Dynamic Regulation of NMDA and AMPA Receptors by Posttranslational Modifications [Neurobiology]

October 9th, 2015 by Lussier, M. P., Sanz-Clemente, A., Roche, K. W.

Many molecular mechanisms underlie the changes in synaptic glutamate receptor content that are required by neuronal networks to generate cellular correlates of learning and memory. During the last decade, posttranslational modifications have emerged as critical regulators of synaptic transmission and plasticity. Notably, phosphorylation, ubiquitination and palmitoylation control the stability, trafficking and synaptic expression of glutamate receptors in the central nervous system. In the current review, we will summarize some of the progress made by the neuroscience community regarding our understanding of phosphorylation, ubiquitination and palmitoylation of the NMDA and AMPA subtypes of glutamate receptors.

An Unprecedented Combination of Serine and Cysteine Nucleophiles in a Split Intein with an Atypical Split Site [Protein Structure and Folding]

October 9th, 2015 by Bachmann, A.-L., Mootz, H. D.

Protein splicing mediated by inteins is a self-processive reaction leading to the excision of the internal intein domain from a precursor protein and the concomitant ligation of the flanking sequences, the extein-N and extein-C parts, thereby reconstituting the host protein. Most inteins employ a splicing pathway in which the upstream scissile peptide bond is consecutively rearranged into two thioester or oxoester intermediates before intein excision and rearrangement into the new peptide bond occurs. The catalytically critical amino acids involved at the two splice junctions are cysteine, serine or threonine. Notably, the only potential combination not observed so far in any of the known or engineered inteins corresponds to the transesterification from an oxoester to a thioester, which suggested that this formal up-hill reaction with regard to the thermodynamic stability might be incompatible with intein-mediated catalysis. We show that corresponding mutations also led to inactive gp41-1 and AceL-TerL inteins. We report the novel GOS-TerL split intein identified from metagenomic databases as the first intein harboring the combination of Ser1 and Cys+1 residues. Mutational analysis showed that its efficient splicing reaction indeed follows the shift from oxoester to thioester and thus represents a rare diversion from the canonical pathway. Furthermore, the GOS-TerL intein has an atypical split site close to the N-terminus. The IntN fragment could be shortened from 37 aa to 28 aa and exchanged with the 25 aa IntN fragment from the AceL-TerL intein, indicating a high degree of promiscuity of the IntC fragment of the GOS-TerL intein.
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Thematic Mini-Review Series: Molecular Mechanisms of Synaptic Plasticity [Signal Transduction]

October 9th, 2015 by Colbran, R. J.

The human brain contains ~86 billion neurons, which are precisely organized in specific brain regions and nuclei. High fidelity synaptic communication between subsets of neurons in specific circuits is required for most human behaviors, and is often disrupted in neuropsychiatric disorders. The presynaptic axon terminals of one neuron release neurotransmitters that activate receptors on multiple postsynaptic neuron targets to induce electrical and chemical responses. Typically, postsynaptic neurons integrate signals from multiple presynaptic neurons at thousands of synaptic inputs in order to control downstream communication to the next neuron in the circuit. Importantly, the strength (or efficiency) of signal transmission at each synapse can be modulated on time scales ranging up to the lifetime of the organism. This "synaptic plasticity" leads to changes in overall neuronal circuit activity, resulting in behavioral modifications. This series of mini-reviews will focus on recent advances in our understanding of the molecular and cellular mechanisms that control synaptic plasticity.

Interaction of the Jhd2 H3K4 demethylase with chromatin is controlled by histone H2A surfaces and restricted by H2B ubiquitination. [Genomics and Proteomics]

