Localization of Proteins to the 1,2-Propanediol Utilization Microcompartment by Non-native Signal Sequences Is Mediated by a Common Hydrophobic Motif [Microbiology]

August 17th, 2015 by

Various bacteria localize metabolic pathways to proteinaceous organelles known as bacterial microcompartments (MCPs), enabling the metabolism of carbon sources to enhance survival and pathogenicity in the gut. There is considerable interest in exploiting bacterial MCPs for metabolic engineering applications, but little is known about the interactions between MCP signal sequences and the protein shells of different MCP systems. We found that the N-terminal sequences from the ethanolamine utilization (Eut) and glycyl radical-generating protein (Grp) MCPs are able to target reporter proteins to the 1,2-propanediol utilization (Pdu) MCP, mediated by a conserved hydrophobic residue motif. Recapitulation of this motif by the addition of a single amino acid confers targeting function on an N-terminal sequence from the ethanol utilization (Etu) MCP system that previously did not act as a Pdu signal sequence. Moreover, the Pdu-localized signal sequences compete with native Pdu targeting sequences for encapsulation in the Pdu MCP. Salmonella enterica natively possesses both the Pdu and Eut operons, and our results suggest that Eut proteins might be localized to the Pdu MCP in vivo. We further demonstrate that S. enterica LT2 retains the ability to grow on 1,2-propanediol as the sole carbon source when a Pdu enzyme is replaced with its Eut homolog. While the relevance of this finding to the native system remains to be explored, we show that the Pdu-localized signal sequences described herein allow control over the ratio of heterologous proteins encapsulated within Pdu MCPs.
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Disruption of nucleotide homeostasis by the antiproliferative drug AICAR (5-aminoimidazole-4-carboxamide-1-{beta}-D-ribofuranoside monophosphate) [Metabolism]

August 17th, 2015 by

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside monophosphate (AICAR) is a natural metabolite with potent anti-proliferative and low-energy mimetic properties. At high concentration, AICAR is toxic for yeast and mammalian cells but the molecular basis of this toxicity is poorly understood. Here, we report the identification of yeast purine salvage pathway mutants which are synthetic lethal with AICAR accumulation. Genetic suppression revealed that this synthetic lethality is in part due to low expression of adenine phosphoribosyl transferase under high AICAR conditions. In addition, metabolite profiling points to the AICAR/nucleotide triphosphate (NTP) balance as crucial for optimal utilization of glucose as a carbon source. Indeed, we found that AICAR toxicity in yeast and human cells is alleviated when glucose is replaced by an alternative carbon source. Together, our metabolic analyses unveil the AICAR/NTP balance as a major factor of AICAR antiproliferative effects.
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Cyclic-di-GMP regulates production of sortase substrates of Clostridium difficile and their surface exposure through ZmpI protease-mediated cleavage [Gene Regulation]

August 17th, 2015 by

In Gram-positive pathogens, surface proteins may be covalently anchored to the bacterial peptidoglycan by sortase, a cysteine transpeptidase enzyme. In contrast to other Gram-positive bacteria, only one single sortase enzyme, SrtB, is conserved between strains of Clostridium difficile. Sortase-mediated peptidase activity has been reported in vitro and seven potential substrates have been identified. Here we demonstrate the functionality of sortase in C. difficile. We identify two sortase-anchored proteins, the putative adhesins CD2831 and CD3246, and determine the cell wall anchor structure of CD2831. The C-terminal PPKTG sorting motif of CD2831 is cleaved between the threonine and glycine residues and the carboxyl group of threonine is amide-linked to the side chain amino group of diaminopimelic acid within the peptidoglycan peptide stem. We show that CD2831 protein levels are elevated in presence of high intracellular cyclic di-GMP (c-di-GMP) concentrations, in agreement with the control of CD2831 expression by a c-di-GMP-dependent type II riboswitch. Low c-di-GMP levels induce the release of CD2831, and presumably CD3246, from the surface of cells. This regulation is mediated by proteolytic cleavage of CD2831 and CD3246 by the zinc metalloprotease ZmpI, whose expression is controlled by a type II c-di-GMP riboswitch. These data reveal a novel regulatory mechanism for expression of two sortase substrates by the secondary messenger c-di-GMP, on which surface anchoring is dependent.
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Regulation of Rubisco Activase: Product Inhibition, Cooperativity, and Magnesium Activation [Plant Biology]

