Increased elastase sensitivity and decreased intramolecular interactions in the more transmissible 501Y.V1 and 501Y.V2 SARS-CoV-2 variants' spike protein-an in silico analysis.

Two SARS-CoV-2 variants of concern showing increased transmissibility relative to the Wuhan virus have recently been identified. Although neither variant appears to cause more severe illness nor increased risk of death, the faster spread of the virus is a major threat. Using computational tools, we found that the new SARS-CoV-2 variants may acquire an increased transmissibility by increasing the propensity of its spike protein to expose the receptor binding domain via proteolysis, perhaps by neutrophil elastase and/or via reduced intramolecular interactions that contribute to the stability of the closed conformation of spike protein. This information leads to the identification of potential treatments to avert the imminent threat of these more transmittable SARS-CoV-2 variants

Peripheral Sensitization Increases Opioid Receptor Expression And Activation By Crotalphine In Rats

Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the μ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE 2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective μ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE 2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids. © 2014 Zambelli et al.93Stein, C., Peripheral mechanisms of opioid analgesia (1993) Anesth Analg, 76, pp. 182-191Obara, I., Parkitna, J.R., Korostynski, M., Makuch, W., Kaminska, D., Local peripheral opioid effects and expression of opioid genes in the spinal cord and dorsal root ganglia in neuropathic and inflammatory pain (2009) Pain, 141, pp. 283-291Puehler, W., Zollner, C., Brack, A., Shaqura, M.A., Krause, H., Schafer, M., Stein, C., Rapid upregulation of mu opioid receptor mRNA in dorsal root ganglia in response to peripheral inflammation depends on neuronal conduction (2004) Neuroscience, 129 (2), pp. 473-479. , DOI 10.1016/j.neuroscience.2004.06.086, PII S030645220400627XMaekawa, K., Minami, M., Masuda, T., Satoh, M., Expression of 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Small‐Molecule Activators of Glucose‐6‐phosephate Dehydrogenase (G6PD) Bridging the Dimer Interface

We have recently identified AG1, a small-molecule activator that functions by promoting oligomerization of glucose-6- phosphate dehydrogenase (G6PD) to the catalytically competent forms. Biochemical experiments indicate activation of G6PD by the original hit molecule (AG1) is noncovalent and that one C2-symmetric region of the G6PD homodimer is important for ligand function. Consequently, the disulfide in AG1 is not required for activation of G6PD and a number of analogs were prepared without this reactive moiety. Our Study supports a mechanism of action whereby AG1 bridges the dimer interface at the structural nicotinamide adenine dinucleotide phosphate (NADP+)-binding sites of two interacting G6PD monomers. Small molecules that promote G6PD oligomerization have the potential to provide a first-in-class treatment for G6PD deficiency. This general strategy could be applied to other enzyme deficiencies where control of oligomerization can enhance enzymatic activity and/or stability

Ischaemic preconditioning improves proteasomal activity and increases the degradation of δPKC during reperfusion

The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilon PKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection. Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished delta PKC translocation by 3.8-fold and increased epsilon PKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of delta PKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilon PKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilon PKC during IPC restored delta PKC levels at the mitochondria while decreasing epsilon PKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a delta PKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol. Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, delta PKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilon PKC.NIH[AA11147]Oklahoma Center for Advancement of Science and Technology[HR05-171S

Protein kinase C in heart failure: a therapeutic target?

Heart failure (HF) afflicts about 5 million people and causes 300 000 deaths a year in the United States alone. An integral part of the pathogenesis of HF is cardiac remodelling, and the signalling events that regulate it are a subject of intense research. Cardiac remodelling is the sum of responses of the heart to causes of HF, such as ischaemia, myocardial infarction, volume and pressure overload, infection, inflammation, and mechanical injury. These responses, including cardiomyocyte hypertrophy, myocardial fibrosis, and inflammation, involve numerous cellular and structural changes and ultimately result in a progressive decline in cardiac performance. Pharmacological and genetic manipulation of cultured heart cells and animal models of HF and the analysis of cardiac samples from patients with HF are all used to identify the molecular and cellular mechanisms leading to the disease. Protein kinase C (PKC) isozymes, a family of serine–threonine protein kinase enzymes, were found to regulate a number of cardiac responses, including those associated with HF. In this review, we describe the PKC isozymes that play critical roles in specific aspects of cardiac remodelling and dysfunction in HF

Scaffold proteins LACK and TRACK as potential drug targets in kinetoplastid parasites: Development of inhibitors

Parasitic diseases cause similar to 500,000 deaths annually and remain a major challenge for therapeutic development. Using a rational design based approach, we developed peptide inhibitors with anti-parasitic activity that were derived from the sequences of parasite scaffold proteins LACK (Leishmania's receptor for activated C-kinase) and TRACK (Trypanosoma receptor for activated C-kinase). We hypothesized that sequences in LACK and TRACK that are conserved in the parasites, but not in the mammalian ortholog, RACK (Receptor for activated C-kinase), may be interaction sites for signaling proteins that are critical for the parasites' viability. One of these peptides exhibited leishmanicidal and trypanocidal activity in culture. Moreover, in infected mice, this peptide was also effective in reducing parasitemia and increasing survival without toxic effects. The identified peptide is a promising new anti-parasitic drug lead, as its unique features may limit toxicity and drug-resistance, thus overcoming central limitations of most anti-parasitic drugs. (C) 2016 The Authors. Published by Elsevier Ltd on behalf of Australian Society for Parasitology.National Institutes of HealthStanford Univ, Sch Med, Dept Chem & Syst Biol, Stanford, CA 94305 USAUniv Sao Paulo, Inst Quim, Dept Bioquim, BR-05508 Sao Paulo, SP, BrazilMcGill Univ, Res Inst, Natl Reference Ctr Parasitol, Montreal, PQ, CanadaUniv Autonoma Yucatan, Ctr Invest Reg Dr Hideyo Noguchi, Parasitol Lab, Merida, Yucatan, MexicoStanford Univ, Biomat & Adv Drug Delivery Lab, Stanford, CA 94305 USAUniv Estadual Campinas, Inst Chem, Campinas, SP, BrazilUniv Fed Sao Paulo, Dept Ciencias Biol, Campus Diadema, Sao Paulo, BrazilMcGill Univ, Inst Parasitol, Quebec City, PQ, CanadaMcGill Univ, Ctr Host Parasite Interact, Quebec City, PQ, CanadaUniv Fed Sao Paulo, Dept Ciencias Biol, Campus Diadema, Sao Paulo, BrazilNIH: TW008781-01C-IDEANIH: AI078505Web of Scienc

Protein kinase Cθ is required for cardiomyocyte survival and cardiac remodeling

Protein kinase Cs (PKCs) constitute a family of serine/threonine kinases, which has distinguished and specific roles in regulating cardiac responses, including those associated with heart failure. We found that the PKCθ isoform is expressed at considerable levels in the cardiac muscle in mouse, and that it is rapidly activated after pressure overload. To investigate the role of PKCθ in cardiac remodeling, we used PKCθ−/− mice. In vivo analyses of PKCθ−/− hearts showed that the lack of PKCθ expression leads to left ventricular dilation and reduced function. Histological analyses showed a reduction in the number of cardiomyocytes, combined with hypertrophy of the remaining cardiomyocytes, cardiac fibrosis, myofibroblast hyper-proliferation and matrix deposition. We also observed p38 and JunK activation, known to promote cell death in response to stress, combined with upregulation of the fetal pattern of gene expression, considered to be a feature of the hemodynamically or metabolically stressed heart. In keeping with these observations, cultured PKCθ−/− cardiomyocytes were less viable than wild-type cardiomyocytes, and, unlike wild-type cardiomyocytes, underwent programmed cell death upon stimulation with α1-adrenergic agonists and hypoxia. Taken together, these results show that PKCθ maintains the correct structure and function of the heart by preventing cardiomyocyte cell death in response to work demand and to neuro-hormonal signals, to which heart cells are continuously exposed

RACK-1 Acts with Rac GTPase Signaling and UNC-115/abLIM in Caenorhabditis elegans Axon Pathfinding and Cell Migration

Migrating cells and growth cones extend lamellipodial and filopodial protrusions that are required for outgrowth and guidance. The mechanisms of cytoskeletal regulation that underlie cell and growth cone migration are of much interest to developmental biologists. Previous studies have shown that the Arp2/3 complex and UNC-115/abLIM act redundantly to mediate growth cone lamellipodia and filopodia formation and axon pathfinding. While much is known about the regulation of Arp2/3, less is known about regulators of UNC-115/abLIM. Here we show that the Caenorhabditis elegans counterpart of the Receptor for Activated C Kinase (RACK-1) interacts physically with the actin-binding protein UNC-115/abLIM and that RACK-1 is required for axon pathfinding. Genetic interactions indicate that RACK-1 acts cell-autonomously in the UNC-115/abLIM pathway in axon pathfinding and lamellipodia and filopodia formation, downstream of the CED-10/Rac GTPase and in parallel to MIG-2/RhoG. Furthermore, we show that RACK-1 is involved in migration of the gonadal distal tip cells and that the signaling pathways involved in this process might be distinct from those involved in axon pathfinding. In sum, these studies pinpoint RACK-1 as a component of a novel signaling pathway involving Rac GTPases and UNC-115/abLIM and suggest that RACK-1 might be involved in the regulation of the actin cytoskeleton and lamellipodia and filopodia formation in migrating cells and growth cones