Genetic modulation of the serotonergic pathway: influence on weight reduction and weight maintenance

The serotonergic pathway plays a major role in the development of obesity. Its activity can be modulated by the 5-HT transporter–linked polymorphic region in the SLC6A4 gene and the upstream variable number of tandem repeats polymorphism in the MAOA gene. We studied whether these genetic modulations have an influence on weight reduction and weight maintenance in a one-year weight reduction program (OPTIFAST^®52). The polymorphisms were genotyped by PCR in a sample of 135 female and 67 male subjects with severe obesity (44 ± 13 years, 122.3 ± 22.2 kg, BMI: 41.7 ± 6.7 kg/m²). The program leads to a total weight loss of 19.9 ± 9.8 kg (16.9 ± 8.3 %) in women and 27.4 ± 13.6 kg (20.4 ± 9.9 %) in men. Anthropometric measurements and blood levels were determined at the start of the program (T0), after the weight reduction phase (T1) and after the subsequent weight maintenance phase at the end of the program (T2). Each polymorphism alone did not significantly influence weight loss or weight maintenance neither in men nor in women. However, women carrying both risk genotypes (SS and 3/3) displayed a lower total weight loss during the program (p = 0.05). This effect derived mainly from difficulties in the weight maintenance phase (p = 0.11), while the weight reduction phase was not affected (p = 0.61). No influence was found in men (p = 0.93). Modulation of the serotonergic pathway by carrying both risk alleles seems to influence success of weight loss programs in women with severe obesity due to problems in stabilizing body weight after weight reduction.     

TLR9 in peritoneal B-1b cells is essential for production of protective self-reactive IgM to control Th17 cells and severe autoimmunity

The role of TLR9 in the development of the autoimmune disease systemic lupus erythematosus is controversial. In different mouse models of the disease, loss of TLR9 abolishes the generation of anti-nucleosome IgG autoantibodies but at the same time exacerbates lupus disease. However, the TLR9-dependent tolerance mechanism is unknown. In this study, we show that loss of TLR9 is associated with low peritoneal B-1b cell numbers and low levels of protective self-reactive IgM serum autoantibodies in lupus-prone FcγRIIB-deficient mice leading to the uncontrolled accumulation of proinflammatory CD4(+) cells and exacerbated autoimmunity. TLR7 signaling was not able to compensate for the loss of TLR9 signaling in peritoneal B-1b cells to induce IgM Abs. Transfer of TLR9-expressing peritoneal B-1b cells from FcγRIIB-deficient mice or of recombinant monoclonal self-reactive IgM Abs was sufficient to reduce the frequency of proinflammatory Th17 cells and lupus disease in FcγRIIB/TLR9 double-deficient mice. Taken together, these data provide evidence for a TLR9-dependent tolerance mechanism of peritoneal B-1b cells generating protective self-reactive IgM in lupus-prone mice to control Th17 cell development and severe autoimmunity.     

The Rickettsia prowazekii ExoU Homologue Possesses Phospholipase A1 (PLA1), PLA2, and Lyso-PLA2 Activities and Can Function in the Absence of Any Eukaryotic Cofactors In Vitro

Here we have characterized the Rickettsia prowazekii RP534 protein, a homologue of the Pseudomonas aeruginosa ExoU phospholipase A (PLA) secreted cytotoxin. Our studies showed that purified recombinant RP534 PLA possessed the predicted PLA₂ and lyso-PLA₂ activities based on what has been published for P. aeruginosa ExoU. RP534 also displayed PLA₁ activity under the conditions tested, whereas ExoU did not. In addition, recombinant RP534 displayed a basal PLA activity that could hydrolyze phosphatidylcholine in the absence of any eukaryotic cofactors. Interestingly, the addition of bovine liver superoxide dismutase 1 (SOD1), a known activator of P. aeruginosa ExoU, resulted in an increased rate of RP534-catalyzed phospholipid hydrolysis, indicating that mechanisms of activation of the ExoU family of PLAs may be evolutionarily conserved. The mechanism of SOD1-dependent stimulation of RP534 was further examined using active site mutants and a fluorogenic phospholipid substrate whose hydrolysis by RP534 over a short time course is measureable only in the presence of SOD1. These studies suggest a mechanism by which SOD1 stimulates RP534 activity once it has bound to the substrate. We also show that antibody raised against RP534 was useful for immunoprecipitating active RP534 from R. prowazekii lysed cell extracts, thus verifying that this protein is expressed and active in rickettsiae isolated from embryonated hen egg yolk sacs.     

