The Exocyst Subunit Sec6 Interacts with Assembled Exocytic SNARE Complexes [Membrane Biology]

October 7th, 2015 by Dubuke, M. L., Maniatis, S., Shaffer, S. A., Munson, M.

In eukaryotic cells, membrane-bound vesicles carry cargo between intracellular compartments, to and from the cell surface, and into the extracellular environment. Many conserved families of proteins are required for properly localized vesicle fusion, including the multisubunit tethering complexes (MTCs) and the SNARE complexes. These protein complexes work together to promote proper vesicle fusion in intracellular trafficking pathways. However, the mechanism by which the exocyst, the exocytosis-specific MTC, interacts with the exocytic SNAREs to mediate vesicle targeting and fusion is currently unknown. We previously demonstrated that the Saccharomyces cerevisiae exocyst subunit Sec6 directly bound the plasma membrane SNARE protein Sec9 in vitro and that Sec6 inhibited the assembly of the binary Sso1:Sec9 SNARE complex. Therefore, we hypothesized that the interaction between Sec6 and Sec9 prevented the assembly of premature SNARE complexes at sites of exocytosis. In order to map the determinants of this interaction, we used cross-linking and mass spectrometry analyses to identify residues required for binding. Mutation of residues identified by this approach resulted in a growth defect when introduced into yeast. Contrary to our previous hypothesis, we discovered that Sec6 does not change the rate of SNARE assembly, but rather binds both the binary Sec9:Sso1 and ternary Sec9:Sso1:Snc2 SNARE complex. Together, these results suggest a new model wherein Sec6 promotes SNARE complex assembly, similar to the role proposed for other tether subunit-SNARE interactions.

Bakuchiol is a Phenolic Isoprenoid with Novel Enantiomer-Selective Anti-Influenza A Virus Activity Involving Nrf2 Activation [Microbiology]

October 7th, 2015 by

Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia Linn.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (-)-(R)-bakuchiol, inhibited influenza A viral infection and growth, and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-β; and myxovirus resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (-)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and a Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.

Mitochondrial Single-stranded DNA-binding Proteins Stimulate the Activity of DNA Polymerase {gamma} by Organization of the Template DNA [Enzymology]

October 7th, 2015 by

The activity of the mitochondrial replicase, DNA polymerase γ (Pol γ) is stimulated by another key component of the mitochondrial replisome, the mitochondrial single-stranded DNA-binding protein (mtSSB). We have performed a comparative analysis of the human and Drosophila Pol γs with their cognate mtSSBs, evaluating their functional relationships using a combined approach of biochemical assays and electron microscopy. We found that increasing concentrations of both mtSSBs led to the elimination of template secondary structure and gradual opening of the template DNA, through a series of visually similar template species. The stimulatory effect of mtSSB on Pol γ is not species-specific. We observed that human mtSSB can be substituted by its Drosophila homologue, and vice versa, finding that a lower concentration of insect mtSSB promotes efficient stimulation of either Pol. Notably, distinct phases of the stimulation by both mtSSBs are distinguishable, and are characterized by a similar organization of the template DNA for both Pol γs. We conclude that organization of the template DNA is the major factor contributing to the stimulation of Pol γ activity. Additionally, we observed that human Pol γ preferentially utilizes compacted templates, whereas the insect enzyme achieves its maximal activity on open templates, emphasizing the relative importance of template DNA organization in modulating Pol γ activity, and the variation among systems.
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Biochemical Characterization and Substrate Specificity of Autophagin-2 from the Parasite Trypanosoma cruzi [Molecular Bases of Disease]

