The role of magnesium for geometry and charge in GTP hydrolysis, revealed by quantum mechanics/molecular mechanics simulations

The coordination of the magnesium ion in proteins by triphosphates plays an important role in catalytic hydrolysis of GTP or ATP, either in signal transduction or energy conversion. For example, in Ras the magnesium ion contributes to the catalysis of GTP hydrolysis. The cleavage of GTP to GDP and P(i) in Ras switches off cellular signaling. We analyzed GTP hydrolysis in water, Ras, and Ras·Ras-GTPase-activating protein using quantum mechanics/molecular mechanics simulations. By comparison of the theoretical IR-difference spectra for magnesium ion coordinated triphosphate to experimental ones, the simulations are validated. We elucidated thereby how the magnesium ion contributes to catalysis. It provides a temporary storage for the electrons taken from the triphosphate and it returns them after bond cleavage and P(i) release back to the diphosphate. Furthermore, the Ras·Mg(2+) complex forces the triphosphate into a stretched conformation in which the β- and γ-phosphates are coordinated in a bidentate manner. In this conformation, the triphosphate elongates the bond, which has to be cleaved during hydrolysis. Furthermore, the γ-phosphate adopts a more planar structure, driving the conformation of the molecule closer to the hydrolysis transition state. GTPase-activating protein enhances these changes in GTP conformation and charge distribution via the intruding arginine finger.     

Rap1a deficiency modifies cytokine responses and MAPK-signaling in vitro and impairs the in vivo inflammatory response

Rap1, which is closely related to ras, plays a key role in T-cell receptor (TCR)-signaling. TCR-stimulation without costimulation leads to constitutively activated rap1, which may mediate T-cell anergy via inhibition of ras-dependent induction of extracellular signal-regulated kinases (ERK). This activation is mediated by a second protein kinase b-Raf. Rap1-GTP is thought to activate ERK in a ras-independent manner by binding b-raf. Generally, T cells do not express b-raf while they express the adaptor protein raf-1, which is usually sequestered by rap1 leading to inhibition of ras-mediated ERK activation. In this study, we demonstrate that in rap1-deficient T cells, signaling by the ERK and p38 kinases is increased following activation by different stimuli leading to increased intracellular accumulation and secretion of cytokines. In addition, in a hypersensitivity model rap1-deficient mice demonstrated reduced contact dermatitis compared to wildtype mice, demonstrating the impact of rap1-deficiency on the inflammatory response in vivo.     

The chromodomain-containing NH<Subscript>2</Subscript>-terminus of Chromator interacts with histone H1 and is required for correct targeting to chromatin

The chromodomain protein, Chromator, can be divided into two main domains, a NH₂-terminal domain (NTD) containing the chromodomain (ChD) and a COOH-terminal domain (CTD) containing a nuclear localization signal. During interphase Chromator is localized to chromosomes; however, during cell division Chromator redistributes to form a macro molecular spindle matrix complex together with other nuclear proteins that contribute to microtubule spindle dynamics and proper chromosome segregation during mitosis. It has previously been demonstrated that the CTD is sufficient for targeting Chromator to the spindle matrix. In this study, we show that the NTD domain of Chromator is required for proper localization to chromatin during interphase and that chromosome morphology defects observed in Chromator hypomorphic mutant backgrounds can be largely rescued by expression of this domain. Furthermore, we show that the ChD domain can interact with histone H1 and that this interaction is necessary for correct chromatin targeting. Nonetheless, that localization to chromatin still occurs in the absence of the ChD indicates that Chromator possesses a second mechanism for chromatin association and we provide evidence that this association is mediated by other sequences residing in the NTD. Taken together these findings suggest that Chromator's chromatin functions are largely governed by the NH₂-terminal domain whereas functions related to mitosis are mediated mainly by COOH-terminal sequences.     

DHTKD1 Mutations Cause 2-Aminoadipic and 2-Oxoadipic Aciduria

Abnormalities in metabolite profiles are valuable indicators of underlying pathologic conditions at the molecular level. However, their interpretation relies on detailed knowledge of the pathways, enzymes, and genes involved. Identification and characterization of their physiological function are therefore crucial for our understanding of human disease: they can provide guidance for therapeutic intervention and help us to identify suitable biomarkers for monitoring associated disorders. We studied two individuals with 2-aminoadipic and 2-oxoadipic aciduria, a metabolic condition that is still unresolved at the molecular level. This disorder has been associated with varying neurological symptoms. Exome sequencing of a single affected individual revealed compound heterozygosity for an initiating methionine mutation (c.1A>G) and a missense mutation (c.2185G>A [p.Gly729Arg]) in DHTKD1. This gene codes for dehydrogenase E1 and transketolase domain-containing protein 1, which is part of a 2-oxoglutarate-dehydrogenase-complex-like protein. Sequence analysis of a second individual identified the same missense mutation together with a nonsense mutation (c.1228C>T [p.Arg410^∗]) in DHTKD1. Increased levels of 2-oxoadipate in individual-derived fibroblasts normalized upon lentiviral expression of the wild-type DHTKD1 mRNA. Moreover, investigation of L-lysine metabolism showed an accumulation of deuterium-labeled 2-oxoadipate only in noncomplemented cells, demonstrating that DHTKD1 codes for the enzyme mediating the last unresolved step in the L-lysine-degradation pathway. All together, our results establish mutations in DHTKD1 as a cause of human 2-aminoadipic and 2-oxoadipic aciduria via impaired turnover of decarboxylation 2-oxoadipate to glutaryl-CoA.     