August 17th, 2015 by

In many photosynthetic organisms, tight-binding Rubisco inhibitors are released by the motor protein Rubisco activase (Rca). In higher plants, Rca plays a pivotal role in regulating CO2 fixation. Here, the ATPase activity of 0.005 mM tobacco Rca was monitored under steady-state conditions, and global curve fitting was utilized to extract kinetic constants. The kcat was best fit by 22.3 +/- 4.9 min-1, the Km for ATP by 0.104 +/- 0.024 mM, and the Ki for ADP by 0.037 +/- 0.007 mM. Without ADP, the Hill coefficient for ATP hydrolysis was extracted to be 1.0 +/- 0.1, indicating non-cooperative behavior of homo-oligomeric Rca assemblies. However, the addition of ADP was shown to introduce positive cooperativity between two or more subunits (Hill coefficient 1.9 +/- 0.2), allowing for regulation via the prevailing ATP/ADP ratio. ADP-mediated activation was not observed, whereas larger amounts led to competitive product inhibition of hydrolytic activity. The catalytic efficiency increased 8.4-fold upon cooperative binding of a second magnesium ion (Hill coefficient 2.5 +/- 0.5), suggesting at least three conformational states (ATP-bound, ADP-bound, empty) within assemblies containing an average of about six subunits. The addition of excess Rubisco (144:1, L8S8:Rca) and crowding agents did not modify catalytic rates. However, high magnesium provided for thermal Rca stabilization. We propose that magnesium mediates the formation of closed hexameric toroids capable of high turnover rates and amenable to allosteric regulation. We suggest that in vivo, the Rca hydrolytic activity is tuned by fluctuating [Mg2+] in response to changes in available light.

MMP Proteolysis of the Extracellular Loop of Voltage-gated Sodium Channels and Potential Alterations in Pain Signaling [Molecular Bases of Disease]

August 17th, 2015 by

Congenital insensitivity to pain (CIP) or congenital analgesia is a rare monogenic hereditary condition. This disorder is characterized by inability to perceive any form of pain. Nonsense mutations in Nav.1.7, the main pain signaling voltage-gated sodium channel, lead to its truncations and, consequently, to the inactivation of the channel functionality. However, a non-truncating homozygously-inherited missense mutation in a Bedouin family with CIP (Nav1.7-R907Q) is also reported. Based on our currently acquired in-depth knowledge of matrix metalloproteinase (MMP) cleavage preferences, we developed the specialized software that predicts the presence of the MMP cleavage sites in the peptide sequences. According to our in silico predictions, the peptide sequence of the exposed extracellular unstructured region linking the S5-S6 transmembrane segments in the DII domain of the human Nav1.7 sodium channel is highly sensitive to MMP-9 proteolysis. Intriguingly, the CIP R907Q mutation overlaps with the predicted MMP-9 cleavage site sequence. Using MMP-9 proteolysis of the wild-type, CIP and control peptides followed by mass-spectrometry of the digests, we demonstrated that the mutant sequence is several-fold more sensitive to MMP-9 proteolysis relative to the wild-type. Because of the substantial level of sequence homology among sodium channels, our data also implicate MMP proteolysis in regulating the cell-surface levels of the Nav1.7, Nav1.6 and Nav1.8 channels, but not Nav1.9. It is likely that the aberrantly accelerated MMP-9 proteolysis during neurogenesis is a biochemical rational for the functional inactivation in Nav1.7 and that the enhanced cleavage of the Nav1.7-R907Q mutant is a cause of CIP in the Bedouin family.
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Role of Pex21p for piggyback import of Gpd1p and Pnc1p into peroxisomes of Saccharomyces cerevisiae [Membrane Biology]