October 7th, 2015 by

The genome of the parasite Trypanosoma cruzi encodes two copies of autophagy-related cysteine proteases, Atg4.1 and Atg4.2. T. cruzi autophagin-2 (TcAtg4.2) carries the majority of proteolytic activity and is responsible for processing of Atg8 proteins near the carboxy-terminus, exposing a conserved glycine. This enables progression of autophagy and differentiation of the parasite, which is required for successful colonization of humans. The mechanism of substrate hydrolysis by Atg4 was found to be highly conserved among the species as critical mutations in the TcAtg4.2, including mutation of the conserved Gly244 residue in the hinge region enabling flexibility of the regulatory loop, and deletion of the regulatory loop, completely abolished processing capacity of the mutants. Using Positional Scanning-Substrate Combinatorial Library (PS-SCL) we determined that TcAtg4.2 tolerates a broad spectrum of amino acids in the P4 and P3 positions, similar to the human orthologue autophagin-1 (HsAtg4B). In contrast, both human and trypanosome Atg4 orthologues exhibited exclusive preference for aromatic amino acid residues in the P2 position, and for Gly in the P1 position, which is absolutely conserved in the natural Atg8 substrates. Using an extended P2 substrate library, which also included the unnatural amino acid cyclohexylalanine (Cha) derivative of Phe, we generated highly selective tetrapeptide substrates acetyl-Lys-Lys-Cha-Gly-AFC (Ac-KKChaG-AFC) and acetyl-Lys-Thr-Cha-Gly-AFC (Ac-KTChaG-AFC). Although these substrates were cleaved by cathepsins, making them unsuitable for analysis of complex cellular systems, they were recognized exclusively by TcAtg4.2, but not by HsAtg4B nor by the structurally related human proteases SENP1, SENP2 and UCH-L3.

A Failure to Communicate: Myosin Residues Involved in Hypertrophic Cardiomyopathy Affect Inter-Domain Interaction [Molecular Bases of Disease]

October 7th, 2015 by Kronert, W. A., Melkani, G. C., Melkani, A., Bernstein, S. I.

Our molecular modeling studies suggest a charge-dependent interaction between residues E497 in the relay domain and R712 in the converter domain of human β-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (~80%) as well as in basal (~67%) and actin-activated (~84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ~73% of wild type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. While sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Further, the same inter-domain interaction is disrupted when modeling human β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.
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Competitive inhibition of the endoplasmic reticulum signal peptidase by non-cleavable mutant preprotein cargos [Enzymology]

October 7th, 2015 by

Upon translocation across the endoplasmic reticulum (ER) membrane, secretory proteins are proteolytically processed to remove their signal peptide (SP) by signal peptidase (SPase). This process is critical for subsequent folding, intracellular trafficking and maturation of secretory proteins. Prokaryotic SPase has been shown to be a promising antibiotic target; in contrast, to date, no eukaryotic SPase inhibitors have been reported. Herein, we report that introducing a proline immediately following the natural SP cleavage site not only blocks preprotein cleavage, but also in trans, impairs processing and maturation of co-expressed preproteins in the ER. Specifically, we find that a variant preproinsulin, pPI-F25P, is translocated across the ER membrane where it binds to the catalytic SPase subunit SEC11A, inhibiting SPase activity in a dose-dependent manner. Similar findings were obtained with an analogous variant of preproparathyroid hormone, demonstrating that inhibition of the SPase does not depend strictly on the sequence or structure of the downstream mature protein. We further show that inhibiting SPase in the ER impairs intracellular processing of viral polypeptides and their subsequent maturation. These observations suggest that eukaryotic SPases (including the human ortholog) are in principle suitable therapeutic targets for antiviral drug design.

Chaperone-Assisted Post-Translational Transport of Plastidic Type I Signal Peptidase 1 [Membrane Biology]

October 7th, 2015 by Endow, J. K., Singhal, R., Fernandez, D. E., Inoue, K.

Type I signal peptidase (SPase I) is an integral membrane Ser/Lys-protease with one or two transmembrane domains (TMDs), cleaving transport signals off translocated precursor proteins. The catalytic domain of SPase I folds to form a hydrophobic surface and inserts into the lipid bilayers at the trans-side of the membrane. In bacteria, SPase I is targeted co-translationally and the catalytic domain remains unfolded until it reaches the periplasm. By contrast, SPases I in eukaryotes are targeted post-translationally, requiring an alternative strategy to prevent premature folding. Here we demonstrate that two distinct stromal components are involved in posttranslational transport of plastidic SPase I (Plsp1) from Arabidopsis thaliana which contains a single TMD. During import into isolated chloroplasts, Plsp1 was targeted to the membrane via a soluble intermediate in an ATP-hydrolysis dependent manner. Insertion of Plsp1 into isolated chloroplast membranes, by contrast, was found to occur by two distinct mechanisms. The first mechanism requires ATP hydrolysis and the protein conducting channel cpSecY1, and was strongly enhanced by exogenously-added cpSecA1. The second mechanism was independent of nucleoside triphosphates and proteinaceous components but with a high frequency of mis-orientation. This unassisted insertion was inhibited by urea and stroma extract. During import-chase assays using intact chloroplasts, Plsp1 was incorporated into a soluble 700-kD complex that co-migrated with the CPN60 complex before inserting into the membrane. The TMD within Plsp1 was required for the cpSecA1-dependent insertion but was dispensable for association with the 700-kD complex and also for unassisted membrane insertion. These results indicate cooperation of Cpn60 and cpSecA1 for proper membrane insertion of Plsp1 by cpSecY1.

