The organophosphate degradation (opd) island borne esterase induced metabolic diversion in E. coli and its influence on p-nitrophenol degradation [Metabolism]

October 9th, 2015 by

In previous studies of the organophosphate degradation gene cluster we showed that expression of an open reading frame (orf306) present within the cluster in E. coli allowed growth on p-nitrophenol (PNP) as sole carbon source. We have now shown that expression of orf306 in E. coli causes a dramatic up-regulation in genes coding for alternative carbon catabolism. The propionate, glyoxylate and methyl citrate cycle (MCC) pathway-specific enzymes are up regulated, along with hca (phenyl propionate) and mhp (hydroxy phenyl propionate) degradation operons. These hca and mhp operons play a key role in degradation of PNP, enabling E. coli to grow using it as sole carbon source. Supporting growth experiments, PNP degradation products entered central metabolic pathways and got incorporated into the carbon backbone. The protein and RNA samples isolated from E. coli (pSDP10) cells grown in C14 labelled PNP indicated incorporation of C14 carbon suggesting Orf306-dependent assimilation of PNP in E. coli cells.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on The organophosphate degradation (opd) island borne esterase induced metabolic diversion in E. coli and its influence on p-nitrophenol degradation [Metabolism]

Hepatic FOXO1 target genes are co-regulated by thyroid hormone via RICTOR deacetylation and MTORC2-AKT inhibition [Metabolism]

October 9th, 2015 by

MTORC2-AKT is a key regulator of carbohydrate metabolism and insulin signaling due to its effects on FOXO1 phosphorylation. Interestingly, both FOXO1 and thyroid hormone (TH) have similar effects on carbohydrate and energy metabolism as well as overlapping transcriptional regulation of many target genes. Currently, little is known about the regulation of MTORC2-AKT or FOXO1 by TH. Accordingly, we performed hepatic transcriptome profiling in mice after FOXO1 knockdown in the absence or presence of TH, and compared these results with hepatic FOXO1 and THRB1 (TRβ1) ChIP-Seq data. We identified a subset of TH-stimulated FOXO1 target genes that required co-regulation by FOXO1 and TH. TH activation of FOXO1 was directly linked to an increase in SIRT1-MTORC2 interaction and RICTOR deacetylation. This, in turn, led to decreased AKT and FOXO1 phosphorylation. Moreover, TH increased FOXO1 nuclear localization, DNA binding, and target gene transcription by reducing AKT-dependent FOXO1 phosphorylation in a THRB1-dependent manner. These events were associated with TH-mediated oxidative phosphorylation and NAD+ production, and suggested that downstream metabolic effects by TH can post-translationally activate other transcription factors. Finally, TH increased glucose output and inhibited the effect of insulin on AKT phosphorylation in hepatic cells. Our results showed that RICTOR/MTORC2-AKT can integrate convergent hormonal and metabolic signals to provide co-ordinated and sensitive regulation of hepatic FOXO1-target gene expression.

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on TRAF5-mediated K63-linked polyubiquitination play essential role in positive regulation of ROR{gamma}t on promoting IL-17A expression [Cell Biology]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Carboxy-terminal Truncations of ClC-Kb Abolish Channel Activation by Barttin Via Modified Common Gating and Trafficking [Molecular Bases of Disease]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on Cardiac myosin binding protein C and Troponin-I phosphorylation independently modulate myofilament length dependent activation [Signal Transduction]

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.
  • Posted in Journal of Biological Chemistry, Publications
  • Comments Off on GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase KRAS4B, Exposing the Effector Binding Site [Molecular Biophysics]

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.