SIRT3 deficiency and mitochondrial protein hyperacetylation accelerate the development of the metabolic syndrome

Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic efficiency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.     

NOA1 is an essential GTPase required for mitochondrial protein synthesis

Nitric oxide associated-1 (NOA1) is an evolutionarily conserved guanosine triphosphate (GTP) binding protein that localizes predominantly to mitochondria in mammalian cells. On the basis of bioinformatic analysis, we predicted its possible involvement in ribosomal biogenesis, although this had not been supported by any experimental evidence. Here we determine NOA1 function through generation of knockout mice and in vitro assays. NOA1-deficient mice exhibit midgestation lethality associated with a severe developmental defect of the embryo and trophoblast. Primary embryonic fibroblasts isolated from NOA1 knockout embryos show deficient mitochondrial protein synthesis and a global defect of oxidative phosphorylation (OXPHOS). Additionally, Noa1–/– cells are impaired in staurosporine-induced apoptosis. The analysis of mitochondrial ribosomal subunits from Noa1–/– cells by sucrose gradient centrifugation and Western blotting showed anomalous sedimentation, consistent with a defect in mitochondrial ribosome assembly. Furthermore, in vitro experiments revealed that intrinsic NOA1 GTPase activity was stimulated by bacterial ribosomal constituents. Taken together, our data show that NOA1 is required for mitochondrial protein synthesis, likely due to its yet unidentified role in mitoribosomal biogenesis. Thus, NOA1 is required for such basal mitochondrial functions as adenosine triphosphate (ATP) synthesis and apoptosis.     

Ancipirhynchus afossalis n. g., n. sp. (Trypanorhyncha: Otobothriidae), from two species of sharks off Indonesian and Malaysian Borneo

A new genus of trypanorhynch cestode is described from two species of sharks, the sliteye shark Loxodon macrorhinus Müller & Henle and the straight-tooth weasel shark Paragaleus tengi (Chen) collected in the Makassar Strait (off Indonesian Borneo) and Sulu Sea (off Malaysian Borneo). Ancipirhynchus afossalis n. g., n. sp. possesses two bothria and a heteroacanthous, heteromorphous tentacular armature with three distinctive files of hooks on the external tentacle surface but lacks prebulbar organs and gland cells within the tentacular bulbs. The hook arrangement of alternating files on the external surface of the tentacle resembles that seen in the superfamily Otobothrioidea Dollfus, 1942 in the genus Fossobothrium Beveridge & Campbell, 2005. However, the new species lacks the defining characteristic of this group, i.e. the paired bothrial pits. A Bayesian inference (BI) analysis of 37 LSU sequences of trypanorhynchs from three superfamilies provided evidence supporting the taxonomic placement of Ancipirhynchus afossalis n. g., n. sp. within the Otobothrioidea.     

Novel yeast bioassay for high-throughput screening of matrix metalloproteinase inhibitors

Diverse malfunctions in the expression and regulation of matrix metalloproteinases (MMPs) are often the cause of severe human diseases, bringing the identification of specific MMP inhibitors into major focus, particularly in anticancer treatment. Here, we describe a novel bioassay based on recombinant yeast cells (Pichia pastoris) that express, deliver, and incorporate biologically active human MMP-2 and MMP-9 at the yeast cell surface. Using Sed1p for cell wall targeting and covalent anchorage, a highly efficient bioassay was established that allows high-throughput screening and subsequent validation of novel MMP inhibitors as potential anticancer drugs. In addition, we developed a straightforward synthesis of a new aspartate-derived MMP inhibitor active in the nM range and bearing an amino functionality that should allow the introduction of a wide range of side chains to modify the properties of these compounds.     

A meta-analysis of the existing knowledge of immunoreactivity against hepatitis C virus (HCV)

Approximately 3% of the world population is infected by HCV, which represents a major global health challenge. Almost 400 different scientific reports present immunological data related to T cell and antibody epitopes derived from HCV literature. Analysis of all HCV-related epitope hosted in the Immune Epitope Database (IEDB), a repository of freely accessible immune epitope data, revealed more than 1500 and 1900 distinct T cell and antibody epitopes, respectively. The inventory of all data revealed specific trends in terms of the host and the HCV genotypes from which sequences were derived. Upon further analysis we found that this large number of epitopes reflects overlapping structures, and homologous sequences derived from different HCV isolates. To access and visualize this information we developed a novel strategy that assembles large sets of epitope data, maps them onto reference genomes and displays the frequency of positive responses. Compilation of the HCV immune reactivity from hundreds of different studies, revealed a complex and thorough picture of HCV immune epitope data to date. The results pinpoint areas of more intense reactivity or research activities at the level of antibody, CD4 and CD8 responses for each of the individual HCV proteins. In general, the areas targeted by the different effector immune functions were distinct and antibody reactivity was positively correlated with hydrophilicity, while T cell reactivity correlated with hydrophobicity. At the sequence level, epitopes frequently recognized by both T cell and B cell correlated with low variability, and our analysis thus highlighted areas of potential interest for practical applications. The human reactivity was further analyzed to pinpoint differential patterns of reactivity associated with acute versus chronic infection, to reveal the apparent impact of glycosylation on T cell, but not antibody responses, and to highlight a paucity of studies involved antibody epitopes associated with virus neutralization.     