October 8th, 2015 by

Histone H3 lysine 4 (K4) methylation is a dynamic modification. In budding yeast, H3K4 methylation is catalyzed by the Set1-COMPASS methyltransferase complex, and removed by Jhd2, a JMJC-domain family demethylase. The catalytic JmjC and JmjN domains of Jhd2 have the ability to remove all three degrees (mono-, di- and tri-) of H3K4 methylation. Jhd2 also contains a PHD finger required for its chromatin association and H3K4 demethylase functions. The Jhd2 PHD finger associates with chromatin independent of H3K4 methylation and the H3 N-terminal tail. Therefore, how Jhd2 associates with chromatin to perform H3K4 demethylation has remained unknown. We report a novel interaction between the Jhd2 PHD finger and histone H2A. Two residues in H2A (F26 and E57) serve as a binding site for Jhd2 in vitro, and mediate its chromatin association and H3K4 demethylase functions in vivo. Using RNA-seq, we have identified the functional target genes for Jhd2 and the H2A F26 and E57 residues. We demonstrate that H2A F26 and E57 residues control Jhd2's chromatin association and H3K4 demethylase functions during positive or negative regulation of transcription at target genes. Importantly, we show that H2BK123 ubiquitination blocks Jhd2 from accessing its binding site on chromatin, and thereby, we have uncovered a second mechanism by which H2B ubiquitination contributes to the trans-histone regulation of H3K4 methylation. Overall, our study provides novel insights into the chromatin binding dynamics and H3K4 demethylase functions of Jhd2.
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Pseudomonas aeruginosa ExoT induces mitochondrial apoptosis in target host cells in a manner that depends on its GAP domain activity. [Molecular Bases of Disease]

October 8th, 2015 by

Pseudomonas aeruginosa is the most common cause of hospital-acquired pneumonia and a killer of immunocompromised patients. Others and we have demonstrated that the type III Secretion System (T3SS) effector protein ExoT plays a pivotal role in facilitating P. aeruginosa pathogenesis. ExoT possesses an N-terminal GTPase activating protein (GAP) domain and a C-terminal ADP ribosyltransferase (ADPRT) domain. Because it targets multiple non-overlapping cellular targets, ExoT performs several distinct virulence functions for P. aeruginosa, including induction of apoptosis in a variety of target host cells. Both the ADPRT and the GAP domain activities contribute to ExoT-induced apoptosis. The ADPRT domain of ExoT induces atypical anoikis by transforming an innocuous cellular protein, Crk, into a cytotoxin, which interferes with integrin survival signaling. However, the mechanism underlying the GAP-induced apoptosis remains unknown. In this report, we demonstrate that the GAP domain activity is both necessary and sufficient to induce mitochondrial (intrinsic) apoptosis. We show that intoxication with GAP domain results in: (i) JNK1/2 activation; (ii) substantial increases in the mitochondrial levels of activated pro-apoptotic proteins Bax and Bid, and to a lesser extent Bim; (iii) loss of mitochondrial membrane potential and cytochrome c release; and (iv) activation of initiator caspase-9 and executioner caspase-3. Further, GAP-induced apoptosis is partially mediated by JNK1/2 but it is completely dependent on caspase-9 activity. Together, the ADPRT and the GAP domains make ExoT into a highly versatile and potent cytotoxin, capable of inducing multiple forms of apoptosis in target host cells.
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Neuropilin Function as an Essential Cell Surface Receptor [Signal Transduction]

October 8th, 2015 by Guo, H.-F., Vander Kooi, C. W.

The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.

Role of Janus Kinase 3 in Predisposition to Obesity associated Metabolic Syndrome [Signal Transduction]

October 8th, 2015 by Mishra, J., Verma, R. K., Alpini, G., Meng, F., Kumar, N.

Obesity, a worldwide epidemic, is a major risk factor for the development of metabolic syndrome (MetS) including diabetes and associated health complications. Recent studies indicate that chronic low-grade inflammation (CLGI) plays a key role in metabolic deterioration in the obese population. Previously, we reported that Jak3 was essential for mucosal differentiation and enhanced colonic barrier functions and its loss in mice resulted in basal CLGI and predisposition to DSS induced colitis. Since CLGI is associated with diabetes, obesity, and metabolic syndrome, present studies determined the role of Jak3 in development of such conditions. Our data show that loss of Jak3 resulted in increased body weight, basal systemic CLGI, compromised glycemic homeostasis, hyperinsulinemia, and early symptoms of liver steatosis. Lack of Jak3 also resulted in exaggerated symptoms of metabolic syndrome by western high-fat diet. Mechanistically, Jak3 was essential for reduced expression and activation of toll like receptors (TLRs) in murine intestinal mucosa and human intestinal epithelial cells where Jak3 interacted with and activated p85, the regulatory sub-unit of the PI3K, through tyrosine phosphorylation of adapter protein insulin receptor substrate (IRS1). These interactions resulted in activation of PI3K-Akt axis, which was essential for reduced TLR expression and TLR associated NFkB activation. Collectively, these results demonstrate the essential role of Jak3 in promoting mucosal tolerance through suppressed expression and limiting activation of TLRs thereby preventing intestinal and systemic CLGI and associated obesity and MetS.