August 15th, 2015 by

Proteins designated for peroxisomal proteins import harbor one of two common peroxisomal targeting signals (PTS). In the yeast Saccharomyces cerevisiae, the oleate-induced PTS2-dependent import of the thiolase Fox3p into peroxisomes is conducted by the soluble import receptor Pex7p in cooperation with the auxiliary Pex18p, one of two supposedly redundant PTS2 co-receptors. Here we report on a novel function for the co-receptor Pex21p, which cannot be fulfilled by Pex18p. The data establish Pex21p as a general co-receptor in PTS2-dependent protein import, while Pex18p is especially important for oleate-induced import of PTS2-proteins. The glycerol-producing PTS2 protein glycerol-phosphate dehydrogenase Gpd1p shows a tripartite localization in peroxisomes, in the cytosol and in the nucleus under osmotic stress conditions. We show (i) that Pex21p is required for peroxisomal import of Gpd1p as well as a key enzyme of the NAD salvage pathway, Pnc1p, and (ii) that Pnc1p, a nicotinamidase without functional PTS2 is co-imported into peroxisomes by piggyback transport via Gpd1p. Moreover, the specific transport of these two enzymes into peroxisomes suggests a novel regulatory role for peroxisomes under various stress conditions.

Mycobacterium tuberculosis RecG but not RuvAB or RecA is efficient at remodeling the stalled replication forks: Implications for multiple mechanisms of replication restart in mycobacteria [Molecular Bases of Disease]

August 14th, 2015 by

Aberrant DNA replication, defects in the protection and restart of stalled replication forks are a major cause of genome instability in all organisms. Replication fork reversal is emerging as an evolutionarily conserved physiological response for restart of stalled forks. Escherichia coli RecG, RuvAB and RecA proteins have been shown to reverse the model replication fork structures in vitro. However, the pathways and the mechanisms by which Mycobacterium tuberculosis, a slow growing human pathogen responds to different types of replication stress and DNA damage is unclear. Here, we show that M. tuberculosis RecG rescues E. coli delta recG cells from replicative stress. The purified M. tuberculosis RecG (MtRecG) and RuvAB (MtRuvAB) proteins catalyze fork reversal of model replication fork structures with and without leading strand ssDNA gap. Interestingly, SSB suppresses the MtRecG and MtRuvAB mediated fork reversal with substrates that contain lagging strand gap. Notably, our comparative studies with fork structures containing template damage and template switching mechanism of lesion bypass reveal that MtRecG but not MtRuvAB or MtRecA is proficient in driving the fork reversal. Finally, unlike MtRuvAB, we find that MtRecG drives efficient reversal of forks when fork structures are tightly bound by protein. These results provide direct evidence and valuable insights into the underlying mechanism of MtRecG catalyzed replication fork remodeling and restart pathways in vivo.
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AMP-activated Protein Kinase Suppresses Arachidonate 15-Lipoxygenase Expression in Interleukin-4-polarized Human Macrophages [Lipids]

August 14th, 2015 by

Macrophages respond to the Th2 cytokine interleukin-4 (IL-4) with elevated expression of arachidonate 15-lipoxygenase (ALOX15). Although IL-4 signaling elicits anti-inflammatory responses, 15-lipoxygenase may either support or inhibit inflammatory processes in a context-dependent manner. AMP-activated protein kinase (AMPK) is a metabolic sensor/regulator that supports an anti-inflammatory macrophage phenotype. How AMPK-activation is linked to IL-4-elicited gene signatures remains unexplored. Using primary human macrophages stimulated with IL-4, we observed elevated ALOX15 mRNA and protein expression, which was attenuated by AMPK activation. AMPK activators, e.g. phenformin and aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) inhibited IL-4-evoked activation of signal transducer and activator of transcription (STAT) 3, while leaving activation of STAT6 and induction of typical IL-4-responsive genes intact. However, phenformin prevented IL-4-induced association of STAT6 and histone H3K9-acetylation at the ALOX15 promoter. Activating AMPK abolished cellular production of 15-lipoxygenase arachidonic acid metabolites in IL-4-stimulated macrophages, which was mimicked by an ALOX15 knockdown. Finally, pre-treatment of macrophages with IL-4 for 48h increased the mRNA expression of pro-inflammatory cytokines IL-6, IL-12, CXCL9, CXCL10 induced by subsequent stimulation with lipopolysaccharide. This response was attenuated by inhibition of ALOX15 or activation of AMPK during incubations with IL-4. In conclusion, limiting ALOX15 expression by AMPK may promote an anti-inflammatory phenotype of IL-4-stimulated human macrophages.

