Structural insight with mutational impact on tyrosinase and PKC-ß interaction from Homo sapiens: Molecular modeling and docking studies for melanogenesis, albinism and increased risk for melanoma.

Human tyrosinase, is an important protein for biosynthetic pathway of melanin. It was studied to be phosphorylated and activated by protein kinase-C, ß-subunit (PKC-ß) through earlier experimentations with in vivo evidences. Documentation documents that mutation in two essentially vital serine residues in C-terminal end of tyrosinase leads to albinism. Due to the deficiency of protective shield like enzyme; melanin, albinos are at an increased peril for melanoma and other skin cancers. So, computational and residue-level insight including a mutational exploration with evolutionary importance into this mechanism lies obligatory for future pathological and therapeutic developments. Therefore, functional tertiary models of the relevant proteins were analyzed after satisfying their stereo-chemical features. Evolutionarily paramount residues for the activation of tyrosinase were perceived via multiple sequence alignment phenomena. Mutant-type tyrosinase protein (S98A and S102A) was thereby modeled, maintaining the wild-type proteins' functionality. Furthermore, this present comparative study discloses the variation in the stable residual participation (for mutant-type and wild-type tyrosinase-PKCß complex). Mainly, an increased number of polar negatively charged residues from the wild-type tyrosinase participated with PKC-ß, predominantly. Fascinatingly supported by evaluation of statistical significances, mutation even led to a destabilizing impact in tyrosinase accompanied by conformational switches with a helix-to-coil transition in the mutated protein. Even the allosteric sites in the protein got poorly hampered upon mutation leading to weaker tendency for binding partners to interact.

Intramolecular C2 Domain-Mediated Autoinhibition of Protein Kinase C ßII.

The signaling output of protein kinase C (PKC) is exquisitely controlled, with its disruption resulting in pathophysiologies. Identifying the structural basis for autoinhibition is central to developing effective therapies for cancer, where PKC activity needs to be enhanced, or neurodegenerative diseases, where PKC activity should be inhibited. Here, we reinterpret a previously reported crystal structure of PKCßII and use docking and functional analysis to propose an alternative structure that is consistent with previous literature on PKC regulation. Mutagenesis of predicted contact residues establishes that the Ca(2+)-sensing C2 domain interacts intramolecularly with the kinase domain and the carboxyl-terminal tail, locking PKC in an inactive conformation. Ca(2+)-dependent bridging of the C2 domain to membranes provides the first step in activating PKC via conformational selection. Although the placement of the C1 domains remains to be determined, elucidation of the structural basis for autoinhibition of PKCßII unveils a unique direction for therapeutically targeting PKC.

Molecular dynamics approach to probe PKCßII-ligand interactions and influence of crystal water molecules on these interactions.

OBJECTIVE: PKCßII is a potential target for therapeutic intervention against pandemic diabetic complications. Present study probes the molecular interactions of PKCßII with its clinically important ligands, viz. ruboxistaurin, enzastaurin and co-crystallized ligand, 2-methyl-1H-indol-3-yl-BIM-1. RESEARCH DESIGN AND METHODS: The essentials of PKCßII-ligand interaction, crystal water-induced alterations in these interactions and key interacting flexible residues are analyzed. Computational methodologies, viz. molecular docking and molecular simulation coupled with molecular mechanics-Poisson-Boltzmann surface area and generalized born surface area (MM-PB[GB]SA) are employed. RESULTS: The structural changes in the presence and absence of crystal water molecules in PKCßII ATP binding site residues, and its interaction with bound ligand, are identified. Difference in interaction of selective and nonselective ligand with ATP binding site residues of PKCßII is reported. CONCLUSIONS: The study showed that the nonbonding interactions contribute significantly in PKCßII-ligand binding and presence of crystal water molecules affects the interactions. The findings of present work may integrate the new aspects in the drug design process of PKCßII inhibitors.

HDAC inhibition through valproic acid modulates the methylation profiles in human embryonic kidney cells.