Oxalic acid supported Si-18F-radiofluorination: one-step radiosynthesis of N-succinimidyl 3-(di-tert-butyl[18F]fluorosilyl)benzoate ([18F]SiFB) for protein labeling

N-Succinimidyl 3-(di-tert-butyl[(18)F]fluorosilyl)benzoate ([(18)F]SiFB), a novel synthon for one-step labeling of proteins, was synthesized via a simple (18)F-(19)F isotopic exchange. A new labeling technique that circumvents the cleavage of the highly reactive active ester moiety under regular basic (18)F-labeling conditions was established. In order to synthesize high radioactivity amounts of [(18)F]SiFB, it was crucial to partially neutralize the potassium oxalate/hydroxide that was used to elute (18)F(-) from the QMA cartridge with oxalic acid to prevent decomposition of the active ester moiety. Purification of [(18)F]SiFB was performed by simple solid-phase extraction, which avoided time-consuming HPLC and yielded high specific activities of at least 525 Ci/mmol and radiochemical yields of 40-56%. In addition to conventional azeotropic drying of (18)F(-) in the presence of [K(+)?2.2.2.]C(2)O(4), a strong anion-exchange (SAX) cartridge was used to prepare anhydrous (18)F(-) for nucleophilic radio-fluorination omitting the vacuum assisted drying of (18)F(-). Using a lyophilized mixture of [K(+)?2.2.2.]OH resolubilized in acetonitrile, the (18)F(-) was eluted from the SAX cartridge and used directly for the [(18)F]SiFB synthesis. [(18)F]SiFB was applied to the labeling of various proteins in likeness to the most commonly used labeling synthon in protein labeling, N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Rat serum albumin (RSA), apo-transferrin, a β-cell-specific single chain antibody, and erythropoietin were successfully labeled with [(18)F]SiFB in good radiochemical yields between 19% and 36%. [(18)F]SiFB- and [(18)F]SFB-derivatized RSA were directly compared as blood pool imaging agents in healthy rats using small animal positron emission tomography. Both compounds demonstrated identical biodistributions in healthy rats, accurately visualizing the blood pool with PET.     

Structural consensus among antibodies defines the antigen binding site

The Complementarity Determining Regions (CDRs) of antibodies are assumed to account for the antigen recognition and binding and thus to contain also the antigen binding site. CDRs are typically discerned by searching for regions that are most different, in sequence or in structure, between different antibodies. Here, we show that ~20% of the antibody residues that actually bind the antigen fall outside the CDRs. However, virtually all antigen binding residues lie in regions of structural consensus across antibodies. Furthermore, we show that these regions of structural consensus which cover the antigen binding site are identifiable from the sequence of the antibody. Analyzing the predicted contribution of antigen binding residues to the stability of the antibody-antigen complex, we show that residues that fall outside of the traditionally defined CDRs are at least as important to antigen binding as residues within the CDRs, and in some cases, they are even more important energetically. Furthermore, antigen binding residues that fall outside of the structural consensus regions but within traditionally defined CDRs show a marginal energetic contribution to antigen binding. These findings allow for systematic and comprehensive identification of antigen binding sites, which can improve the understanding of antigenic interactions and may be useful in antibody engineering and B-cell epitope identification.     

Membrane interactions of two arginine-rich peptides with different cell internalization capacities

Cell penetrating peptides (CPPs) can cross cell membranes in a receptor independent manner and transport cargo molecules inside cells. These peptides can internalize through two independent routes: energy dependent endocytosis and energy independent translocation across the membrane, but the exact mechanisms are still unknown. The interaction of the CPP with different membrane components is certainly a preliminary key point that triggers internalization, such as the interaction with lipids to lead to the translocation process. In this study, we used two arginine-rich peptides, RW9 (RRWWRRWRR-NH(2)), which is a potent CPP, and RL9 (RRLLRRLRR-NH(2)) that, although binding tightly and accumulating on membranes, does not enter into cells. Using a set of experimental and theoretical techniques, we studied the binding, insertion and orientation of the peptides into different model membranes as well as the subsequent membrane reorganization. Herein we show that although the two peptides had rather similar behavior regarding lipid membrane interaction, subtle differences were found concerning the depth of peptide insertion, effect on the lipid chain ordering and kinetics of peptide insertion in the membrane, which altogether might explain their different cell internalization capacities. Molecular dynamics simulation studies show that some peptide molecules flipped their orientation over the course of the simulation such that the hydrophobic residues penetrated deeper in the lipid core region while Arg-residues maintained H-bonds with the lipid headgroups, serving as a molecular hinge in a conformation that appeared to correspond to the equilibrium one.     

Integrating epitope data into the emerging web of biomedical knowledge resources

The recognition of immune epitopes is an important molecular mechanism of the vertebrate immune system to discriminate between self and non-self. Increasing amounts of data on immune epitopes are becoming available due to technological advances in epitope-mapping techniques and the availability of genomic information for pathogens. Organizing this data poses a challenge that is similar to the successful effort that was required to organize genomic data, which needed the establishment of centralized databases that complement the primary literature to make the data readily accessible and searchable by researchers. As described in this Innovation article, the Immune Epitope Database and Analysis Resource aims to achieve the same for the more complex and context-dependent information on immune epitopes, and to integrate this data with existing and emerging knowledge resources.