High-level expression of Rhodotorula gracilis d-amino acid oxidase in Pichia pastoris

By combining gene design and heterologous over-expression of Rhodotorula gracilis D-amino acid oxidase (RgDAO) in Pichia pastoris, enzyme production was enhanced by one order of magnitude compared to literature benchmarks, giving 350 kUnits/l of fed-batch bioreactor culture with a productivity of 3.1 kUnits/l h. P. pastoris cells permeabilized by freeze-drying and incubation in 2-propanol (10% v/v) produce a highly active (1.6 kUnits/g dry matter) and stable oxidase preparation. Critical bottlenecks in the development of an RgDAO catalyst for industrial applications have been eliminated.     

Hybridization and restricted gene flow between native and introduced stocks of Alpine whitefish (Coregonus sp.) across multiple environments

Translocations of Baltic whitefish (Coregonus sp.) into Austrian Alpine lakes have created 'artificial hybrid zones', threatening the genetic integrity of native lineages. We evaluate the genetic structure of Coregonus in Austrian lakes and characterize hybridization and introgression between native and introduced lineages. Fifteen populations (N=747) were assessed for allelic variation at eight microsatellite loci and a reduced set (N=253) for variation across two mtDNA genes (cyt b and NADH-3). Bayesian approaches were used to estimate individual admixture proportions (q-values) and classify genotypes as native, introduced or hybrids. q-value distributions varied among populations highlighting differential hybridization and introgression histories. Many lakes revealed a clear distinction between native and introduced genotypes despite hybridization, whereas some locations revealed hybrid swarms. Genetic structure among lakes was congruent with morphological divergence and novelty raising speculation of multiple taxa, including a population south of the Alps, outside the putative native range of Coregonus. Although statistically congruent with inferences based on nuclear markers, mitochondrial haplotype data was not diagnostic with respect to native and non-native lineages, supporting that the Alpine region was colonized post-glacially by an admixture of mtDNA lineages, which coalesce >1 Ma. Mechanisms promoting or eroding lineage isolation are discussed, as well as a high potential to conserve native Alpine lineages despite the extensive historical use of introduced Baltic stocks.     

Structure and Dynamics of a Compact State of a Multidomain Protein,the Mercuric Ion Reductase

The functional efficacy of colocalized, linked protein domains is dependent on linker flexibility and system compaction. However, the detailed characterization of these properties in aqueous solution presents an enduring challenge. Here, we employ a novel, to our knowledge, combination of complementary techniques, including small-angle neutron scattering, neutron spin-echo spectroscopy, and all-atom molecular dynamics and coarse-grained simulation, to identify and characterize in detail the structure and dynamics of a compact form of mercuric ion reductase (MerA), an enzyme central to bacterial mercury resistance. MerA possesses metallochaperone-like N-terminal domains (NmerA) tethered to its catalytic core domain by linkers. The NmerA domains are found to interact principally through electrostatic interactions with the core, leashed by the linkers so as to subdiffuse on the surface over an area close to the core C-terminal Hg(II)-binding cysteines. How this compact, dynamical arrangement may facilitate delivery of Hg(II) from NmerA to the core domain is discussed.     

Conformational State Distributions and Catalytically Relevant Dynamics of a Hinge-Bending Enzyme Studied by Single-Molecule FRET and a Coarse-Grained Simulation

Over the last few decades, a view has emerged showing that multidomain enzymes are biological machines evolved to harness stochastic kicks of solvent particles into highly directional functional motions. These intrinsic motions are structurally encoded, and Nature makes use of them to catalyze chemical reactions by means of ligand-induced conformational changes and states redistribution. Such mechanisms align reactive groups for efficient chemistry and stabilize conformers most proficient for catalysis. By combining single-molecule Förster resonance energy transfer measurements with normal mode analysis and coarse-grained mesoscopic simulations, we obtained results for a hinge-bending enzyme, namely phosphoglycerate kinase (PGK), which support and extend these ideas. From single-molecule Förster resonance energy transfer, we obtained insight into the distribution of conformational states and the dynamical properties of the domains. The simulations allowed for the characterization of interdomain motions of a compact state of PGK. The data show that PGK is intrinsically a highly dynamic system sampling a wealth of conformations on timescales ranging from nanoseconds to milliseconds and above. Functional motions encoded in the fold are performed by the PGK domains already in its ligand-free form, and substrate binding is not required to enable them. Compared to other multidomain proteins, these motions are rather fast and presumably not rate-limiting in the enzymatic reaction. Ligand binding slightly readjusts the orientation of the domains and feasibly locks the protein motions along a preferential direction. In addition, the functionally relevant compact state is stabilized by the substrates, and acts as a prestate to reach active conformations by means of Brownian motions.