Cuticular lipid profiles of fertile and non-fertile Cardiocondyla ant queens

Both mating and reproduction strongly affect the physiology of insect females. In the ant Cardiocondyla obscurior, a comparison among virgin queens, mated queens, and queens mated with sterilized males ("sham-mated") allows to separate the different effects of mating and egg laying. Here, we investigate whether and how different mating status is reflected in the cuticular lipid profiles of queens, i.e., the blend of chemicals that is thought to signal a queen's fertility. Surprisingly, discriminant analyses failed to reliably distinguish among virgin, mated, and sham-mated queens. A generalized linear model on individual substances showed only very subtle differences. While mating appeared to be positively associated with the proportions of 3-MeC(25,) 11-/13-MeC(27), 5-MeC(27), 3-MeC(27), and 12-/14-MeC(28) and negatively with C(27:1), fecundity was negatively associated with C(29:1), C(31:1), and a sterol derivative. We discuss these results in the light of the special life history of C. obscurior, with completely sterile workers and low egg laying rates in queens.     

Lipidomic analysis of lipid droplets from murine hepatocytes reveals distinct signatures for nutritional stress

Liver steatosis can be induced by fasting or high-fat diet. We investigated by lipidomic analysis whether such metabolic states are reflected in the lipidome of hepatocyte lipid droplets (LDs) from mice fed normal chow diet (FED), fasted (FAS), or fed a high-fat diet (HFD). LC-MS/MS at levels of lipid species profiles and of lipid molecular species uncovered a FAS phenotype of LD enriched in triacylglycerol (TG) molecular species with very long-chain (VLC)-PUFA residues and an HFD phenotype with less unsaturated TG species in addition to characteristic lipid marker species. Nutritional stress did not result in dramatic structural alterations in diacylglycerol (DG) and phospholipid (PL) classes. Moreover, molecular species of bulk TG and of DG indicated concomitant de novo TG synthesis and lipase-catalyzed degradation to be active in LDs. DG species with VLC-PUFA residues would be preferred precursors for phosphatidylcholine (PC) species, the others for TG molecular species. In addition, molecular species of PL classes fitted the hepatocyte Kennedy and phosphatidylethanolamine methyltransferase pathways. We demonstrate that lipidomic analysis of LDs enables phenotyping of nutritional stress. TG species are best suited for such phenotyping, whereas structural analysis of TG, DG, and PL molecular species provides metabolic insights.     

Metabolic adaptation of human pathogenic and related nonpathogenic bacteria to extra- and intracellular habitats

Most bacteria pathogenic for humans have closely related nonpathogenic counterparts that live as saprophytes, commensals or even symbionts (mutualists) in similar or different habitats. The knowledge of how these bacteria adapt their metabolism to the preferred habitats is critical for our understanding of pathogenesis, commensalism and symbiosis, and - in the case of bacterial pathogens - could help to identify targets for new antimicrobial agents. The focus of this review is on the metabolic potentials and adaptations of three different groups of human extra- and intracellular bacterial pathogens and their nonpathogenic relatives. All bacteria selected have the potential to reach the interior of mammalian host cells. However, their ability to replicate intracellularly differs significantly. The question therefore arises whether there are specific metabolic requirements that support stable intracellular replication. Furthermore, we discuss - whenever relevant data for the pathogenic representatives are available - the possible effect of the metabolism on the expression of virulence genes.     

Microbial CO2 fixation potential in a tar-oil-contaminated porous aquifer

CO(2) fixation is one of the most important processes on the Earth's surface, but our current understanding of the occurrence and importance of chemolithoautotrophy in the terrestrial subsurface is poor. Groundwater ecosystems, especially at organically polluted sites, have all the requirements for autotrophic growth processes, and CO(2) fixation is thus suggested to contribute significantly to carbon flux in these environments. We explored the potential for autotrophic CO(2) fixation in microbial communities of a petroleum hydrocarbon-contaminated aquifer by detection of functional marker genes (cbbL, cbbM), encoding different forms of the key enzyme RubisCO of the Calvin-Benson-Bassham cycle. Quantification of (red-like) cbbL genes revealed highest numbers at the upper fringe of the contaminant plume and the capillary fringe where reduced sulphur and iron species are regularly oxidized in the course of groundwater table changes. Functional gene sequences retrieved from this area were most closely related to sequences of different thiobacilli. Moreover, several cultures could be enriched from fresh aquifer material, all of which are able to grow under chemolithoautotrophic conditions. A novel, nitrate-reducing, thiosulfate-oxidizing bacterial strain, recently described as Thiobacillus thiophilus D24TN(T) sp. nov., was shown to carry and transcribe RubisCO large-subunit genes of form I and II. Enzyme tests proved the actual activity of RubisCO in this strain.