Troponin I Mutations R146G and R21C Alter Cardiac Troponin Function, Contractile Properties and Modulation by PKA-mediated Phosphorylation [Molecular Biophysics]

September 21st, 2015 by

Two HCM-associated cardiac troponin I (cTnI) mutations, R146G and R21C, are located in different regions of cTnI, the inhibitory-peptide and the cardiac-specific N-terminus. We recently reported these regions may interact when Ser23/Ser24 are phosphorylated, weakening cTnI interaction with cardiac TnC (cTnC). Little is known about how these mutations influence the affinity of cTnC for cTnI (KC-I) or contractile kinetics during β-adrenergic stimulation. Here, we tested how cTnIR146G or cTnIR21C influence contractile activation and relaxation and their response to protein kinase A (PKA). Both mutations significantly increased Ca2+ binding affinity to cTn (KCa) and KC-I. PKA phosphorylation resulted in a similar reduction of KCa for all complexes, but KC-I was reduced only with cTnIWT. cTnIWT, cTnIR146G and cTnIR21C were complexed into cTn and exchanged into rat ventricular myofibrils, and contraction/relaxation kinetics were measured ± PKA phosphorylation. Maximal tension (TMAX) was maintained for cTnIR146G and cTnIR21C exchanged myofibrils, and Ca2+ sensitivity of tension (pCa50) was increased. PKA phosphorylation decreased pCa50 for cTnIWT exchanged myofibrils, but not for either mutation. PKA phosphorylation accelerated the early, slow-phase relaxation for cTnIWT myofibrils, especially at Ca2+-levels that the heart operates in vivo. Importantly, this effect was blunted for cTnIR146G and cTnIR21C exchanged myofibrils. Molecular dynamics simulations suggest both mutations inhibit formation of intra-subunit contacts between the N-terminus and the inhibitory-peptide of cTnI that is normally seen with WT-cTn upon PKA phosphorylation. Together our results suggest that cTnIR146G and cTnIR21C blunt PKA modulation of activation and relaxation kinetics by prohibiting cardiac-specific N-terminal interaction with the cTnI inhibitory-peptide.
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Phosphatidylcholine affects the role of the sorting and assembly machinery in the biogenesis of mitochondrial {beta}-barrel proteins [Protein Synthesis and Degradation]

September 18th, 2015 by

Two protein translocases drive the import of β-barrel precursor proteins into the mitochondrial outer membrane: The translocase of the outer membrane (TOM complex) promotes transport of the precursor to the intermembrane space, whereas the sorting and assembly machinery (SAM complex) mediates subsequent folding of the β-barrel and its integration into the target membrane. The non-bilayer forming phospholipids phosphatidylethanolamine (PE) and cardiolipin (CL) are required for the biogenesis of β-barrel proteins. Whether bilayer-forming phospholipids such as phosphatidylcholine (PC), the most abundant phospholipid of the mitochondrial outer membrane, play a role in the import of β-barrel precursors is unclear. In this study we show that PC is required for stability and function of the SAM complex during the biogenesis of β-barrel proteins. PC further promotes the SAM-dependent assembly of the TOM complex, indicating a general role of PC for the function of the SAM complex. In contrast to PE-deficient mitochondria precursor accumulation at the TOM complex is not affected by depletion of PC. We conclude that PC and PE affect the function of distinct protein translocases in mitochondrial β-barrel biogenesis.
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Novel {beta}-1,4-mannanase belonging to a new glycoside hydrolase family in Aspergillus nidulans [Enzymology]

September 18th, 2015 by

Many filamentous fungi produce β-mannan-degrading β-1,4-mannanases that belong to the glycoside hydrolase (GH) 5 and GH26 families. Here we identified a novel β-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase (GH) family (GH134) in Aspergillus nidulans. Blast analysis of the amino acid sequence using the NCBI protein database revealed that this enzyme had no similarity to any sequences and no putative conserved domains. Protein homologs of the enzyme were distributed to limited fungal and bacterial species. Man134A released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free β-mannan was the substrate from the initial stage of the reaction, suggesting that Man134A preferentially reacts with β-mannan via a unique catalytic mode. Man134A had high catalytic efficiency (kcat/Km) towards mannohexaose (M6) compared with the endo-β-1,4-mannanase Man5C, and notably converted M6 to M2, M3 and M4, with M3 being the predominant reaction product. The action of Man5C towards β-mannans was synergistic. The growth phenotype of a Man134A disruptant was poor when β-mannans comprised the sole carbon source, indicating that Man134A is involved in β-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of β-mannan degradation that is enhanced by the novel β-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various endo-β-1,4-mannanases.