Porphyrin Binding to Gun4 protein, Facilitated by a Flexible Loop, Controls Metabolite Flow through the Chlorophyll Biosynthetic Pathway [Computational Biology]

October 7th, 2015 by

In oxygenic phototrophs, chlorophylls, hemes and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated and an important regulatory role is attributed to Mg-chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the Mg-chelatase activity but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for Mg-protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding and the magnesium ion held within the porphyrin is coordinated by Asn211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in Mg-chelatase assay showing that tight porphyrin-binding in Gun4 facilitates its interaction with the Mg-chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.
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MAPK Activated by Prostaglandin E2 Phosphorylates Connexin 43 and Closes Osteocytic Hemichannels in Response to Continuous Flow Shear Stress [Cell Biology]

October 6th, 2015 by Riquelme, M. A., Burra, S., Kar, R., Lampe, P. D., Jiang, J. X.

Cx43 hemichannels serve as a portal for the release of prostaglandins, a critical process in mediating biological responses of mechanical loading on bone formation and remodeling. We have previously observed that fluid flow shear stress (FFSS) opens hemichannels; however sustained FFSS results in hemichannel closure, as continuous opening of hemichannels is detrimental to cell viability and bone remodeling. However, the mechanism that regulates the closure of the hemichannels is unknown. Here, we show that activation of p44/42 ERK upon continuous FFSS leads to Cx43 phosphorylation at S279/282, sites known to be phosphorylated sites by p44/42 MAPK. Incubation of osteocytic MLO-Y4 cells with conditioned media (CM) collected after continuous FFSS increased MAPK-dependent phosphorylation of Cx43. CM treatment inhibited hemichannel opening and this inhibition was reversed when cells were pre-treated with MAPK pathway inhibitor. We found that PGE2 accumulates in the CM in a time-dependent manner. Treatment with PGE2 increased phospho-p44/42 ERK levels and also Cx43 phosphorylation at S279/282 sites. Depletion of PGE2 from CM and pre-treatment with a p44/42 ERK pathway specific inhibitor, resulted in a complete inhibition of ERK-dependent Cx43 phosphorylation and attenuated the inhibition of hemichannels by CM and PGE2. Consistently, the opening of hemichannels by FFSS was blocked by PGE2 and CM and this blockage was reversed by U0126 and the CM depleted of PGE2. Similar observation was also obtained in isolated primary osteocytes. Together, results from this study suggest that extracellular PGE2 accumulated after continuous FFSS is responsible for activation of p44/42 ERK signaling and subsequently, direct Cx43 phosphorylation by activated ERK leads to hemichannel closure.
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Tumor suppressive function of p21-activated kinase 6 in hepatocellular carcinoma [Gene Regulation]

October 6th, 2015 by Liu, W., Liu, Y., Liu, H., Zhang, W., Fu, Q., Xu, J., Gu, J.

Our previous studies identified the oncogenic role of p21-activated kinase 1 (PAK1) in hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Contrarily, PAK6 was found to predict favorable prognosis in RCC patients. Nevertheless, the ambiguous tumor suppressive function of PAK6 in hepatocarcinogenesis remains obscure. Herein, decreased PAK6 expression was found to be associated with Tumor Node Metastasis (TNM) stage progression and unfavorable overall survival (OS) in HCC patients. Additionally, overexpression and silence of PAK6 experiments showed that PAK6 inhibited xenografted tumor growth in vivo, and restricted cell proliferation, colony formation, migration and invasion and promoted cell apoptosis and anoikis in vitro. Moreover, overexpression of kinase dead and nuclear localization signal (NLS) deletion mutants of PAK6 experiments indicated tumor suppressive function of PAK6 was dependent on its kinase activity and nuclear translocation partially. Furthermore, gain or loss of function in polycomb repressive complex 2 (PRC2) components including EZH2, SUZ12 and EED elucidated that epigenetic control of H3K27me3 arbitrated PAK6 downregulation in hepatoma cells. More importantly, negative correlation between PAK6 and EZH2 expression was observed in hepatoma tissues from HCC patients. These data identified the tumor suppressive role and potential underlying mechanism of PAK6 in hepatocarcinogenesis.