The Regulation of Synaptic Protein Turnover [Protein Synthesis and Degradation]

October 9th, 2015 by Alvarez-Castelao, B., Schuman, E. M.

Emerging evidence indicates that protein synthesis and degradation are necessary for the remodeling of synapses. These two processes govern cellular protein turnover, are tightly regulated, and neuronal activity modulates them in time and space. The anisotropic anatomy of the neurons presents a challenge for the study of protein turnover, but understanding protein turnover in neurons and its modulation in response to activity can help us to unravel the fine-tuned changes that occur at synapses in response to activity. Here we review the key experimental evidence demonstrating the role of protein synthesis and degradation in synaptic plasticity, and the turnover rates of specific neuronal proteins

TRAF5-mediated K63-linked polyubiquitination play essential role in positive regulation of ROR{gamma}t on promoting IL-17A expression [Cell Biology]

October 9th, 2015 by

Retinoid-related orphan nuclear receptor γt (RORγt) is a key transcription factor for the de-velopment and function of Th17 cells. In this study, we showed that tumor necrosis factor receptor as-sociated factor 5 (TRAF5), known as an E3 ubiq-uitin-protein ligase and signal transducer, interacts with and ubiquitinates RORγt via K63-linked polyubiquitination. TRAF5 stabilizes RORγt pro-tein level depending on its RING-finger domain. And depletion of TRAF5 in Th17 cells destabilizes RORγt protein and down-regulates Th17-related genes, including IL-17A, an inflammatory cytokine involved in pathogenic mechanisms of several au-toimmune diseases, such as systemic lupus erythe-matosus. Moreover, up-regulation of TRAF5 mRNA level was found in SLE patient CD4+ T cells. Our findings reveal a direct link between TRAF5-mediated ubiquitination and RORγt pro-tein regulation, which may aggravate inflamma-tory progress and provide new therapeutic drug targets for autoimmune diseases.
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Actin Out: Regulation of the Synaptic Cytoskeleton [Cell Biology]

October 9th, 2015 by Spence, E. F., Soderling, S. H.

The small size of dendritic spines belies the elaborate role they play in excitatory synaptic transmission and ultimately complex behaviors. The cytoskeletal architecture of the spine is predominately composed of actin filaments. These filaments, which at first glance might appear simple, are also surprisingly complex. They dynamically assemble into different structures and serve as a platform for orchestrating the elaborate responses of the spine during spinogenesis and experience-dependent plasticity. Multiple mutations associated with human neurodevelopmental and psychiatric disorders involve genes which encode regulators of the synaptic cytoskeleton. A major, unresolved question is how the disruption of specific actin filament structures leads to the onset and progression of complex synaptic and behavioral phenotypes. This review will cover established and emerging mechanisms of actin cytoskeletal remodeling and how this influences specific aspects of spine biology that are implicated in disease.

Neutrophil elastase differentially regulates IL-8 and VEGF production by cigarette smoke extract [Signal Transduction]

October 9th, 2015 by Lee, K.-H., Lee, C.-H., Jeong, J., Jang, A.-H., Yoo, C.-G.

Inflammation by IL-8-induced neutrophil recruitment and apoptosis of epithelial cells by decreased expression of VEGF have been suggested as one of the complicated pathogenic mechanisms of COPD. The role of neutrophil elastase (NE) in the development of COPD is also well-known. However, little is known about how they interact. The objective of this study was to elucidate the effect of NE on the cigarette smoke extract (CSE)-induced IL-8 and VEGF production, and its molecular mechanism in bronchial epithelial cells. CSE increased both IL-8 and VEGF productions in human bronchial epithelial cell (BEAS-2B). While NE significantly enhanced CSE-induced IL-8 production, it suppressed VEGF production. This differential regulation was not CSE-specific. The effect of NE on IL-8 production, but not VEGF, was ERK-dependent. Interestingly, in contrast to decreased VEGF protein expression, NE accelerated VEGF transcription by CSE, suggesting post-translational modification. When cells were incubated with purified NE, it was detected in the cytoplasm, suggesting the intracellular translocation of NE. Furthermore, NE fragmented rhVEGF in vitro, but not rhIL-8. These results indicate that VEGF down-regulation is due to direct degradation by NE which is translocated into cells. Similar to in vitro cell experiments, elastase treatment increases CSE-induced IL-8, however, it suppresses VEGF production in bronchoalveolar lavage (BAL) fluid of CSE-treated mice. Moreover, elastase treatment enhances CSE-induced emphysema in mice. Considering the actions of IL-8 and VEGF, our results suggest that NE contributes to the pathogenesis of COPD by enhancing inflammation and apoptosis.

Carboxy-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking [Molecular Bases of Disease]

October 9th, 2015 by Stolting, G., Bungert–Plumke, S., Franzen, A., Fahlke, C.

ClC-K chloride channels are crucial for auditory transduction and urine concentration. Mutations in CLCNKB, the gene encoding the renal chloride channel hClC-Kb, cause Bartter syndrome type III, a human genetic condition characterized by polyuria, hypokalemia and alkalosis. In recent years, several Bartter syndromeassociated mutations have been described that result in truncations of the intracellular carboxy-terminus of hClC-Kb. We here used a combination of whole-cell patch clamp, confocal imaging, co-immunoprecipitation and surface biotinylation to study the functional consequences of a frequent CLCNKB mutation that creates a premature stop codon at W610. We found that W610X leaves the association of hClC-Kb and the accessory subunit barttin unaffected, but impairs its regulation by barttin. W610X attenuates hClC-Kb surface membrane insertion. Moreover, W610X results in hClCKb channel opening in the absence of barttin and prevents further barttin-mediated activation. To describe how the carboxyterminus modifies the regulation by barttin we used V166E rClC-K1. V166E rClC-K1 is active without barttin and exhibits prominent, barttin-regulated voltagedependent gating. Electrophysiological characterization of truncated V166E rClC-K1 demonstrated that the distal carboxyterminus is necessary for slow cooperative gating. Since barttin modifies this particular gating process, channels lacking the distal carboxy-terminal domain are no longer regulated by the accessory subunit. Our results demonstrate that the carboxyterminus of hClC-Kb is not part of the binding site for barttin, but functionally modifies the interplay with barttin. The loss-of-activation of truncated hClC-Kb channels in heterologous expression systems fully explains the reduced basolateral chloride conductance in affected kidneys and the clinical symptoms of Bartter syndrome patients.
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Cardiac myosin binding protein C and Troponin-I phosphorylation independently modulate myofilament length dependent activation [Signal Transduction]

