Asn-150 of murine erythroid 5-aminolevulinate synthase modulates the catalytic balance between the rates of the reversible reaction [Protein Structure and Folding]

October 28th, 2015 by Stojanovski, B. M., Ferreira, G. C.

5-Aminolevulinate synthase (ALAS) catalyzes the first step in mammalian heme biosynthesis, the pyridoxal 5-phosphate (PLP)-dependent and reversible reaction between glycine and succinyl-CoA to generate CoA, CO2, and 5-aminolevulinate (ALA). Apart from coordinating the positioning of succinyl-CoA, Rhodobacter capsulatus ALAS Asn85 has a proposed role in regulating the opening of an active site channel. Here, we constructed a library of murine erythroid ALAS variants with substitutions at the position occupied by the analogous bacterial asparagine, screened for ALAS function and characterized the catalytic properties of the N150H and N150F variants. Quinonoid intermediate formation occurred with a significantly reduced rate for either the N150H- or N150F-catalyzed condensation of glycine with succinyl-CoA during a single turnover. The introduced mutations caused modifications in the ALAS active site such that the resulting variants tipped the balance between the forward- and reverse-catalyzed reactions. While wild-type ALAS catalyzes the conversion of ALA into the quinonoid intermediate at a rate 6.3-fold slower than the formation of the same quinonoid intermediate from glycine and succinyl-CoA, the N150F variant catalyzes the forward reaction at a mere 1.2-fold faster rate than that of the reverse reaction, and the N150H variant reverses the rate values with a 1.7-fold faster rate for the reverse reaction than that for the forward reaction. We conclude that the evolutionary selection of Asn150 was significant for optimizing the forward enzymatic reaction at the expense of the reverse, and thus, ensuring that ALA is predominantly available for heme biosynthesis.
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Hsp90-Cdc37 complexes with protein kinases form cooperatively with multiple distinct interaction sites [Protein Structure and Folding]

October 28th, 2015 by

Protein kinases are the most prominent group of Hsp90 (heat shock protein 90) clients and are recruited to the molecular chaperone by the kinase specific cochaperone Cdc37 (cell division cycle 37). The interaction between Hsp90 and nematode Cdc37 is mediated by binding of the Hsp90 middle domain to an N-terminal region of C. elegans Cdc37 (CeCdc37). Here we map the binding site by NMR spectroscopy and define amino acids relevant for the interaction between CeCdc37 and the middle domain of Hsp90. Apart from these distinct Cdc37/Hsp90 interfaces binding of the B-Raf protein kinase to the cochaperone is conserved between mammals and nematodes. In both cases, the C-terminal part of Cdc37 is relevant for kinase binding, whereas the N-terminal domain displaces the nucleotide from the kinase. This interaction leads to a cooperative formation of the ternary complex of Cdc37 and kinase with Hsp90. For the MAP-kinase (mitogen activated protein kinase) Erk2 (extracellular-signal regulated kinase 2) we observe that certain features of the interaction with Cdc37-Hsp90 are conserved, but the contribution of Cdc37 domains varies slightly, implying that different kinases may utilize distinct variations of this binding mode to interact with the Hsp90 chaperone machinery.

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: Evidence for a Common Reaction Mechanism [Protein Structure and Folding]

October 28th, 2015 by Booth, E. S., Basran, J., Lee, M., Handa, S., Raven, E. L.

The kynurenine pathway is the major route of L-tryptophan (L-Trp) catabolism in biology, leading ultimately to the formation of NAD+. The initial and rate-limiting step of the kynurenine pathway involves oxidation of L-Trp to N-formylkynurenine (NFK). This is an O2-dependent process and catalyzed by indoleamine 2,3 dioxygenase (IDO). More than sixty years after these enzymes were first isolated (1), the mechanism of their reaction is not established. We have examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observe formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of L-Trp, 1-Me-L-Trp and a number of other substrate analogues. The data are consistent with a common reaction mechanism for IDO-catalyzed oxidation of tryptophan and other tryptophan analogues.

Insulin dissociates the effects of Liver X Receptor on lipogenesis, endoplasmic reticulum stress and inflammation [Metabolism]

October 28th, 2015 by

Diabetes is characterized by increased lipogenesis as well as increased endoplasmic reticulum (ER) stress and inflammation. The nuclear hormone receptor Liver X Receptor is induced by insulin and is a key regulator of lipid metabolism. It promotes lipogenesis and cholesterol efflux, but suppresses endoplasmic reticulum stress and inflammation. The goal of these studies was to dissect the effects of insulin on LXR action. We used antisense oligonucleotides to knockdown Lxrα in mice with hepatocyte-specific deletion of the insulin receptor and their controls. We found, surprisingly, that knockout of the insulin receptor and knockdown of Lxrα produced equivalent, non-additive effects on the lipogenic genes. Thus, insulin was unable to induce the lipogenic genes in the absence of Lxrα, and LXRα was unable to induce the lipogenic genes in the absence of insulin. However, insulin was not required for LXRα to modulate the phospholipid profile, or to suppress genes in the ER stress or inflammation pathways. These data show that insulin is required specifically for the lipogenic effects of LXRα and that manipulation of the insulin signaling pathway could dissociate the beneficial effects of LXR on cholesterol efflux, inflammation and ER stress, from the negative effects on lipogenesis.

In Vivo Studies in Rhodospirillum rubrum Indicate that Ribulose-1,5,bisphosphate carboxylase/oxygenase (Rubisco) Catalyzes Two Obligatorily Required and Physiologically Significant Reactions for Distinct Carbon and Sulfur Metabolic Pathways [Microbiology]

October 28th, 2015 by Dey, S., North, J. A., Sriram, J., Evans, B. S., Tabita, F. R.

All organisms possess fundamental metabolic pathways to insure that needed carbon and sulfur compounds are provided to the cell in the proper chemical form and oxidation state. For most organisms capable of using CO2 as sole source of carbon, ribulose-1,5-bisphosphate (RuBP) carboxylase/oxy-genase (Rubisco) catalyzes primary carbon dioxide assimilation. In addition, sulfur salvage pathways are necessary to insure that key sulfur-containing compounds are both available and, where necessary, detoxified in the cell. Using knockout mutations and metabolomics in the bacterium Rhodospirillum rubrum, we show here that Rubisco concurrently catalyzes key and essential reactions for seemingly unrelated but physiologically essential central carbon and sulfur salvage metabolic pathways of the cell. In this study, complementation and mutagenesis studies indicated representatives of all extant known functional Rubisco forms found in nature are capable of simultaneously catalyzing reactions required for both CO2-dependent growth as well as growth using 5-methylthioadenosine (MTA) as sole sulfur source under anaerobic photosynthetic conditions. Moreover, specific inactivation of the CO2 fixation reaction did not affect the ability of Rubisco to support anaerobic MTA metabolism, suggesting that the active site of Rubisco has evolved to insure that this enzyme maintains both key functions. Thus, despite the coevolution of both functions, the active site of this protein may be differentially modified to affect only one of its key functions
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Plasticity in repressor-DNA interactions neutralizes loss of symmetry in bipartite operators [Gene Regulation]

October 28th, 2015 by Jain, D., Narayanan, N., Nair, D. T.

Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. In order to understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators- ORE1, ORX1, ORA1 and ORR3- the AraR-DBD as well as full length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07Å and 1.97Å resolution respectively. These structures are compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with symmetric operator, ORE1 are identical, but their interaction with non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. Chip-Seq data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits.

The Disulfide Bond, but not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu-Zn Superoxide Dismutase (SOD1) Fibrillation [Protein Structure and Folding]

October 28th, 2015 by

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of posttranslational modifications: Formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact C57-C146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above posttranslational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.
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Hyperosmotic Shock Engages Two Positive Feedback Loops Through Caspase-3 Dependent Proteolysis of JNK1-2 and Bid [Signal Transduction]

October 28th, 2015 by Yue, J., Ben Messaoud, N., Lopez, J. M.

