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Protein kinase M zeta, PKMζ, is a brain enriched kinase with a well characterized role in Long-Term Potentiation (LTP), the activity-dependent strengthening of synapses involved in long-term memory formation. However, little is known about the molecular mechanisms that maintain the tissue specificity of this kinase. Here, we characterized the epigenetic factors, mainly DNA methylation, regulating PKMζ expression in the human brain. The PRKCZ gene has an upstream promoter regulating Protein kinase C ζ (PKCζ), and an internal promoter driving PKMζ expression. A demethylated region, including a canonical CREB binding site, situated at the internal promoter was only observed in human CNS tissues. The induction of site-specific hypermethylation of this region resulted in decreased CREB1 binding and downregulation of PKMζ expression. Noteworthy, CREB binding sites were absent in the upstream promoter of PRKCZ locus, suggesting a specific mechanism for regulating PKMζ expression. These observations were validated using a system of human neuronal differentiation from induced pluripotent stem cells (iPSCs). CREB1 binding at the internal promoter was detected only in differentiated neurons, where PKMζ is expressed. The same epigenetic mechanism in the context of CREB binding site was identified in other genes involved in neuronal differentiation and LTP. Additionally, aberrant DNA hypermethylation at the internal promoter was observed in cases of Alzheimer's disease, correlating with decreased expression of PKMζ in patient brains. Altogether, we present a conserved epigenetic mechanism regulating PKMζ expression and other genes enhanced in the CNS with possible implications in neuronal differentiation and Alzheimer's disease., Competing Interests: Declaration of competing interest The authors no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Chronic pain is a major health issue, and the search for new analgesics has become increasingly important because of the addictive properties and unwanted side effects of opioids. To explore potentially new drug targets, we investigated mutations in the NTRK1 gene found in individuals with congenital insensitivity to pain with anhidrosis (CIPA). NTRK1 encodes tropomyosin receptor kinase A (TrkA), the receptor for nerve growth factor (NGF) and that contributes to nociception. Molecular modeling and biochemical analysis identified mutations that decreased the interaction between TrkA and one of its substrates and signaling effectors, phospholipase Cγ (PLCγ). We developed a cell-permeable phosphopeptide derived from TrkA (TAT-pQYP) that bound the Src homology domain 2 (SH2) of PLCγ. In HEK-293T cells, TAT-pQYP inhibited the binding of heterologously expressed TrkA to PLCγ and decreased NGF-induced, TrkA-mediated PLCγ activation and signaling. In mice, intraplantar administration of TAT-pQYP decreased mechanical sensitivity in an inflammatory pain model, suggesting that targeting this interaction may be analgesic. The findings demonstrate a strategy to identify new targets for pain relief by analyzing the signaling pathways that are perturbed in CIPA.

It is well accepted that treatment of chronic pain with morphine leads to μ opioid receptor (MOR) desensitization and the development of morphine tolerance. MOR activation by the selective peptide agonist, D-Ala2, N-MePhe4, Gly-ol]-enkephalin(DAMGO), leads to robust G protein receptor kinase activation, β-arrestin recruitment, and subsequent receptor endocytosis, which does not occur in an activation by morphine. However, MOR activation by morphine induces receptor desensitization, in a Protein kinase C (PKC) dependent manner. PKC inhibitors have been reported to decrease receptor desensitization, reduce opiate tolerance, and increase analgesia. However, the exact role of PKC in these processes is not clearly delineated. The difficulties in establishing a particular role for PKC have been, in part, due to the lack of reagents that allow the selective identification of PKC targets. Recently, we generated a conformation state-specific anti-PKC antibody that preferentially recognizes the active state of this kinase. Using this antibody to selectively isolate PKC substrates and a proteomics strategy to establish the identity of the proteins, we examined the effect of morphine treatment on the PKC targets. We found an enhanced interaction of a number of proteins with active PKC, in the presence of morphine. In this article, we discuss the role of these proteins in PKC-mediated MOR desensitization and analgesia. In addition, we posit a role for some of these proteins in mediating pain by TrKA activation, via the activation of transient receptor potential cation channel subfamily V member 1 (TRPV1). Finally, we discuss how these new PKC interacting proteins and pathways could be targeted for the treatment of pain., Competing Interests: The authors declare no conflict of interest.

