Cyclic AMP induces reversible EPAC1 condensates that regulate histone transcription.

The second messenger cyclic AMP regulates many nuclear processes including transcription, pre-mRNA splicing and mitosis. While most functions are attributed to protein kinase A, accumulating evidence suggests that not all nuclear cyclic AMP-dependent effects are mediated by this kinase, implying that other effectors may be involved. Here we explore the nuclear roles of Exchange Protein Activated by cyclic AMP 1. We find that it enters the nucleus where forms reversible biomolecular condensates in response to cyclic AMP. This phenomenon depends on intrinsically disordered regions present at its amino-terminus and is independent of protein kinase A. Finally, we demonstrate that nuclear Exchange Protein Activated by cyclic AMP 1 condensates assemble at genomic loci on chromosome 6 in the proximity of Histone Locus Bodies and promote the transcription of a histone gene cluster. Collectively, our data reveal an unexpected mechanism through which cyclic AMP contributes to nuclear spatial compartmentalization and promotes the transcription of specific genes.

Alpha-melanocyte stimulating hormone (α-MSH): biology, clinical relevance and implication in melanoma.

Alpha-melanocyte stimulating hormone (α-MSH) and its receptor, melanocortin 1 receptor (MC1R), have been proposed as potential target for anti-cancer strategies in melanoma research, due to their tissue specific expression and involvement in melanocyte homeostasis. However, their role in prevention and treatment of melanoma is still debated and controversial. Although a large body of evidence supports α-MSH in preventing melanoma development, some preclinical findings suggest that the α-MSH downstream signalling may promote immune escape and cancer resistance to therapy. Additionally, in metastatic melanoma both MC1R and α-MSH have been reported to be overexpressed at levels much higher than normal cells. Furthermore, targeted therapy (e.g. BRAF inhibition in BRAFV600E mutant tumours) has been shown to enhance this phenomenon. Collectively, these data suggest that targeting MC1R could serve as an approach in the treatment of metastatic melanoma. In this review, we explore the molecular biology of α-MSH with particular emphasis into its tumor-related properties, whilst elaborating the experimental evidence currently available regarding the interplay between α-MSH/MC1R axis, melanoma and antitumor strategies.

The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain.

AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine331 and serine335, located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser335 is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser335 was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser335 in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis.

cGMP signals modulate cAMP levels in a compartment-specific manner to regulate catecholamine-dependent signaling in cardiac myocytes.

RATIONALE: cAMP and cGMP are intracellular second messengers involved in heart pathophysiology. cGMP can potentially affect cAMP signals via cGMP-regulated phosphodiesterases (PDEs). OBJECTIVE: To study the effect of cGMP signals on the local cAMP response to catecholamines in specific subcellular compartments. METHODS AND RESULTS: We used real-time FRET imaging of living rat ventriculocytes expressing targeted cAMP and cGMP biosensors to detect cyclic nucleotides levels in specific locales. We found that the compartmentalized, but not the global, cAMP response to isoproterenol is profoundly affected by cGMP signals. The effect of cGMP is to increase cAMP levels in the compartment where the protein kinase (PK)A-RI isoforms reside but to decrease cAMP in the compartment where the PKA-RII isoforms reside. These opposing effects are determined by the cGMP-regulated PDEs, namely PDE2 and PDE3, with the local activity of these PDEs being critically important. The cGMP-mediated modulation of cAMP also affects the phosphorylation of PKA targets and myocyte contractility. CONCLUSIONS: cGMP signals exert opposing effects on local cAMP levels via different PDEs the activity of which is exerted in spatially distinct subcellular domains. Inhibition of PDE2 selectively abolishes the negative effects of cGMP on cAMP and may have therapeutic potential.

A semi-immobilizing system associated with microspectrofluorimetric and videoimaging analysis for intracytoplasmic calcium measurement in individual viable spermatozoa.

We describe a fast and simple method to monitor variations of intracytoplasmic calcium concentrations in very small and motile cells such as mammalian spermatozoa during measurement time. The method combines a procedure of sperm cells semi-immobilization with microspectrofluorimetric measurement of intracytoplasmic calcium concentrations supported by videoimaging that allows also a constant monitoring of viability during the time of calcium recording. In this paper we show that a semi-immobilization of individual viable spermatozoa can be obtained preparing the agar matrix in a two step procedure. The cell immobilizing system proposed, associated with the microspectrofluorimetric analysis supported by videoimaging, is a simple, rapid and useful tool for those studies having the goal of correlating the presence and cellular distribution of ion channels with their functional status and their response to physiologic and/or pharmacological molecules.

In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects.

BACKGROUND: Abnormalities of pre-mRNA splicing are increasingly recognized as an important mechanism through which gene mutations cause disease. However, apart from the mutations in the donor and acceptor sites, the effects on splicing of other sequence variations are difficult to predict. Loosely defined exonic and intronic sequences have been shown to affect splicing efficiency by means of silencing and enhancement mechanisms. Thus, nucleotide substitutions in these sequences can induce aberrant splicing. Web-based resources have recently been developed to facilitate the identification of nucleotide changes that could alter splicing. However, computer predictions do not always correlate with in vivo splicing defects. The issue of unclassified variants in cancer predisposing genes is very important both for the correct ascertainment of cancer risk and for the understanding of the basic mechanisms of cancer gene function and regulation. Therefore we aimed to verify how predictions that can be drawn from in silico analysis correlate with results obtained in an in vivo splicing assay. RESULTS: We analysed 99 hMLH1 and hMSH2 missense mutations with six different algorithms. Transfection of three different cell lines with 20 missense mutations, showed that a minority of them lead to defective splicing. Moreover, we observed that some exons and some mutations show cell-specific differences in the frequency of exon inclusion. CONCLUSION: Our results suggest that the available algorithms, while potentially helpful in identifying splicing modulators especially when they are located in weakly defined exons, do not always correspond to an obvious modification of the splicing pattern. Thus caution must be used in assessing the pathogenicity of a missense or silent mutation with prediction programs. The variations observed in the splicing proficiency in three different cell lines suggest that nucleotide changes may dictate alternative splice site selection in a tissue-specific manner contributing to the widely observed phenotypic variability in inherited cancers.