Lecithin:Cholesterol Acyltransferase (LCAT) Deficiency Promotes Differentiation of Satellite Cells to Brown Adipocytes in a Cholesterol-Dependent Manner [Lipids]

October 22nd, 2015 by Nesan, D., Tavallaee, G., Koh, D., Bashiri, A., Abdin, R., Ng, D. S.

Our lab previously reported that lecithin:cholesterol acyltransferase (LCAT) and LDL receptor double knockout mice (Ldlr-/-xLcat-/- or DKO) spontaneously develop functioning ectopic brown adipose tissue (BAT) in skeletal muscle, putatively contributing to the protection from diet-induced obesity phenotype. Here we further investigated their developmental origin and the mechanistic role of LCAT deficiency. Gene profiling of skeletal muscle in DKO newborns and adults revealed a classical lineage. Primary quiescent satellite cells (SC) from chow-fed DKO mice, not in Ldlr-/-xLcat+/+ single-knockout (SKO) or C57BL/6 wild type, were found to (i) express exclusively classical BAT-selective genes, (ii) be primed to express key functional BAT genes and (iii) exhibit markedly increased ex vivo adipogenic differentiation into brown adipocytes. This gene priming effect was abrogated upon feeding the mice a 2% high cholesterol diet (HCD) in association with accumulation of excess intracellular cholesterol. Ex vivo cholesterol loading of chow-fed DKO SC recapitulated the effect, indicating that cellular cholesterol is a key regulator of SC-to-BAT differentiation. Comparing adipogenicity of Ldlr+/+xLcat-/- (LCAT-KO) SC to those of DKO mice identified a specific role for LCAT deficiency in priming of SC to express BAT genes, and that cellular cholesterol depletion is important for adipogenic differentiation, as evidenced by comparable induction of adipogenesis by depleting cholesterol in SC from both LCAT-KO and SKO mice. Taken together, we conclude that ectopic BAT in DKO cells are classical in origin and their development begins in utero. We further showed complementary roles of LCAT deficiency and cellular cholesterol reduction in the SC-to-BAT adipogenesis.
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Structural and Functional highlights of Vacuolar Soluble Protein 1 from pathogen T. brucei. brucei [Microbiology]

October 22nd, 2015 by

Trypanosoma brucei (T. brucei) is responsible for the fatal human disease called African trypanosomiasis or sleeping sickness. The causative parasite Trypanosoma encodes soluble versions of inorganic pyrophosphatases (PPase), also called vacuolar soluble proteins (VSPs), which are localized to its acidocalcisomes. Latter are acidic membrane enclosed organelles rich in polyphosphate chains and divalent cations whose significance in these parasites remains unclear. We here report the crystal structure of T. brucei brucei acidocalcisomal PPases in a ternary complex with Mg2+ and imidodiphosphate. The crystal structure reveals a novel structural architecture distinct from known class I PPases in its tetrameric oligomeric state in which a fused EF-hand domain arranges around the catalytic PPase domain. This unprecedented assembly evident from TbbVSP1 crystal structure is further confirmed by small angle X-ray scattering (SAXS) and electron microscopy data. Solution scattering data suggests structural flexibility in EF-hand domains indicative of conformational plasticity within TbbVSP1.

The {beta}5-loop and lid-domain contribute to the substrate specificity of pancreatic lipase related protein 2 (PNLIPRP2) [Enzymology]

October 21st, 2015 by Xiao, X., Lowe, M. E.

Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults whereas a homologue, pancreatic lipase related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIP cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the lid-domain, β5- and β9-loops, the lid-domain and the β5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid-domain or β5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP β5-loop and decreased by the PNLIP lid-domain. The source of the β9-loop had minimal effect on activity. We conclude that the lid-domain and β5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2.