Post-translational modifications on the tails of core and linker histones dictate transcription and have vital roles in disease and development. Acetylation and deacetylation events enabled by histone acetyl transferases and histone deacetylases (HDACs) on the chromatin milieu are intricately involved in gene regulation. Inhibition of HDACs is emerging as a powerful strategy in regenerative therapy, transplantation, development and in nuclear reprogramming events. Valproic acid (VPA), belonging to the short-chain fatty acid group of HDAC inhibitors, modulates the epigenome altering gene expression profiles across cell lines. This work attempts to explore the methylation profiles triggered by VPA treatment on human embryonic kidney cells (HEK 293) through a biochemical and computational approach. VPA treatment (for 48 h) has been observed to hypermethylate lysine 4 on the core histone H3 and confers a hypomethylation status of H3 lysine 27 in HEK 293 cells leaving the nuclear area and nuclear contour unaltered. Our structural docking and Binding Free Energy (BFE) calculations establish an active role for VPA in inhibiting the demethylase JARID1A (Jumonji, AT Rich Interactive Domain 1A) and the methyl-transferase EZH2 (Enhancer of Zeste Homologue 2). This work has also proven that VPA can inhibit the activity of proteins like GSK3ß and PKCßII involved in developmental disorders. This work establishes a dynamic correlation between histone methylation events and HDAC inhibition and may define newer epigenetic strategies for treating neurodevelopmental and oncological disorders.

Structural basis of conformational variance in phosphorylated and non-phosphorylated states of PKCßII.

PKCßII activation is achieved by primary phosphorylation at three phosphorylation sites, followed by the addition of secondary messengers for full activation. Phosphorylation is essential for enzyme maturation, and the associated conformational changes are known to modulate the enzyme activation. To probe into the structural basis of conformational changes on phosphorylation of PKCßII, a comprehensive study of the changes in its complexes with ATP and ruboxistaurin was performed. ATP is a phosphorylating agent in its phosphorylation reaction, and ruboxistaurin is its specific inhibitor. This study provides insight into the differences in the important structural features in phosphorylated and non-phosphorylated states of PKCßII. Less conformational changes when PKCßII is bound to inhibitor in comparison to when it is bound to its phosphorylating agent in both states were observed. The interactions of ruboxistaurin significant in restricting PKCßII to attain the conformational state competent for full activation are reported.

Interactions of protein kinase C-α C1A and C1B domains with membranes: a combined computational and experimental study.

Protein kinase C-α (PKCα) has been studied widely as a paradigm for conventional PKCs, with two C1 domains (C1A and C1B) being important for the regulation and function of the kinase. However, it is challenging to explore these domains in membrane-bound environments with either simulations or experiments alone. In this work, we have combined modeling, simulations, and experiments to understand the molecular basis of the PKCα C1A and C1B domain interactions with membranes. Our atomistic simulations of the PKCα C1 domains reveal the dynamic interactions of the proteins with anionic lipids, as well as the conserved hydrogen bonds and the distinct nonpolar contacts formed with lipid activators. Corroborating evidence is obtained from additional simulations and experiments in terms of lipid binding and protein diffusion. Overall, our study, for the first time, explains with atomistic detail how the PKCα C1A and C1B domains interact differently with various lipids. On the molecular level, the information provided by our study helps to shed light on PKCα regulation and activation mechanism. The combined computational/experimental approach demonstrated in this work is anticipated to enable further studies to explore the roles of C1 domains in many signaling proteins and to better understand their molecular mechanisms in normal cellular function and disease development.

Protein kinase C isoforms in atherosclerosis: pro- or anti-inflammatory?

Atherosclerosis is a pathologic condition caused by chronic inflammation in response to lipid deposition in the arterial wall. There are many known contributing factors such as long-term abnormal glucose levels, smoking, hypertension, and hyperlipidemia. Under the influence of such factors, immune and non-immune effectors cells are activated and participate during the progression of atherosclerosis. Protein kinase C (PKC) family isoforms are key players in the signal transduction pathways of cellular activation and have been associated with several aspects of the atherosclerotic vascular disease. This review article summarizes the current knowledge of PKC isoforms functions during atherogenesis, and addresses differential roles and disputable observations of PKC isoforms. Among PKC isoforms, both PKCß and PKCδ are the most attractive and potential therapeutic targets. This commentary discusses in detail the outcomes and current status of clinical trials on PKCß and PKCδ inhibitors in atherosclerosis-associated disorders like diabetes and myocardial infarction. The risk and benefit of these inhibitors for clinical purposes will be also discussed. This review summarizes what is already being done and what else needs to be done in further targeting PKC isoforms, especially PKCß and PKCδ, for therapy of atherosclerosis and atherosclerosis-associated vasculopathies in the future.

Scaffold hopping for identification of novel PKCßII inhibitors based on ligand and structural approaches, virtual screening and molecular dynamics study.

Protein Kinase C ßII (PKCßII) overexpression has been linked to various diabetic microvascular complications viz. retinopathy, neuropathy, and cardiomyopathy. Novel and potent small molecules with preferential selective inhibitory property of PKCßII will be helpful in treatment as well as understanding insight of PKCßII involvement in these complications. Robust 3D hypotheses were developed using both the crystal structure and available PKCßII ligands, and were validated by feature mapping and screening in-house database of reported PKCßII compounds. The best hypothesis from both methods consists of six features viz. one hydrogen bond donor (D), two hydrogen bond acceptors (A1, A2), two hydrophobic-aromatics (H1, H2) and one ring aromatic (R). A synergistic approach of virtual screening using both ligand and receptor based pharmacophore model was used for the flexible search of ligands from chemical databases. The hits obtained were screened by molecular docking and their binding affinity was predicted using MMPBSA calculations. The first receptor based query of PKCßII and new scaffold of its inhibitors with good estimated activities, favorable binding interactions, and high docking score were identified.

Hyperglycaemia promotes cerebral barrier dysfunction through activation of protein kinase C-ß.

AIM: To examine whether protein kinase C (PKC) and associated downstream mechanisms are involved in hyperglycaemia (HG)-evoked blood-brain barrier (BBB) damage. METHODS: The activities of total PKC (Peptag assay), NADPH oxidase (lucigenin assay) and matrix metalloproteinase-2 (MMP-2; gelatin zymography) were measured in human brain microvascular endothelial cells (HBMEC) exposed to normoglycaemia (5.5 mM) or HG (25 mM) using the specific assays indicated in parentheses. The integrity and function of the in vitro models of human BBB were assessed by measurements of transendothelial electrical resistance and paracellular flux of permeability markers, respectively. Occludin protein expression was studied by immunoblotting. RESULTS: HG significantly compromised the BBB integrity and enhanced total PKC activity to which increases in PKC-ß and PKC-ßII isoforms contributed the most. Elevations in NADPH oxidase and MMP-2 activities and decreases in occludin levels contributed to barrier dysfunction. Selective inhibition of PKC-ß isoform prevented the changes observed in occludin expression and the aforementioned enzyme activities and thus effectively preserved barrier integrity. Similarly, apocynin, a specific NADPH oxidase inhibitor, also effectively neutralized the effects of HG on barrier integrity, MMP-2 activity, occludin expression and PKC-ß activity. CONCLUSION: HG promotes cerebral-barrier dysfunction through activation of PKC-ß and consequent stimulations of oxidative stress and tight junction dissolution.

Heterozygous mis-sense mutations in Prkcb as a critical determinant of anti-polysaccharide antibody formation.

To identify rate-limiting steps in T cell-independent type 2 antibody production against polysaccharide antigens, we performed a genome-wide screen by immunizing several hundred pedigrees of C57BL/6 mice segregating N-ethyl-N-nitrosurea-induced mis-sense mutations. Two independent mutations, Tilcara and Untied, were isolated that semi-dominantly diminished antibody against polysaccharide but not protein antigens. Both mutations resulted from single-amino-acid substitutions within the kinase domain of protein kinase C-ß (PKCß). In Tilcara, a Ser552>Pro mutation occurred in helix G, in close proximity to a docking site for the inhibitory N-terminal pseudosubstrate domain of the enzyme, resulting in almost complete loss of active, autophosphorylated PKCßI, whereas the amount of alternatively spliced PKCßII protein was not markedly reduced. Circulating B cell subsets were normal and acute responses to B-cell receptor stimulation such as CD25 induction and initiation of DNA synthesis were only measurably diminished in Tilcara homozygotes, whereas the fraction of cells that had divided multiple times was decreased to an intermediate degree in heterozygotes. These results, coupled with evidence of numerous mis-sense PRKCB mutations in the human genome, identify Prkcb as a genetically sensitive step likely to contribute substantially to population variability in anti-polysaccharide antibody levels.