Molecular Dynamics Characterization of the C2 Domain of Protein Kinase Cβ

Protein kinase C (PKC) isozymes comprise a family of related enzymes that play a central role in many intracellular eukaryotic signaling events. Isozyme specificity is mediated by association of each PKC isozyme with specific anchoring proteins, termed RACKs. The C2 domain of betaPKC contains at least part of the RACK-binding sites. Because the C2 domain contains also a RACK-like sequence (termed pseudo-RACK), it was proposed that this pseudo-RACK site mediates intramolecular interaction with one of the RACK-binding sites in the C2 domain itself, stabilizing the inactive conformation of betaPKC. BetaPKC depends on calcium for its activation, and the C2 domain contains the calcium-binding sites. The x-ray structure of the C2 domain of betaPKC shows that three Ca(2+) ions can be coordinated by two opposing loops at one end of the domain. Starting from this x-ray structure, we have performed molecular dynamics (MD) calculations on the C2 domain of betaPKC bound to three Ca(2+) ions, to two Ca(2+) ions, and in the Ca(2+)-free state, in order to analyze the effect of calcium on the RACK-binding sites and the pseudo-RACK sites, as well as on the loops that constitute the binding site for the Ca(2+) ions. The results show that calcium stabilizes the beta-sandwich structure of the C2 domain and thus affects two of the three RACK-binding sites within the C2 domain. Also, the interactions between the third RACK-binding site and the pseudo-RACK site are not notably modified by the removal of Ca(2+) ions. On that basis, we predict that the pseudo-RACK site within the C2 domain masks a RACK-binding site in another domain of betaPKC, possibly the V5 domain. Finally, the MD modeling shows that two Ca(2+) ions are able to interact with two molecules of O-phospho-l-serine. These data suggest that Ca(2+) ions may be directly involved in PKC binding to phosphatidylserine, an acidic lipid located exclusively on the cytoplasmic face of membranes, that is required for PKC activation

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

Evidence of ζ Protein Kinase C Involvement in Polymorphonuclear Neutrophil Integrin-dependent Adhesion and Chemotaxis

Classical chemoattractants and chemokines trigger integrin-dependent adhesion of blood leukocytes to vascular endothelium and also direct subsequent extravasation and migration into tissues. In studies of human polymorphonuclear neutrophil responses to formyl peptides and to interleukin 8, we show evidence of involvement of the atypical zeta protein kinase C in the signaling pathway leading to chemoattractant-triggered actin assembly, integrin-dependent adhesion, and chemotaxis. Selective inhibitors of classical and novel protein kinase C isozymes do not prevent chemoattractant-induced neutrophil adhesion and chemotaxis. In contrast, chelerythrine chloride and synthetic myristoylated peptides with sequences based on the endogenous zeta protein kinase C pseudosubstrate region block agonist-induced adhesion to fibrinogen, chemotaxis and F-actin accumulation. Biochemical analysis shows that chemoattractants trigger rapid translocation of zeta protein kinase C to the plasma membrane accompanied by rapid but transient increase of the kinase activity. Moreover, pretreatment with C3 transferase, a specific inhibitor of Rho small GTPases, blocks zeta but not alpha protein kinase C plasma membrane translocation. Synthetic peptides from zeta protein kinase C also inhibit phorbol ester-induced integrin-dependent adhesion but not NADPH-oxidase activation, and C3 transferase pretreatment blocks phorbol ester-triggered translocation of zeta but not alpha protein kinase C. These data suggest the involvement of zeta protein kinase C in chemoattractant-induced leukocyte integrin-dependent adhesion and chemotaxis. Moreover, they highlight a potential link between atypical protein kinase C isozymes and Rho signaling pathways leading to integrin-activation

Characterization of the binding and phosphorylation of cardiac calsequestrin by ɛ protein kinase C

AbstractIn this study, we report the cloning of the rat cardiac isoform of calsequestrin on the basis of its interaction with an ɛprotein kinase C-unique sequence (ɛV1) derived from the ɛprotein kinase C regulatory domain. Calsequestrin binds activated ɛprotein kinase C holoenzyme better than the inactive enzyme and nearly three times better than other protein kinase C isozymes. The interaction between ɛprotein kinase C and calsequestrin is mediated by sequences in both the regulatory and kinase domains of the ɛprotein kinase C. Finally, we show that calsequestrin is an ɛprotein kinase C substrate in vitro and protein kinase C phosphorylation of calsequestrin leads to a decreased binding of ɛprotein kinase C to calsequestrin

Protein Quality Control Disruption by PKCβII in Heart Failure; Rescue by the Selective PKCβII Inhibitor, βIIV5-3