October 9th, 2015 by

β-adrenergic stimulation in heart leads to increased contractility and lusitropy via activation of protein kinase A (PKA). In the cardiac sarcomere, both myosin binding protein C (cMyBP-C) and troponin-I (cTnI) are prominent myofilament targets of PKA. Treatment of permeabilized myocardium with PKA induces enhanced myofilament length dependent activation (LDA), the cellular basis of the Frank-Starling cardiac regulatory mechanism. It is not known, however, which of these targets mediates the altered LDA, and to what extent. Here, we employed two genetic mouse models in which the three PKA sites in cMyBP-C were replaced with either phospho-mimic (DDD) or phospho-null (AAA) residues. AAA or DDD permeabilized myocytes (n=12-17) were exchanged (~93%) for recombinant cTnI in which the two PKA sites were mutated to either phospho-mimic (DD) or phospho-null (AA) residues. Force-[Ca2+] relationships were determined at two sarcomere lengths (SL=1.9 um and SL=2.3 um). Data were fit to a modified Hill equation for each individual cell preparation at each SL. LDA was indexed as ΔEC50, the difference in [Ca2+] required to achieve 50% force activation at the two SL. We found that PKA mediated phosphorylation of cMyBP-C and cTnI each independently contribute to enhance myofilament length-dependent activation properties of the cardiac sarcomere, with relative contributions of ~67% and ~33% for cMyBP-C for cTnI, respectively. We conclude that β-adrenergic stimulation enhances the Frank-Starling regulatory mechanism predominantly via cMyBP-C PKA mediated phosphorylation. We speculate that this molecular mechanism enhances cross-bridge formation at long SL, while accelerating cross-bridge detachment and relaxation at short SL.
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GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase KRAS4B, Exposing the Effector Binding Site [Molecular Biophysics]

October 9th, 2015 by

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, while the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate HVR's disassociation in a nucleotide-independent manner, thereby upregulate oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way - abolishing GTP hydrolysis and facilitating effector binding.
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Concerted Trafficking Regulation of Kv2.1 and KATP Channels by Leptin in Pancreatic {beta}-cells [Signal Transduction]

October 9th, 2015 by Wu, Y., Shyng, S.-L., Chen, P.-C.

In pancreatic β-cells, Kv2.1 channels are the dominant delayed rectifier potassium channels responsible for action potential repolarization. Here, we report that leptin, a hormone secreted by adipocytes known to inhibit insulin secretion, causes a transient increase in surface expression of Kv2.1 channels in rodent and human β -cells. The effect of leptin on Kv2.1 surface expression is mediated by the AMP-activated protein kinase AMPK. Activation of AMPK mimics, whereas inhibition of AMPK occludes the effect of leptin. Inhibition of CaMKKβ;, a known upstream kinase of AMPK, also blocks the effect of leptin. In addition, the cAMP-dependent protein kinase PKA is involved in Kv2.1 channel trafficking regulation. Inhibition of PKA prevents leptin or AMPK activators from increasing Kv2.1 channel density, while stimulation of PKA is sufficient to promote Kv2.1 channel surface expression. The increased Kv2.1 surface expression by leptin is dependent on actin depolymerization; and pharmacologically-induced actin depolymerization is sufficient to enhance Kv2.1 surface expression. The signaling and cellular mechanisms underlying Kv2.1 channel trafficking regulation by leptin mirror those reported recently for ATP-sensitive potassium (KATP) channels, which are critical for coupling glucose stimulation with membrane depolarization. We show that the leptin-induced increase in surface KATP channels results in more hyperpolarized membrane potentials than control cells at stimulating glucose concentrations, and the increase in Kv2.1 channels leads to a more rapid repolarization of membrane potential in cells firing action potentials. The study supports a model in which leptin exerts concerted trafficking regulation of KATP and Kv2.1 channels to coordinately inhibit insulin secretion.

Coordination of Protein Phosphorylation and Dephosphorylation in Synaptic Plasticity [Signal Transduction]

October 9th, 2015 by Woolfrey, K. M., Dell'Acqua, M. L.

A central theme in nervous system function is equilibrium: synaptic strengths wax and wane, neuronal firing rates adjust up and down, and neural circuits balance excitation with inhibition. This push/pull regulatory theme carries through to the molecular level at excitatory synapses, where protein function is controlled through phosphorylation and dephoshorylation by kinases and phosphatases. However, these opposing enzymatic activities are only part of the equation, as scaffolding interactions and assembly of multi-protein complexes are further required for efficient, localized synaptic signaling. This review will focus on coordination of postsynaptic serine/threonine kinase and phosphatase signaling by scaffold proteins during synaptic plasticity.

A Disintegrin and Metalloprotease 10 is Indispensable for Maintenance of the Muscle Satellite Cell Pool [Cell Biology]

October 9th, 2015 by

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10, a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared to wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool.