Mechanistic insight into allosteric activation of human pyruvate carboxylase by acetyl-CoA

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Mechanistic insight into light-dependent recognition of Timeless by Drosophila Cryptochrome

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Mechanistic insight into mycobacterial MmpL protein function

Mycobacterial cell walls are complex structures containing a broad range of unusual lipids, glycolipids and other polymers, some of which act as immunomodulators or virulence determinants. Better understanding of the enzymes involved in export processes would enlighten cell wall biogenesis. Bernut et al. ( 2015 ) present the findings of a structural and functional investigation of one of the most important transporter families, the MmpL proteins, members of the resistance‐nodulation‐cell division (RND) superfamily. A Tyr842His missense mutation in the mmpL4a gene was shown to be responsible for the smooth‐to‐rough morphotype change of the near untreatable opportunistic pathogen Mycobacterium bolletii due to its failure to export a glycopeptidolipid (GPL). This mutation was pleiotropic and markedly increased virulence in infection models. Tyr842 is well conserved in all actinobacterial MmpL proteins suggesting that it is functionally important and this was confirmed by several approaches including replacing the corresponding residue in MmpL3 of Mycobacterium tuberculosis. Structural modelling combined with experimental results showed Tyr842 to be a critical residue for mediating the proton motive force required for GPL export. This mechanistic insight applies to all MmpL proteins and probably to all RND transporters.     

Mechanistic insight into the photosensory versatility of DXCF cyanobacteriochromes

Cyanobacteriochromes (CBCRs) are photosensory proteins related to the red/far-red phytochromes. Like phytochromes, CBCRs use linear tetrapyrrole (bilin) chromophores covalently attached via a thioether linkage to a conserved Cys residue also found in plant and cyanobacterial phytochromes. Unlike almost all phytochromes, CBCRs require only an isolated GAF domain to undergo efficient, reversible photocycles that are responsible for their broad light sensing range, spanning the visible to the near ultraviolet (UV). Sensing of blue, violet, and near-UV light by CBCRs requires another Cys residue proposed to form a second linkage to the bilin precursor. Light triggers 15,16-double bond isomerization as in phytochromes. After photoisomerization, elimination of the second linkage frequently occurs, thus yielding a large red shift of the stable photoproducts. Here we examine this process for representative DXCF CBCRs, a large subfamily named for the conserved Asp-Xaa-Cys-Phe motif that contains their second Cys residue. DXCF CBCRs with such dual-Cys photocycles yield a wide diversity of photoproducts absorbing teal, green, or orange light. Using a combination of CD spectroscopy, chemical modification, and bilin substitution experiments with recombinant CBCRs from Thermosynechococcus elongatus and Nostoc punctiforme expressed in Escherichia coli, we establish that second-linkage elimination is required for all of these photocycles. We also identify deconjugation of the D-ring as the mechanism for specific detection of teal light, at approximately 500 nm. Our studies thus provide new mechanistic insight into the photosensory versatility of this important family of photosensory proteins.     

Mechanistic insight into how Shh patterns the vertebrate limb

The hands and feet of a newborn baby are a beautiful reminder of the complexity of embryonic patterning. Classical studies on how these structures form have led to a theoretical framework for understanding, in general, how discrete groups of cells can instruct differential fates across a wider field through the action of long-range signals. The discovery just more than a decade ago that localized expression of Sonic hedgehog (Shh) differentially patterns structures across the limb field, resulting in digits with unique characteristics, provided a starting point for readdressing these models at a molecular level. Current research has revealed unexpected complexity in how a gradient of Shh activity is both established and received, prompting re-evaluation of the nature of patterning mechanisms within the limb.     

Mechanistic insight into the catalytic inhibition by nitroxides of tyrosine oxidation and nitration

BackgroundNitroxide antioxidants (RNO^•) protect from injuries associated with oxidative stress. Tyrosine residues in proteins are major targets for oxidizing species giving rise to irreversible cross-linking and protein nitration, but the mechanisms underlying the protective activity of RNO^• on these processes are not sufficiently clear.MethodsTyrosine oxidation by the oxoammonium cation (RN⁺=O) was studied by following the kinetics of RNO^• formation using EPR spectroscopy. Tyrosine oxidation and nitration were investigated using the peroxidase/H₂O₂ system without and with nitrite. The inhibitory effect of RNO^• on these processes was studied by following the kinetics of the evolved O₂ and accumulation of tyrosine oxidation and nitration products.ResultsTyrosine ion is readily oxidized by RN⁺=O, and the equilibrium constant of this reaction depends on RNO^• structure and reduction potential. RNO^• catalytically inhibits tyrosine oxidation and nitration since it scavenges both tyrosyl and ^•NO₂ radicals while recycling through RN⁺=O reduction by H₂O₂, tyrosine and nitrite. The inhibitory effect of nitroxide on tyrosine oxidation and nitration increases as its reduction potential decreases where the 6-membered ring nitroxides are better catalysts than the 5-membered ones.ConclusionsNitroxides catalytically inhibit tyrosine oxidation and nitration. The proposed reaction mechanism adequately fits the results explaining the dependence of the nitroxide inhibitory effect on its reduction potential and on the concentrations of the reducing species present in the system.General significanceNitroxides protect against both oxidative and nitrative damage. The proposed reaction mechanism further emphasizes the role of the reducing environment to the efficacy of these catalysts.     

Mechanistic insight into the procoagulant activity of tumor-derived apoptotic vesicles

BackgroundChemotherapy induces the release of apoptotic vesicles (ApoV) from the tumor plasma membrane. Tumor ApoV may enhance the risk of thrombotic events in cancer patients undergoing chemotherapy. However, the relative contribution of ApoV to coagulation and the pathways involved remain poorly characterized. In addition, this study sets out to compare the procoagulant activity of chemotherapy-induced ApoV with their cell of origin and to determine the mechanisms of ApoV-induced coagulation.MethodsWe utilized human and murine cancer cell lines and chemotherapeutic agents to determine the requirement for the coagulation factors (tissue factor; TF, FII, FV, FVII, FVIII, FIX and phosphatidylserine) in the procoagulant activity of ApoV. The role of previously identified ApoV-associated FV was determined in a FV functional assay.ResultsApoV were significantly more procoagulant per microgram of protein compared to parental living or dying tumor cells. In the phase to peak fibrin generation, procoagulant activity was dependent on phosphatidylserine, TF expression, FVII and the prothrombinase complex. However, the intrinsic coagulation factors FIX and FVIII were dispensable. ApoV-associated FV could not support coagulation in the absence of supplied, exogenous FV.ConclusionsApoV are significantly more procoagulant than their parental tumor cells. ApoV require the extrinsic tenase and prothrombinase complex to activate the early phase of coagulation. Endogenous FV identified on tumor ApoV is serum-derived and functional, but is non-essential for ApoV-mediated fibrin generation.General significanceThis study clarifies the mechanisms of procoagulant activity of vesicles released from dying tumor cells.     

Mechanistic insight into the catechol oxidase activity by a biomimetic dinuclear copper complex

The biomimetic catalytic oxidation of 3,5-di-tert-butylcatechol by the dicopper(II) complex of the ligand ,-bis{bis[1-(1-methyl-2-benzimidazolyl)methyl]amino}-m-xylene in the presence of dioxygen has been investigated as a function of temperature and pH in a mixed aqueous/organic solvent. The catalytic cycle occurs in two steps, the first step being faster than the second step. In the first step, one molecule of catechol is oxidized by the dicopper(II) complex, and the copper(II) centers are reduced. From the pH dependence, it is deduced that the active species of the process is the monohydroxo form of the dinuclear complex. In the second step, the second molecule of catechol is oxidized by the dicopper(I)-dioxygen complex formed upon oxygenation of the reduced complex. In both cases, catechol oxidation is an inner-sphere electron transfer process involving binding of the catechol to the active species. The binary catechol-dicopper(II) complex formed in the first step could be characterized at very low temperature (–90 °C), where substrate oxidation is blocked. On the contrary, the ternary complex of dicopper(I)-O₂-catechol relevant to the second step does not accumulate in solution and could not be characterized, even at low temperature. The investigation of the biphasic kinetics of the catalytic reaction over a range of temperatures allowed the thermodynamic (H° and S°) and activation parameters (H and S) connected with the key steps of the catecholase process to be obtained.     

Structural insight into the mechanisms of Wnt signaling antagonism by Dkk

Dickkopf (Dkk) proteins are antagonists of the canonical Wnt signaling pathway and are crucial for embryonic cell fate and bone formation. Wnt antagonism of Dkk requires the binding of the C-terminal cysteine-rich domain of Dkk to the Wnt coreceptor, LRP5/6. However, the structural basis of the interaction between Dkk and low density lipoprotein receptor-related protein (LRP) 5/6 is unknown. In this study, we examined the structure of the Dkk functional domain and elucidated its interactions with LRP5/6. Using NMR spectroscopy, we determined the solution structure of the C-terminal cysteine-rich domain of mouse Dkk2 (Dkk2C). Then, guided by mutagenesis studies, we docked Dkk2C to the YWTD beta-propeller domains of LRP5/6 and showed that the ligand binding site of the third LRP5/6 beta-propeller domain matches Dkk2C best, suggesting that this domain binds to Dkk2C with higher affinity. Such differential binding affinity is likely to play an essential role in Dkk function in the canonical Wnt pathway.     

Mechanistic insight into the RNA-stimulated ATPase activity of tick-borne encephalitis virus helicase

The helicase domain of nonstructural protein 3 (NS3H) unwinds the double-stranded RNA replication intermediate in an ATP-dependent manner during the flavivirus life cycle. While the ATP hydrolysis mechanism of Dengue and Zika viruses NS3H has been extensively studied, little is known in the case of the tick-borne encephalitis virus NS3H. We demonstrate that ssRNA binds with nanomolar affinity to NS3H and strongly stimulates the ATP hydrolysis cycle, whereas ssDNA binds only weakly and inhibits ATPase activity in a noncompetitive manner. Thus, NS3H is an RNA-specific helicase, whereas DNA might act as an allosteric inhibitor. Using modeling, we explored plausible allosteric mechanisms by which ssDNA inhibits the ATPase via nonspecific binding in the vicinity of the active site and ATP repositioning. We captured several structural snapshots of key ATP hydrolysis stages using X-ray crystallography. One intermediate, in which the inorganic phosphate and ADP remained trapped inside the ATPase site after hydrolysis, suggests that inorganic phosphate release is the rate-limiting step. Using structure-guided modeling and molecular dynamics simulation, we identified putative RNA-binding residues and observed that the opening and closing of the ATP-binding site modulates RNA affinity. Site-directed mutagenesis of the conserved RNA-binding residues revealed that the allosteric activation of ATPase activity is primarily communicated via an arginine residue in domain 1. In summary, we characterized conformational changes associated with modulating RNA affinity and mapped allosteric communication between RNA-binding groove and ATPase site of tick-borne encephalitis virus helicase.     

Mechanistic insight into the ribosome biogenesis functions of the ancient protein KsgA

While the general blueprint of ribosome biogenesis is evolutionarily conserved, most details have diverged considerably. A striking exception to this divergence is the universally conserved KsgA/Dim1p enzyme family, which modifies two adjacent adenosines in the terminal helix of small subunit ribosomal RNA (rRNA). While localization of KsgA on 30S subunits [small ribosomal subunits (SSUs)] and genetic interaction data have suggested that KsgA acts as a ribosome biogenesis factor, mechanistic details and a rationale for its extreme conservation are still lacking. To begin to address these questions we have characterized the function of Escherichia coli KsgA in vivo using both a ksgA deletion strain and a methyltransferase-deficient form of this protein. Our data reveal cold sensitivity and altered ribosomal profiles are associated with a DeltaksgA genotype in E. coli. Our work also indicates that loss of KsgA alters 16S rRNA processing. These findings allow KsgAs role in SSU biogenesis to be integrated into the network of other identified factors. Moreover, a methyltransferase-inactive form of KsgA, which we show to be deleterious to cell growth, profoundly impairs ribosome biogenesis-prompting discussion of KsgA as a possible antimicrobial drug target. These unexpected data suggest that methylation is a second layer of function for KsgA and that its critical role is as a supervisor of biogenesis of SSUs in vivo. These new findings and this proposed regulatory role offer a mechanistic explanation for the extreme conservation of the KsgA/Dim1p enzyme family.     

Mechanistic insight into the peroxidase catalyzed nitration of tyrosine derivatives by nitrite and hydrogen peroxide.