Hyperosmotic shock induces early calpain activation, Smac/DIABLO release from the mitochondria and p38/JNK activation in Xenopus oocytes. These pathways regulate late cytochrome c release and caspase-3 activation. Here we show that JNK1-1 and JNK1-2 are early activated by osmostress and sustained activation of both isoforms accelerates the apoptotic program. When caspase-3 is activated JNK1-2 is proteolyzed at Asp385 increasing the release of cytochrome c and caspase-3 activity, and therefore creating a positive feedback loop. Expression of Bcl-xL markedly reduces hyperosmotic shock-induced apoptosis. In contrast, expression of Bid induces rapid caspase-3 activation, even in the absence of osmostress, which is blocked by Bcl-xL co-expression. In these conditions a significant amount of Bid in the cytosol is mono- and biubiquitinated. Caspase-3 activation by hyperosmotic shock induces proteolysis of Bid and mono-ubiquitinated Bid at Asp52 increasing the release of cytochrome c and caspase-3 activation, and thus creating a second positive feedback loop. Revealing the JNK isoforms and the loops activated by osmostress could help to design better treatments for human diseases caused by perturbations in fluid osmolarity.

Structure-function analysis of a mixed-linkage Beta-glucanase/xyloglucanase from key ruminal Bacteroidetes Prevotella bryantii B14 [Glycobiology and Extracellular Matrices]

October 27th, 2015 by

The recent classification of Glycoside Hydrolase Family 5 (GH5) members into subfamilies enhances the prediction of substrate specificity by phylogenetic analysis. However, the small number of well-characterized members is a current limitation to understanding the molecular basis of the diverse specificity observed across individual GH5 subfamilies. GH5 Subfamily 4 (GH5_4) is one of the largest, with known activities comprising (carboxymethyl)cellulases, mixed-linkage endo-glucanases, and endo-xyloglucanases. Through detailed structure-function analysis, we have revisited the characterization of a classic GH5_4 carboxymethylcellulase, PbGH5A (also known as Orf4, CMCase, and Cel5A) from the symbiotic rumen Bacteroidetes Prevotella bryantii B14. We demonstrate that CMC and phosphoric acid-swollen cellulose (PASC) are in fact strikingly poor substrates for PbGH5A, which instead exhibits clear primary specificity for the plant storage and cell wall polysaccharide, mixed-linkage β-glucan. Significant activity toward the plant cell wall polysaccharide xyloglucan was also observed. Determination of PbGH5A crystal structures in the apo form and in complex with (xylo)glucan oligosaccharides and an active-site affinity label, together with detailed kinetic analysis using a variety of well-defined oligosaccharide substrates, revealed the structural determinants of polysaccharide substrate specificity. In particular, this analysis highlighted the PbGH5A active-site motifs which engender predominant mixed-linkage endo-glucanase activity vis-a-vis predominant endo-xyloglucanases in GH5_4. However the detailed phylogenetic analysis of GH5_4 members did not delineate particular clades of enzymes sharing these sequence motifs; the phylogeny was instead dominated by bacterial taxonomy. Nonetheless, our results provide key enzyme functional and structural reference data for future bioinformatics analyses of meta(genomes) to elucidate the biology of complex gut ecosystems.
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Unmasking of CD22 on germinal center B-cells occurs by alternative mechanisms in mouse and man [Glycobiology and Extracellular Matrices]

October 27th, 2015 by

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here we show in both mouse and man that loss of high-affinity ligands on GC B-cells unmask the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking since high-affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has Neu5Ac as the Sia, and the high affinity ligand on naive B cells contains 6-O-sulfate on the GlcNAc. On human GC cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mCD22 has Neu5Gc as the Sia, which is replaced on GC B cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high-affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high-affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high-affinity CD22 ligands on GC B-cells occurs in both mouse and man through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells.