gene. Nevertheless, OADH/DHTKD1 are linked to impaired insulin sensitivity, cardiovascular disease risks, and Charcot-Marie-Tooth neuropathy. We hypothesize that systemic significance of OADH relies on its generation of glutaryl residues for protein glutarylation. Using pharmacological inhibition of OADH and the animal model of spinal cord injury (SCI), we explore this hypothesis.MethodsThe weight-drop model of SCI, a single intranasal administration of an OADH-directed inhibitor trimethyl adipoyl phosphonate (TMAP), and quantification of the associated metabolic changes in the rat brain employ established methods.ResultsThe TMAP-induced metabolic changes in the brain of the control, laminectomized (LE) and SCI rats are long-term and (patho)physiology-dependent. Increased glutarylation of the brain proteins, proportional to OADH expression in the control and LE rats, represents a long-term consequence of the OADH inhibition. The proportionality suggests autoglutarylation of OADH, supported by our mass-spectrometric identification of glutarylated K155 and K818 in recombinant human OADH. In SCI rats, TMAP increases glutarylation of the brain proteins more than OADH expression, inducing a strong perturbation in the brain glutathione metabolism. The redox metabolism is not perturbed by TMAP in LE animals, where the inhibition of OADH increases expression of deglutarylase sirtuin 5. The results reveal the glutarylation-imposed control of the brain glutathione metabolism. Glutarylation of the ODP2 subunit of pyruvate dehydrogenase complex at K451 is detected in the rat brain, linking the OADH function to the brain glucose oxidation essential for the redox state. Short-term inhibition of OADH by TMAP administration manifests in increased levels of tryptophan and decreased levels of sirtuins 5 and 3 in the brain.ConclusionPharmacological inhibition of OADH affects acylation system of the brain, causing long-term, (patho)physiology-dependent changes in the expression of OADH and sirtuin 5, protein glutarylation and glutathione metabolism. The identified glutarylation of ODP2 subunit of pyruvate dehydrogenase complex provides a molecular mechanism of the OADH association with diabetes.

Abstract Among the mechanisms of suppression that T regulatory (Treg) cells exert to control the immune responses, the secretion of small extracellular vesicles (sEV) has been recently proposed as a novel contact‐independent immunomodulatory mechanism. Previous studies have demonstrated that Treg cells produce sEV, including exosomes, able to modulate the effector function of CD4+ T cells, and antigen presenting cells (APCs) such as dendritic cells (DCs) through the transfer of microRNA, cytokines, the production of adenosine, among others. Previously, we have demonstrated that Neuropilin‐1 (Nrp1) is required for Tregs‐mediated immunosuppression mainly by impacting on the phenotype and function of effector CD4+ T cells. Here, we show that Foxp3+ Treg cells secrete sEV, which bear Nrp1 in their membrane. These sEV modulate effector CD4+ T cell phenotype and proliferation in vitro in a Nrp1‐dependent manner. Proteomic analysis indicated that sEV obtained from wild type (wt) and Nrp1KO Treg cells differed in proteins related to immune tolerance, finding less representation of CD73 and Granzyme B in sEV obtained from Nrp1KO Treg cells. Likewise, we show that Nrp1 is required in Treg cell‐derived sEV for inducing skin transplantation tolerance, since a reduction in graft survival and an increase on M1/M2 ratio were found in animals treated with Nrp1KO Treg cell‐derived sEV. Altogether, this study describes for the first time that Treg cells secrete sEV containing Nrp1 and that this protein, among others, is necessary to promote transplantation tolerance in vivo via sEV local administration.

Abstract Cell death by glutamate excitotoxicity, mediated by N-methyl-d-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-κB transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence, and luciferase reporter assays, we found that NMDA-stimulated NF-κB activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-κB inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-κB transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

Chronic stress can trigger several pathologies including mood disorders for which no clear diagnostic molecular markers have been established yet. Attractive biomarker sources are extracellular vesicles (EVs). Evs are released by cells in health and disease and contain genetic material, proteins and lipids characteristic of the cell state. Here we show that Evs recovered from the blood of animals exposed to a repeated interrupted stress protocol (RIS) have a different protein profile compared to those obtained from control animals. Proteomic analysis indicated that proteins differentially present in bulk serum Evs from stressed animals were implicated in metabolic and inflammatory pathways and several of them were previously related to psychiatric disorders. Interestingly, these serum Evs carry brain-enriched proteins including the stress-responsive neuronal protein M6a. Then, we used an in-utero electroporation strategy to selectively overexpress M6a-GFP in brain neurons and found that M6a-GFP could also be detected in bulk serum Evs suggesting a neuronal origin. Finally, to determine if these Evs could have functional consequences, we administered Evs from control and RIS animals intranasally to naïve mice. Animals receiving stress EVs showed changes in behavior and brain M6a levels similar to those observed in physically stressed animals. Such changes could therefore be attributed, or at least in part, to EV protein transfer. Altogether these findings show that EVs may participate in stress signaling and propose proteins carried by EVs as a valuable source of biomarkers for stress-induced diseases. [ABSTRACT FROM AUTHOR]