Evaluating the use of antibody variable region (Fv) charge as a risk assessment tool for predicting typical cynomolgus monkey pharmacokinetics [Protein Structure and Folding]

October 21st, 2015 by

The pharmacokinetic (PK) behavior of monoclonal antibodies in cynomolgus monkeys (cynos) is generally translatable to that in humans. Unfortunately, about 39% of the antibodies evaluated for PK in cynos have fast non-specific (or non-target mediated) clearance (in-house data). An empirical model relating variable region (Fv) charge and hydrophobicity to cyno non-specific clearance was developed to gauge the risk an antibody would have for fast non-specific clearance in the monkey. The purpose of this study was to evaluate the predictability of this empirical model on cyno non-specific clearance with antibodies specifically engineered to have either high or low Fv charge. These amino acid changes were made in the Fv region of two test antibodies, humAb4D5-8 and anti-LTα. HumAb4D5-8 has a typical non-specific clearance in cynos and by making it more positively charged, the antibody acquires fast non-specific clearance, while making it less positively charged did not impact its clearance. Anti-LTα has fast non-specific clearance in cynos and making it more positively charged caused it to clear even faster, while making it less positively charged caused it to clear slower and within the typical range. These trends in clearance were also observed in two other preclinical species, mice and rats. The effect of modifying Fv charge on subcutaneous bioavailability was also examined and in general bioavailability was inversely related to the direction of Fv charge change. Thus, modifying Fv charge appears to impact antibody PK and the changes tended to correlate with those predicted by the empirical model.
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Gene Expression Profiling Reveals a Novel Regulatory Role for Sox21 in Mouse Trophoblast Stem Cell Differentiation. [Gene Regulation]

October 21st, 2015 by Moretto Zita, M., Soncin, F., Natale, D., Pizzo, D., Parast, M.

Appropriate self-renewal and differentiation of trophoblast stem cells (TSC) are key factors for proper placental development and function, and in turn, for appropriate in utero fetal growth. In order to identify novel TSC-specific genes, we performed genome-wide expression profiling of TSCs, embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), and mouse embryo fibroblasts (MEFs), derived from mice of the same genetic background. Our analysis revealed a high expression of Sox21 in TSCs compared to other cell types. Sox21 levels were high in undifferentiated TSCs and were dramatically reduced upon differentiation. In addition, modulation of Sox21 expression in TSCs affected lineage-specific differentiation, based on both marker analysis and functional assessment. Our results implicate Sox21 specifically in the promotion of spongiotrophoblast and giant cell differentiation and establish a new mechanism through which trophoblast sublineages are specified

A second {beta}-hexosaminidase encoded in the Streptococcus pneumoniae genome provides an expanded biochemical ability to degrade host glycans [Protein Structure and Folding]

October 21st, 2015 by

An important facet of the interaction between the pathogen Streptococcus pneumoniae (pneumococcus) and its human host is the ability of this bacterium to process host glycans. To achieve cleavage of the glycosidic bonds in host glycans, S. pneumoniae deploys a wide array of glycoside hydrolases. Here we identify and characterize a new family 20 glycoside hydrolase, GH20C, from S. pneumoniae. Recombinant GH20C possessed the ability to hydrolyze the β-linkages joining either N-acetylglucosamine or N-acetylgalactosamine to a wide variety of aglycon residues, thus revealing this enzyme to be a generalist N-acetylhexosaminidase in vitro. X-ray crystal structures were determined for GH20C in apo-form, in complex with the N-acetylglucosamine and N-acetylgalactosamine products of catalysis, and in complex with both gluco- and galacto-configured inhibitors (PUGNAc, GalPUGNAc, NGT, and GalNGT) at resolutions from 1.84 Å to 2.7 Å. These structures showed N-acetylglucosamine and N-acetylgalactosamine to be recognized via identical sets of molecular interactions. Though the same sets of interaction were maintained with the gluco- and galacto-configured inhibitors, the inhibition constants suggested preferred recognition of the axial O4 when an aglycon moiety was present (Ki for PUGNAc > GalPUGNAc) but preferred recognition of an equatorial O4 when the aglycon was absent (Ki for GalNGT > NGT). Overall, this study reveals GH20C to be another tool that is unique in the arsenal of S. pneumoniae and that it may implement in the effort of the bacterium to utilize and/or destroy the wide array of host glycans that it may encounter.
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Evidence for a functional O-GlcNAc system in the thermophilic bacterium Thermobaculum terrenum [Protein Structure and Folding]

October 21st, 2015 by

Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. While isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signalling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc transferase are co-expressed and, like their mammalian orthologues, localise to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase-peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc transferase could not be detected, treatment of T. terrenum with an O-GlcNAc transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system.