Peroxidases perform the nitration of tyrosine and tyrosyl residues in proteins, in the presence of nitrite and hydrogen peroxide. The nitrating species is still unknown but it is usually assumed to be nitrogen dioxide. In the present investigation, the nitration of phenolic compounds derived from tyrosine by lactoperoxidase and horseradish peroxidase was studied, with the aim of elucidating the mechanism of the reaction. The results indicate that nitrogen dioxide cannot be the only nitrating species and suggest the presence of two simultaneously operative pathways, one proceeding through enzyme-generated nitrogen dioxide and another through a more reactive species, assumed to be complexed peroxynitrite, which is generated by reaction of hydrogen peroxide with the enzyme-nitrite complex. The importance of the two pathways depends on peroxide and nitrite concentrations. With lactoperoxidase, nitration through the highly reactive intermediate is preferred except at very low nitrite concentration, while with horseradish peroxidase, the nitrogen dioxide driven mechanism is preferred except at very high nitrite concentration. The preferred mechanism for the two enzymes is that operative in the physiological nitrite concentration range.     

Mechanistic insight into exclusive nitric oxide recovery from a carbon-bound diazeniumdiolate.

We report that NaON=N(O)-X-N(O)=NONa (1), where X is para-disubstituted benzene, hydrolyzes to 2 mol of nitric oxide (NO) with concurrent production of 1 mol of p-benzoquinone dioxime at physiological pH. The reaction is acid catalyzed, with a rate that slows as the substrate concentration is increased. The results demonstrate that a carbon-bound diazeniumdiolate can be quantitatively hydrolyzed to produce NO as the only gaseous nitrogen-containing product. The data also suggest that N-N bond cleavage is the rate-determining step in NO release, since C-N cleavage followed by dissociation of O=N-N=O to two NO molecules cannot be operative in this case. The finding that this oxime can absorb NO in organic media and regenerate it quantitatively at physiological pHs extends the potential pharmacological implications of the carbon-bound diazeniumdiolates.     

Mechanistic insight into heterogeneity of trans-plasma membrane electron transport in cancer cell types

Trans-plasma membrane electron transfer (tMPET) is a process by which reducing equivalents, either electrons or reductants like ascorbic acid, are exported to the extracellular environment by the cell. TPMET is involved in a number of physiological process and has been hypothesised to play a role in the redox regulation of cancer metabolism. Here, we use a new electrochemical assay to elucidate the ‘preference’ of cancer cells for different trans tPMET systems. This aids in proving a biochemical framework for the understanding of tPMET role, and for the development of novel tPMET-targeting therapeutics. We have delineated the mechanism of tPMET in 3 lung cancer cell models to show that the external electron transfer is orchestrated by ascorbate mediated shuttling via tPMET. In addition, the cells employ a different, non-shuttling-based mechanism based on direct electron transfer via Dcytb. Results from our investigations indicate that tPMETs are used differently, depending on the cell type. The data generated indicates that tPMETs may play a fundamental role in facilitation of energy reprogramming in malignant cells, whereby tPMETs are utilised to supply the necessary energy requirement when mitochondrial stress occurs. Our findings instruct a deeper understanding of tPMET systems, and show how different cancer cells may preferentially use distinguishable tPMET systems for cellular electron transfer processes.     

MAPK activation by radio waves

In this issue of the Biochemical Journal, Freidman et al. report the findings of a study to look at the potential of mobile phones to activate intracellular signalling cascades. They found that radio waves corresponding to the frequency commonly used by mobile phones are able to activate ERK1/2 (extracellular-signal-regulated kinases 1 and 2). This effect was observed even at intensities lower than those emitted by mobile phones that are unable to cause any measurable heating effects. This study provides evidence that radio waves induce ERK1/2 activation downstream of the EGF (epidermal growth factor) receptor, which is in turn activated by the release of reactive oxygen species.     

Memory-improving effect of formulation-MSS by activation of hippocampal MAPK/ERK signaling pathway in rats

MSS, a comprising mixture of maesil (Prunus mume Sieb. et Zucc) concentrate, disodium succinate and Span80 (3.6:4.6 :1 ratio) showed a significant improvement of memory when daily administered (460 mg/kg day, p.o.) into the normal rats for 3 weeks. During the spatial learning of 4 days in Morris water maze test, both working memory and short-term working memory index were significantly increased when compared to untreated controls. We investigated a molecular signal transduction mechanism of MSS on the behaviors of spatial learning and memory. MSS treatment increased hippocampal mRNA levels of NR2B and TrkB without changes of NR1, NR2A, ERK1, ERK2 and CREB. However, the protein levels of pERK/ERK and pCREB/CREB were all significantly increased to 1.5+/-0.17 times. These results suggest that the improving effect of spatial memory for MSS is linked to MAPK/ERK signaling pathway that ends up in the phosphorylation of CREB through TrkB and/or NR2B of NMDA receptor.