A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation—measured by changes in DAPI uptake rate—was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

C-Jun N-terminal kinase post-translational regulation of pain-related Acid-Sensing Ion Channels 1b and 3

Neuronal proton-gated Acid-Sensing Ion Channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathological diseases. Such conditions often involve in parallel the activation of various signaling pathways such as the Mitogen Activated Protein Kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal Kinase (JNK), as a new post-translational positive regulator of ASIC channels in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit and species dependent manner, targeting both rat and human ASIC1b and ASIC3 subunits but only mouse ASIC1b subunit. The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost upon mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNFα. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons, and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.

Molecular Characterization and Phylogenetic Analysis of MADS-Box Gene VroAGL11 Associated with Stenospermocarpic Seedlessness in Muscadine Grapes

Reduced expression of MADS-box gene AGAMOUS-LIKE11 (VviAGL11) is responsible for stenospermocarpic seedlessness in bunch grapes. This study is aimed to characterize the VviAGL11 orthologous gene (VroAGL11) in native muscadine grapes (Vitis rotundifolia) at the molecular level and analyze its divergence from other plants. The VroAGL11 transcripts were found in all muscadine cultivars tested and highly expressed in berries while barely detectable in leaves. RT-PCR and sequencing of predicted ORFs from diverse grape species showed that AGL11 transcripts were conservatively spliced. The encoded VroAGL11 protein contains highly conserved MADS-MEF2-like domain, MADS domain, K box, putative phosphorylation site and two sumoylation motifs. The muscadine VroAGL11 proteins are almost identical (99%) to that of seeded bunch cultivar, Chardonnay, except in one amino acid (A79G), but differs from mutant protein of seedless bunch grape, Sultanina, in two amino acids, R197L and T210A. Phylogenetic analysis showed that AGL11 gene of muscadine and other Vitis species formed a separate clade than that of other eudicots and monocots. Muscadine grape cultivar “Jane Bell” containing the highest percentage of seed content in berry (7.2% of berry weight) had the highest VroAGL11 expression, but almost none to nominal expression in seedless cultivars Fry Seedless (muscadine) and Reliance Seedless (bunch). These findings suggest that VroAGL11 gene controls the seed morphogenesis in muscadine grapes like in bunch grape and can be manipulated to induce stenospermocarpic seedlessness using gene editing technology.

Phosphorylation of a chronic pain mutation in the voltage-gated sodium channel Nav1.7 increases voltage sensitivity

Mutations in voltage-gated sodium channels (Navs) can cause alterations in pain sensation, such as chronic pain diseases like inherited erythromelalgia (IEM). The IEM-causing mutation Nav1.7 p.I848T is known to induce a hyperpolarized shift in the voltage dependence of activation in Nav1.7. So far, however, the mechanism to explain this increase in voltage sensitivity remains unknown. In the present study, we show that phosphorylation of the newly introduced Thr residue explains the functional change. We expressed either wild type human Nav1.7, the I848T mutant, or other mutations in HEK293T cells and performed whole-cell patch-clamp electrophysiology. As the insertion of a Thr residue potentially creates a novel phosphorylation site for Ser/Thr kinases and because Nav1.7 had been shown in Xenopus oocytes to be affected by protein kinases C (PKC) and A (PKA), we used different non-selective and selective kinase inhibitors and activators to test the effect of phosphorylation on Nav1.7 in a human system. We identify PKC, but not PKA, to be responsible for the phosphorylation of T848 and thereby for the shift in voltage sensitivity. Introducing a negatively charged amino acid instead of the putative phosphorylation site mimics the effect on voltage gating to a lesser extent. 3D modelling using the published cryo-EM structure of human Nav1.7 showed that introduction of this negatively charged site seems to alter the interaction of this residue with surrounding amino acids and thus to influence channel function. These results could provide new opportunities for the development of novel treatment options for chronic pain patients.

Evidences of a Direct Relationship between Cellular Fuel Supply and Ciliogenesis Regulated by Hypoxic VDAC1-ΔC

Metabolic flexibility is the ability of a cell to adapt its metabolism to changes in its surrounding environment. Such adaptability, combined with apoptosis resistance provides cancer cells with a survival advantage. Mitochondrial voltage-dependent anion channel 1 (VDAC1) has been defined as a metabolic checkpoint at the crossroad of these two processes. Here, we show that the hypoxia-induced cleaved form of VDAC1 (VDAC1-ΔC) is implicated in both the up-regulation of glycolysis and the mitochondrial respiration. We demonstrate that VDAC1-ΔC, due to the loss of the putative phosphorylation site at serine 215, concomitantly with the loss of interaction with tubulin and microtubules, reprograms the cell to utilize more metabolites, favoring cell growth in hypoxic microenvironment. We further found that VDAC1-ΔC represses ciliogenesis and thus participates in ciliopathy, a group of genetic disorders involving dysfunctional primary cilium. Cancer, although not representing a ciliopathy, is tightly linked to cilia. Moreover, we highlight, for the first time, a direct relationship between the cilium and cancer cell metabolism. Our study provides the first new comprehensive molecular-level model centered on VDAC1-ΔC integrating metabolic flexibility, ciliogenesis, and enhanced survival in a hypoxic microenvironment.

Requirement for Serine-384 in Caspase-2 processing and activity

Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.

Distinct roles for extracellular and intracellular domains in neuroligin function at inhibitory synapses

Neuroligins (NLGNs) are postsynaptic cell adhesion molecules that interact trans-synaptically with neurexins to mediate synapse development and function. NLGN2 is only at inhibitory synapses while NLGN3 is at both excitatory and inhibitory synapses. We found that NLGN3 function at inhibitory synapses in rat CA1 depends on the presence of NLGN2 and identified a domain in the extracellular region that accounted for this functional difference between NLGN2 and 3 specifically at inhibitory synapses. We further show that the presence of a cytoplasmic tail (c-tail) is indispensible, and identified two domains in the c-tail that are necessary for NLGN function at inhibitory synapses. These domains point to a gephyrin-dependent mechanism that is disrupted by an autism-associated mutation at R705 and a gephyrin-independent mechanism reliant on a putative phosphorylation site at S714. Our work highlights unique and separate roles for the extracellular and intracellular regions in specifying and carrying out NLGN function respectively.

A fragmented alignment method detects a putative phosphorylation site and a putative BRC repeat in the Drosophila melanogaster BRCA2 protein

Mutations in the BRCA2 tumor suppressor protein leave individuals susceptible to breast, ovarian and other cancers. The BRCA2 protein is a critical component of the DNA repair pathways in eukaryotes, and also plays an integral role in fostering genomic variability through meiotic recombination. Although present in many eukaryotes, as a whole the BRCA2 gene is weakly conserved. Conserved fragments of 30 amino acids (BRC repeats), which mediate interactions with the recombinase RAD51, helped detect orthologs of this protein in other organisms. The carboxy-terminal of the human BRCA2 has been shown to be phosphorylated by checkpoint kinases (Chk1/Chk2) at T3387, which regulate the sequestration of RAD51 on DNA damage. However, apart from three BRC repeats, the Drosophila melanogaster gene has not been annotated and associated with other functionally relevant sequence fragments in human BRCA2. In the current work, the carboxy-terminal phosphorylation threonine site (E=9.1e-4) and a new BRC repeat (E=17e-4) in D. melanogaster has been identified, using a fragmented alignment methodology (FRAGAL). In a similar study, FRAGAL has also identified a novel half-a- tetratricopeptide (HAT) motif (E=11e-4), a helical repeat motif implicated in various aspects of RNA metabolism, in Utp6 from yeast. The characteristic three aromatic residues with conserved spacing are observed in this new HAT repeat, further strengthening my claim. The reference and target sequences are sliced into overlapping fragments of equal parameterized lengths. All pairs of fragments in the reference and target proteins are aligned, and the gap penalties are adjusted to discourage gaps in the middle of the alignment. The results of the best matches are sorted based on differing criteria to aid the detection of known and putative sequences. The source code for FRAGAL results on these sequences is available at https://github.com/sanchak/FragalCode, while the database can be accessed at www.sanchak.com/fragal.html.

A Single Amino Acid Substitution in the Renal Betaine/GABA Transporter Prevents Trafficking to the Plasma Membrane

One response to hypertonic stress in the renal medulla and MDCK cells is the upregulation of betaine transporter (BGT1) synthesis, followed by trafficking to the plasma membrane (PM) and an increase in betaine transport. Upregulation of BGT1 was enhanced by inhibitors of phosphatases PP1 and PP2A and was attenuated by inhibitors of protein kinase C, suggesting an important role for phosphorylation reactions. This was tested using mutants of BGT1 tagged with EGFP. The PM trafficking motifs of BGT1 reside near the C terminus, and truncation at lysine560 resulted in a protein that remained intracellular during hypertonic stress. This K560Δ mutant colocalized with endoplasmic reticulum (ER). Substitution of alanine at Thr40, a putative phosphorylation site, also prevented trafficking to the PM during hypertonic stress. Live-cell imaging showed that T40A was not retained in the ER and colocalized with markers for Golgi and endosomes. In contrast, substitution of aspartate or glutamate at Thr40, to mimic phosphorylation, restored normal trafficking to the PM. HEK293 cells transfected with K560Δ or T40A mutants had 10% of the GABA transport activity of native BGT1, but normal transport activity was restored in cells expressing T40E. Normal BGT1 trafficking likely requires phosphorylation at Thr40 in addition to C-terminal motifs.