Klotho reduction in alveolar macrophages contributes to CSE-induced inflammation in chronic obstructive pulmonary disease [Metabolism]

September 18th, 2015 by

Abnormal inflammation and accelerated decline in lung function occur in patients with chronic obstructive pulmonary disease (COPD).Klotho, an anti-aging protein, has an anti-inflammatory function. However, the role of Klotho has never been investigated in COPD. The aim of this study is to investigate the possible role of Klotho by alveolar macrophages in airway inflammation in COPD.Klotho levels were assessed in the lung samples and peripheral blood mononuclear cells (PBMCs) of non-smokers, smokers and patients with COPD.The regulation of Klotho expression by cigarette smoke extract (CSE) was studied in vitro, and small interfering RNA (siRNA) and recombinant Klotho were employed to investigate the role of Klotho on CSE-induced inflammation.Klotho expression was reduced in alveolar macrophages in the lungs and PBMCs of COPD patients.CSE decreased Klotho expression and release from alveolar macrophages. Knockdown of endogenous Klotho augmented the expression of the inflammatory mediators, such as MMP-9, IL-6 and TNF-alpha by alveolar macrophages.Exogenous Klotho inhibited the expression of CSE-induced the inflammatory mediators. Furthermore, we showed that Klotho interacts with IkappaBalpha of the NF-kappaB pathway.Dexamethasone treatment increased expression and release level of Klotho in alveolar macrophages. Our findings suggest that Klotho plays a role in sustained inflammation of the lungs, which in turn may have therapeutic implications in COPD.

Anti-oncogenic and oncogenic properties of Nrf2 in arsenic-induced carcinogenesis [Cell Biology]

September 18th, 2015 by

Arsenic (As3+) is a carcinogen with considerable environmental and occupational relevancy. The present study shows that As3+-transformed human lung bronchial epithelial BEAS-2B cells (AsT cells) exhibit a property of apoptosis resistance. The level of basal reactive oxygen species (ROS) is very low in AsT cells in correlation with elevated expressions of both anti-oxidant enzymes and anti-apoptotic proteins. Nuclear factor erythroid 2-related factor (Nrf2) and p62 are constitutively expressed. These two proteins up-regulate anti-oxidant enzymes and anti-apoptotic proteins. The knockdown of Nrf2 or p62 by small interference RNA (siRNA) enhanced both ROS levels and As3+-induced apoptosis in transformed cells. AsT cells have autophagy deficiency as evidenced by reduced formation of LC3-II, GFP-LC3 puncta, and autophagy flux. Results obtained using soft agar assay and shRNA Nrf2 transfected cells show that Nrf2 plays an anti-oncogenic role before transformation, whereas this transcription factor plays an oncogenic role after transformation. In addition, depletion of Nrf2 by shRNA dramatically inhibited growth and proliferation of transformed cells. Furthermore, the Nrf2 protein levels and anti-apoptotic and anti-oxidant enzyme levels are higher in lung adenocarcinoma than in normal tissues. Collectively, this study demonstrates that constitutively high level of Nrf2 up-regulates the anti-oxidant proteins catalase and SOD, as well as the anti-apoptotic proteins Bcl-2 and Bcl-xL. The final consequences are decreased ROS generation and increased apoptotic resistance, cell survival and proliferation, and tumorigenesis.

Calcium Regulates the Activity and Structural Stability of Tpr, a Bacterial Calpain-like Peptidase [Enzymology]

September 18th, 2015 by

Porphyromonas gingivalis is a peptide-fermenting asaccharolytic periodontal pathogen. Its genome contains several genes encoding cysteine peptidases other than gingipains. One of these genes (PG1055) encodes a protein called Tpr (thiol protease), which has sequence similarity to cysteine peptidases of the papain and calpain families. In this study, we biochemically characterize Tpr. We found that the 55 kDa Tpr inactive zymogen proteolytically processes itself into active forms of 48 kDa, 37 kDa, and 33 kDa via sequential truncations at the N-terminus. These processed molecular forms of Tpr are associated with the bacterial outer membrane, where they are likely responsible for the generation of metabolic peptides required for survival of the pathogen. Both autoprocessing and activity were dependent on calcium concentrations greater than 1 mM, consistent with the protein's activity within the intestinal and inflammatory milieus. Calcium also stabilized the Tpr structure and rendered the protein fully resistant to proteolytic degradation by gingipains. Together, our findings suggest that Tpr is an example of a bacterial calpain, a calcium-responsive peptidase that may generate substrates required for the peptide-fermenting metabolism of P. gingivalis. Aside from nutrient generation, Tpr may also be involved in evasion of host immune response through degradation of the antimicrobial peptide LL-37 and complement proteins C3, C4 and C5. Taken together, these results indicate that Tpr likely represents an important pathogenesis factor for P. gingivalis.