Polarized Effect of Intracellular Calcium on the Renal Epithelial Sodium Channel Occurs as a Result of Subcellular Calcium Signaling Domains Maintained by Mitochondria. [Cell Biology]

October 8th, 2015 by

The renal epithelial sodium channel (ENaC) provides regulated sodium transport in the distal nephron. The effects of intracellular calcium ([Ca2+]i) on this channel are only beginning to be elucidated. It appears from previous studies that the [Ca2+]i increases downstream of ATP administration may have a polarized effect on ENaC where apical application of ATP and subsequent [Ca2+]i increase has an inhibitory effect on the channel whereas basolateral ATP and [Ca2+]i have a stimulatory effect. We asked if this polarized effect of ATP is in fact reflective of a polarized effect of increased [Ca2+]i on ENaC and what underlying mechanism is responsible. We began by performing patch clamp experiments in which ENaC activity was measured during apical or basolateral application of ionomycin to increase [Ca2+]i near the apical or basolateral membrane, respectively. We found that ENaC does indeed respond to increased [Ca2+]i in a polarized fashion, with apical increases being inhibitory and basolateral stimulating channel activity. In other epithelial cell types, mitochondria sequester [Ca2+]i, creating [Ca2+]i signaling microdomains within the cell that are dependent on mitochondrial localization. We found that mitochondria localize in bands just beneath the apical and basolateral membranes in two different cortical collecting duct principal cell lines and in cortical collecting duct principal cells in mouse kidney tissue. We found that inhibiting mitochondrial [Ca2+]i uptake destroyed the polarized response of ENaC to [Ca2+]i. Overall, our data suggest that ENaC is regulated by [Ca2+]i in a polarized fashion and that this polarization is maintained by mitochondrial [Ca2+]i sequestration.
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The Orphan G Protein-Coupled Receptor Gpr175 (TPRA40) Enhances Hedgehog Signaling by Modulating cAMP Levels [Signal Transduction]

October 8th, 2015 by Singh, J., Wen, X., Scales, S. J.

The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as TPRA1 or TPRA40: transmembrane protein, adipocyte associated 1 or of 40 kDa) that also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway.

Mechanism of assembly of a substrate-transfer complex during tail-anchored protein targeting [Membrane Biology]

October 7th, 2015 by

Tail-anchored (TA) proteins, defined as having a single transmembrane helix at their C-terminus, are post-translationally targeted to the endoplasmic reticulum (ER) membrane by the GET (Guided Entry of TA proteins) pathway. In yeast, the handover of TA substrates is mediated by the heterotetrameric Get4/Get5 (Get4/5) complex, which tethers the co-chaperone Sgt2 to the targeting factor, the Get3 ATPase. Binding of Get4/5 to Get3 is critical for efficient TA targeting; however, questions remain about the formation of the Get3-Get4/5 complex. Here we report crystal structures of a Get3-Get4/5 complex from Saccharomyces cerevisiae (Sc) at 2.8 Å and 6.0 Å, which reveal a novel interface between Get3 and Get4 dominated by electrostatic interactions. Kinetic and mutational analyses strongly suggest that these structures represent an on-pathway intermediate that rapidly assembles and then rearranges to the final Get3-Get4/5 complex. Furthermore, we provide evidence that the Get3-Get4/5 complex is dominated by a single Get4/5 heterotetramer bound to one monomer of a Get3 dimer, uncovering an intriguing asymmetry in the Get4/5 heterotetramer upon Get3 binding. Ultrafast diffusion-limitd electrostatically driven Get3-Get4/5 association enables Get4/5 to rapidly sample and capture Get3 at different stages of the GET pathway.