Epigenetic dysregulation is an important emerging hallmark of cutaneous melanoma development. The global loss of DNA methylation in gene-poor regions and transposable DNA elements of cancer cells contributes to increased genomic instability. Long interspersed element-1 (LINE-1) sequences are the most abundant repetitive sequence of the genome and can be evaluated as a surrogate marker of the global level of DNA methylation. In this work, LINE-1 methylation levels were evaluated in cutaneous melanomas and normal melanocyte primary cell cultures to investigate their possible association with both distinct clinicopathological characteristics and tumor mutational profile. A set of driver mutations frequently identified in cutaneous melanoma was assessed by sequencing (actionable mutations in BRAF, NRAS, and KIT genes, and mutations affecting the TER T promoter) or multiplex ligation-dependent probe amplification (MLPA) (CDKN2A deletions). Pyrosequencing was performed to investigate the methylation level of LINE-1 and CDKN2A promoter sequences. The qualitative analysis showed a trend toward an association between LINE-1 hypomethylation and CDKN2A inactivation (p=0.05). In a quantitative approach, primary tumors, mainly the thicker ones (>4 mm), exhibited a trend toward LINE-1 hypomethylation when compared with control melanocytes. To date, this is the first study reporting in cutaneous melanomas a possible link between the dysregulation of LINE-1 methylation and the presence of driver mutations., (Copyright © 2016 American Federation for Medical Research.)

Despite the efforts of pharmaceutical companies to develop specific kinase modulators, few drugs targeting kinases have been completely successful in the clinic. This is primarily due to the conserved nature of kinases, especially in the catalytic domains. Consequently, many currently available inhibitors lack sufficient selectivity for effective clinical application. Kinases phosphorylate their substrates to modulate their activity. One of the important steps in the catalytic reaction of protein phosphorylation is the correct positioning of the target residue within the catalytic site. This positioning is mediated by several regions in the substrate binding site, which is typically a shallow crevice that has critical subpockets that anchor and orient the substrate. The structural characterization of this protein-protein interaction can aid in the elucidation of the roles of distinct kinases in different cellular processes, the identification of substrates, and the development of specific inhibitors. Because the region of the substrate that is recognized by the kinase can be part of a linear consensus motif or a nonlinear motif, advances in technology beyond simple linear sequence scanning for consensus motifs were needed. Cost-effective bioinformatics tools are already frequently used to predict kinase-substrate interactions for linear consensus motifs, and new tools based on the structural data of these interactions improve the accuracy of these predictions and enable the identification of phosphorylation sites within nonlinear motifs. In this Review, we revisit kinase-substrate interactions and discuss the various approaches that can be used to identify them and analyze their binding structures for targeted drug development., (Copyright © 2016, American Association for the Advancement of Science.)

In melanoma development, oncogenic process is mediated by genetic and epigenetic mutations, and few studies have so far explored the role of DNA methylation either as predisposition factor or biomarker. We tested patient samples for germline CDKN2A methylation status and found no evidence of inactivation by promoter hypermethylation. We have also investigated the association of clinical characteristics of samples with the DNA methylation pattern of twelve genes relevant for melanomagenesis. Five genes (BAP1, MGMT, MITF, PALB2, and POT1) presented statistical association between blood DNA methylation levels and either CDKN2A-mutation status, number of lesions, or Breslow thickness. In tumors, five genes (KIT, MGMT, MITF, TERT, and TNF) exhibited methylation levels significantly different between tumor groups including acral compared to nonacral melanomas and matched primary lesions and metastases. Our data pinpoint that the methylation level of eight melanoma-associated genes could potentially represent markers for this disease both in peripheral blood and in tumor samples.