Structural Basis for the ATP-Dependent Configuration of Adenylation Active Site in Bacillus subtilis o-Succinylbenzoyl-CoA Synthetase [Protein Structure and Folding]

August 14th, 2015 by Chen, Y., Sun, Y., Song, H., Guo, Z.

o-Succinylbenzoyl-CoA synthetase, or MenE, is an essential adenylate-forming enzyme targeted for development of novel antibiotics in the menaquinone biosynthesis. Using its crystal structures in a ligand-free form or in complex with nucleotides, a conserved pattern is identified in the interaction between ATP and adenylating enzymes of the family including acyl/aryl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and luciferases. It involves tight gripping interactions of the phosphate-binding loop (P-loop) with the ATP triphosphate moiety and an open-closed conformational change to form a compact adenylation active site. In MenE catalysis, this ATP-enzyme interaction creates a new binding site for the carboxylate substrate, allowing revelation of the determinants of substrate specificities and inline alignment of the two substrates for backside nucleophilic substitution reaction by molecular modeling. In addition, the ATP-enzyme interaction is suggested to play a crucial catalytic role by mutation of the P-loop residues hydrogen-bonded to ATP. Moreover, the ATP-enzyme interaction has also clarified the positioning and catalytic role of a conserved lysine residue in stabilization of the transition state. These findings provide new insights into the adenylation half-reaction in the domain alteration catalytic mechanism of the adenylate-forming enzymes.
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The IVVY Motif and Tumor Necrosis Factor Receptor Associated Factor (TRAF) Sites in the Cytoplasmic Domain of the Receptor Activator of Nuclear Factor kappa B (RANK) Cooperate to Induce Osteoclastogenesis [Cell Biology]

August 14th, 2015 by Jules, J., Wang, S., Shi, Z., Liu, J., Wei, S., Feng, X.

RANK activation by RANK ligand (RANKL) mediates osteoclastogenesis by recruiting TRAFs via three cytoplasmic motifs (Motif 1: PFQEP369-373; Motif 2: PVQEET559-564; and Motif 3: PVQEQG604-609) to activate the NF-κB and MAPK signaling pathways. RANK also has a TRAF-independent motif (IVVY535-538) which is dispensable for the activation of TRAF-induced signaling pathways but is essential for osteoclast lineage commitment by inducing the expression of the nuclear factor of activated T-cells, c1 (NFATc1) to regulate osteoclast gene expression. Notably, TNF-/IL-1-mediated osteoclastogenesis requires RANKL assistance, and the IVVY motif is also critical for TNF-/IL-1-mediated osteoclastogenesis by rendering osteoclast genes responsive to these two cytokines. Here, we show that the two types of RANK cytoplasmic motifs have to be on the same RANK molecule to mediate osteoclastogenesis, suggesting a functional cooperation between them. Subsequent osteoclastogenesis assays with TNF or IL-1 revealed that while all three TRAF motifs play roles in TNF-/IL-1-mediated osteoclastogenesis, Motifs 2 and 3 are more potent than Motif 1. Accordingly, inactivation of Motifs 2 and 3 blocks TNF-/IL-1-mediated osteoclastogenesis. Mechanistically, double mutation of Motifs 2 and 3, similarly to inactivation of IVVY motif, abrogates the expression of NFATc1and osteoclast genes in assays reflecting RANK-initiated and TNF-/IL-1-mediated osteoclastogenesis. In contrast, double inactivation of Motifs 2 and 3 did not affect the ability of RANK to activate the NF-κB and MAPK signaling pathways. Collectively, these results indicate that the RANK IVVY motif cooperates with the TRAF-binding motifs to promote osteoclastogenesis, which provide a novel insight into the molecular mechanism of RANK signaling in osteoclastogenesis.
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