Senescence-associated long non-coding RNA (SALNR) delays oncogene-induced senescence through NF90 regulation [RNA]

October 21st, 2015 by Wu, C. L., Wang, Y., Jin, B., Chen, H., Xie, B.-S., Mao, Z.-B.

Long non-coding RNAs (lncRNAs) have recently emerged as key players in many physiologic and pathologic processes. Although many lncRNAs have been identified, few lncRNA has been characterized functionally in aging. In this study, we used human fibroblast cell to investigate genome-wide lncRNA expression during cellular senescence. We identified 968 down-regulated lncRNAs and 899 up-regulated lncRNAs in senescent cells compared with young cells. Among these lncRNAs, we characterized a senescence-associated lncRNA (SALNR), whose expression was reduced during cellular senescence and in pre-malignant colon adenomas. Overexpression of SALNR delayed cellular senescence in fibroblast cells. Furthermore, we found that SALNR interacts with nuclear factor of activated T-cells 90 kDa (NF90), a RNA binding protein suppressing miRNA biogenesis. We demonstrated that NF90 is a SALNR downstream target, whose inhibition led to premature senescence and enhanced expressions of senescence-associated miRNAs (SA-miRNAs). Moreover, our data showed that Ras-induced stress promotes NF90 nucleolus translocation and suppresses its ability to suppress SA-miRNA biogenesis, which could be rescued by SALNR overexpression. These data suggest that lncRNA SALNR modulate cellular senescence at least partly through changing NF90 activity.

Analysis of ribonucleotide removal from DNA by human nucleotide excision repair [RNA]

October 21st, 2015 by Lindsey-Boltz, L. A., Kemp, M. G., Hu, J., Sancar, A.

Ribonucleotides are incorporated into the genome during DNA replication. The enzyme RNase H2 plays a critical role in targeting the removal of these ribonucleotides from DNA, and defects in RNase H2 activity are associated with both genomic instability and the human autoimmune/inflammatory disorder Aicardi-Goutieres syndrome. Whether additional general DNA repair mechanisms contribute to ribonucleotide removal from DNA in human cells is not known. Because of its ability to act on a wide variety of substrates, we examined a potential role for canonical nucleotide excision repair in the removal of ribonucleotides from DNA. However, using highly sensitive dual incision/excision assays we find that ribonucleotides are not efficiently targeted by the human nucleotide excision repair system in vitro or in vivo. These results suggest that nucleotide excision repair is unlikely to play a major role in the cellular response to ribonucleotide incorporation in genomic DNA in human cells.

{alpha}/{beta} Hydrolase Domain-Containing 6 (ABHD6) Degrades the Late Endosomal/Lysosomal Lipid Bis(monoacylglycero)phosphate [Metabolism]

October 21st, 2015 by

α/β Hydrolase domain-containing 6 (ABHD6) can act as monoacylglycerol (MG) hydrolase and is believed to play a role in endocannabinoid signaling as well as in the pathogenesis of obesity and liver steatosis. Yet, the mechanistic link between gene function and disease is incompletely understood. Here, we aimed to further characterize the role of ABHD6 in lipid metabolism. We show that mouse and human ABHD6 degrade bis(monoacylglycero)phosphate (BMP) with high specific activity. BMP, also known as lysobisphosphatidic acid (LBPA), is enriched in late endosomes/lysosomes, where it plays a key role in the formation of intraluminal vesicles and in lipid sorting. Up to now, little is known about the catabolism of this lipid. Our data demonstrate that ABHD6 is responsible for ~ 90% of the BMP hydrolase activity detected in liver and that knockdown of ABHD6 increases hepatic BMP levels. Tissue fractionation and live cell imaging experiments revealed that ABHD6 co-localizes with late endosomes/lysosomes. The enzyme is active at cytosolic pH and lacks acid hydrolase activity implicating that it degrades BMP exported from acidic organelles or de novo formed BMP. In conclusion, our data suggest that ABHD6 controls BMP catabolism and is therefore part of the late endosomal/lysosomal lipid sorting machinery.