Generation, release and uptake of the NAD precursor nicotinic acid riboside by human cells [Enzymology]

September 18th, 2015 by

NAD is essential for cellular metabolism and has also a key role in various signaling pathways in human cells. To ensure proper control of vital reactions, NAD must be permanently resynthesized. Nicotinamide (Nam) and nicotinic acid (NA) as well as nicotinamide riboside (NR) and nicotinic acid riboside (NAR) are the major precursors for NAD biosynthesis in humans. In this study we explored whether the ribosides NR and NAR can be generated in human cells. We demonstrate that purified, recombinant human cytosolic 5'-nucleotidases (5'-NTs) CN-II and CN-III, but not CN-IA, can dephosphorylate the mononucleotides NMN and NAMN and thus catalyze NR and NAR formation in vitro. Similar to their counterpart from yeast, Sdt1, the human 5'-NTs require high (millimolar) concentrations of NMN or NAMN for efficient catalysis. Overexpression of FLAG-tagged CN-II and CN-III in HEK293 and HepG2 cells resulted in the formation and release of NAR. However, NAR accumulation in the culture medium of these cells was only detectable under conditions that led to increased NAMN production from NA. The amount of NAR released from cells engineered for increased NAMN production was sufficient to maintain viability of surrounding cells unable to use any other NAD precursor. Moreover, we found that untransfected HeLa cells produce and release sufficient amounts of NAR and NR under normal culture conditions. Collectively, our results indicate that cytosolic 5'-NTs participate in the conversion of NAD precursors and establish NR and NAR as integral constituents of human NAD metabolism. In addition, they point to the possibility that different cell types might facilitate each other's NAD supply by providing alternative precursors.

Structural Insights into Mitochondrial Antiviral-Signaling Protein (MAVS)-Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6) Signaling [Signal Transduction]

September 18th, 2015 by

In response to viral infection, cytosolic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) sense viral RNA and promote oligomerization of mitochondrial antiviral-signaling protein (MAVS), which then recruits tumor necrosis factor receptor-associated factor (TRAF) family proteins including TRAF6 to activate antiviral response. Currently, the interaction between MAVS and TRAF6 is only partially understood lacking atomic details. Here, we demonstrated that MAVS directly interacts with TRAF6 through its potential TRAF6-binding motif 2 (T6BM2, amino acids 455-460). Further, we solved the crystal structure of MAVS T6BM2 in complex with TRAF6 TRAF_C domain at 2.95-Å resolution. T6BM2 of MAVS binds to the canonical adaptor-binding groove of the TRAF_C domain. Structure-directed mutational analyses in vitro and in cells revealed that MAVS binding to TRAF6 via T6BM2 instead of T6BM1 is essential but not sufficient for an optimal antiviral response. Particularly, a MAVS mutant Y460E retained its TRAF6-binding ability as predicted, but showed significantly impaired signaling activity, highlighting the functional importance of this tyrosine. Moreover, these observations were further confirmed in MAVS-/- mouse embryonic fibroblast (MEF) cells. Collectively, our work provides a structural basis for understanding the MAVS-TRAF6 antiviral response.
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The RNA-binding Complexes, NF45-NF90 and NF45-NF110, Associate Dynamically with the c-fos Gene and Function as Transcriptional Coactivators [Gene Regulation]

September 17th, 2015 by

The c-fos gene is rapidly induced to high levels by various extracellular stimuli. We used a defined in vitro transcription system that utilizes the c-fos promoter to purify a coactivator activity in an unbiased manner. We report here that NF45-NF90 and NF45-NF110, which possess archetypical double-stranded RNA binding motifs, have a direct function as transcriptional coactivators. The transcriptional activities of the NF complexes (NF45-NF90 and NF45-NF110) are mediated by both the upstream enhancer and core promoter regions of the c-fos gene and do not require their double-stranded RNA-binding activities. The NF complexes cooperate with general coactivators, PC4 and Mediator, to elicit a high level of transcription and display multiple interactions with activators and the components of the general transcriptional machinery. Knockdown of the endogenous NF90/NF110 in mouse cells shows an important role for the NF complexes in inducing c-fos transcription. Chromatin immunoprecipitation assays demonstrate that the NF complexes occupy the c-fos enhancer/promoter region before and after serum induction and that their occupancies within the coding region of the c-fos gene increase in parallel to that of RNAPII upon serum induction. In light of their dynamic occupancy on the c-fos gene as well as direct functions in both transcription and posttranscriptional processes, the NF complexes appear to serve as multifunctional coactivators that coordinate different steps of gene expression to facilitate rapid response of inducible genes.
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Globins Scavenge Sulfur Trioxide Anion Radical [Metabolism]

September 17th, 2015 by Gardner, P. R., Gardner, D. P., Gardner, A. P.

Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600 and ≥ 7,500 x 106 M-1 s-1, respectively, and correlate with redox potentials. Measured rate constants for O2, GSH, ascorbate and NAD(P)H are also large at approximately 100, 10, 130 and 30 x 106 M-1 s-1, respectively, but nevertheless allow for favorable competition by globins, and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O2-dependent growth impairment with non-fermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O2 exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP+-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.