Myocardial remodeling and heart failure (HF) are common sequelae of many forms of cardiovascular disease and a leading cause of mortality worldwide. Accumulation of damaged cardiac proteins in heart failure has been described. However, how protein quality control (PQC) is regulated and its contribution to HF development are not known. Here, we describe a novel role for activated protein kinase C isoform βII (PKCβII) in disrupting PQC. We show that active PKCβII directly phosphorylated the proteasome and inhibited proteasomal activity in vitro and in cultured neonatal cardiomyocytes. Importantly, inhibition of PKCβII, using a selective PKCβII peptide inhibitor (βIIV5-3), improved proteasomal activity and conferred protection in cultured neonatal cardiomyocytes. We also show that sustained inhibition of PKCβII increased proteasomal activity, decreased accumulation of damaged and misfolded proteins and increased animal survival in two rat models of HF. Interestingly, βIIV5-3-mediated protection was blunted by sustained proteasomal inhibition in HF. Finally, increased cardiac PKCβII activity and accumulation of misfolded proteins associated with decreased proteasomal function were found also in remodeled and failing human hearts, indicating a potential clinical relevance of our findings. Together, our data highlights PKCβII as a novel inhibitor of proteasomal function. PQC disruption by increased PKCβII activity in vivo appears to contribute to the pathophysiology of heart failure, suggesting that PKCβII inhibition may benefit patients with heart failure. (218 words

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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10.1124/mol.64.2.202Shaqura, M.A., Zollner, C., Mousa, S.A., Stein, C., Schafer, M., Characterization of mu Opioid Receptor Binding and G Protein Coupling in Rat Hypothalamus, Spinal Cord, and Primary Afferent Neurons during Inflammatory Pain (2004) Journal of Pharmacology and Experimental Therapeutics, 308 (2), pp. 712-718. , DOI 10.1124/jpet.103.057257Antonijevic, I., Mousa, S.A., Schafer, M., Stein, C., Perineurial defect and peripheral opioid analgesia in inflammation (1995) J Neurosci, 15, pp. 165-172Mousa, S.A., Zhang, Q., Sitte, N., Ji, R.-R., Stein, C., beta-endorphin-containing memory-cells and mu-opioid receptors undergo transport to peripheral inflamed tissue (2001) Journal of Neuroimmunology, 115 (1-2), pp. 71-78. , DOI 10.1016/S0165-5728(01)00271-5, PII S0165572801002715Konno, K., Picolo, G., Gutierrez, V.P., Brigatte, P., Zambelli, V.O., Crotalphine, a novel potent analgesic peptide from the venom of the South American rattlesnake Crotalus durissus terrificus (2008) 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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

14-3-3 proteins. a highly conserved, widespread family of eukaryotic proteins

A family of proteins known as 14-3-3 is currently receiving increased attention by investigators studying a broad range of biological systems, including plants and invertebrates. The outstanding feature of this family is the extraordinarily high sequence conservation observed. Current thinking indicates that these proteins may function as regulators in signal tranduction/phosphorylation mechanisms

Immunoglobulin Y for Potential Diagnostic and Therapeutic Applications in Infectious Diseases

Antiviral, antibacterial, and antiparasitic drugs and vaccines are essential to maintaining the health of humans and animals. Yet, their production can be slow and expensive, and efficacy lost once pathogens mount resistance. Chicken immunoglobulin Y (IgY) is a highly conserved homolog of human immunoglobulin G (IgG) that has shown benefits and a favorable safety profile, primarily in animal models of human infectious diseases. IgY is fast-acting, easy to produce, and low cost. IgY antibodies can readily be generated in large quantities with minimal environmental harm or infrastructure investment by using egg-laying hens. We summarize a variety of IgY uses, focusing on their potential for the detection, prevention, and treatment of human and animal infections

Correcting Glucose-6-Phosphate Dehydrogenase Deficiency with a Small-Molecule Activator

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common human genetic enzymopathies, is caused by over 160 different point mutations and contributes to the severity of many acute and chronic diseases associated with oxidative stress, including hemolytic anemia and bilirubin-induced neurological damage particularly in newborns. As no medications are available to treat G6PD deficiency, here we seek to identify a small molecule that corrects it. Crystallographic study and mutagenesis analysis identify the structural and functional defect of one common mutant (Canton, R459L). Using high-throughput screening, we subsequently identify AG1, a small molecule that increases the activity of the wild-type, the Canton mutant and several other common G6PD mutants. AG1 reduces oxidative stress in cells and zebrafish. Furthermore, AG1 decreases chloroquine- or diamide-induced oxidative stress in human erythrocytes. Our study suggests that a pharmacological agent, of which AG1 may be a lead, will likely alleviate the challenges associated with G6PD deficiency