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.

Lactate contributes to glyceroneogenesis and glyconeogenesis in skeletal muscle by reversal of pyruvate kinase [Glycobiology and Extracellular Matrices]

October 21st, 2015 by Jin, E. S., Sherry, A. D., Malloy, C. R.

Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase while another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol and lactate under a fed or fasted state. Only one of the three substrates was 13C-labeled in each experiment. After 3 hours of normal awake activity, the animals were sacrificed and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of 13C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected.
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Retinoblastoma Binding Protein 4-Regulated Classical Nuclear Transport Is Involved in Cellular Senescence [Molecular Bases of Disease]

October 21st, 2015 by

Nucleocytoplasmic trafficking is a fundamental cellular process in eukaryotic cells. Here, we demonstrated that retinoblastoma binding protein 4 (RBBP4) functions as a novel regulatory factor to increase the efficiency of importin α/β-mediated nuclear import. RBBP4 accelerates the release of importin β1 from importin α via competitive binding to the importin β-binding (IBB) domain of importin α in the presence of RanGTP. Therefore, it facilitates importin α/β-mediated nuclear import. We showed that the importin α/β pathway is downregulated in replicative senescent cells, concomitant with a decrease in RBBP4 level. Knockdown of RBBP4 caused both suppression of nuclear transport and induction of cellular senescence. This is the first report to identify a factor that competes with importin β1 to bind to importin α, and demonstrates that the loss of this factor can trigger cellular senescence.

Mitochondrial respiratory defect causes dysfunctional lactate turnover via AMP-activated protein kinase activation in human induced pluripotent stem cell-derived hepatocytes [Metabolism]

October 21st, 2015 by

A defective mitochondrial respiratory chain complex (DMRC) causes various metabolic disorders in humans. However, the pathophysiology of DMRC in the liver remains unclear. To understand DMRC pathophysiology in vitro, DMRC-induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts of a DMRC patient who had a homoplasmic mutation (m.3398T>C) in mitochondrial-encoded NADH dehydrogenase 1 (MTND1) gene, and differentiated into hepatocytes (DMRC-hepatocytes) in vitro. DMRC-hepatocytes showed abnormalities in mitochondrial characteristics, the NAD+/NADH ratio, the glycogen storage level, the lactate turnover rate, and the AMPK activity. Intriguingly, low glycogen storage and transcription of lactate turnover-related genes in DMRC-hepatocytes were recovered by inhibition of AMPK activity. Thus, AMPK activation led to metabolic changes in terms of glycogen storage and lactate turnover in DMRC-hepatocytes. These data demonstrate for the first time that energy depletion may lead to lactic acidosis in the DMRC patient by reduction of lactate uptake via AMPK in liver.
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Genetic Control of Replication through N1-methyladenine in Human Cells [DNA and Chromosomes]

October 21st, 2015 by

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick (W-C) base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesions is mediated via three different pathways in which Pols iota and theta function in one pathway, and Pols eta and zeta, respectively, function in the other two pathways. Our biochemical studies indicate that in the Pol iota/Pol theta; pathway, Pol iota would carry out nucleotide (nt) insertion opposite 1-MeA from which Pol theta would extend synthesis. In the Pol eta pathway, this Pol alone would function at both the nt insertion and extension steps of TLS, and in the third pathway, Pol zeta would extend from the nt inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Pol iota would carry out TLS opposite 1-MeA, the ability of Pol eta to replicate through 1-MeA suggests that in spite of its need for W-C hydrogen bonding, Pol eta can stabilize the adduct in its active site. Remarkably, even though Pols eta and iota are quite error-prone at inserting nts opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.

Proteomic Analysis Identifies Ribosome Reduction as an Effective Proteotoxic Stress Response [Cell Biology]

October 21st, 2015 by

Stress responses are adaptive cellular programs that identify and mitigate potentially dangerous threats. Misfolded proteins are a ubiquitous and clinically relevant stress. Trivalent metalloids, such as arsenic, have been proposed to cause protein misfolding. Using tandem mass tag-based mass spectrometry, we show that trivalent arsenic results in widespread reorganization of the cell from an anabolic to a catabolic state. Both major pathways of protein degradation, the proteasome and autophagy, show increased abundance of pathway components, increased functional output, and are required for survival. Remarkably, cells also showed a downregulation of ribosomes at the protein level. That this represented an adaptive response, and not an adverse toxic effect, was indicated by enhanced survival of ribosome mutants after arsenic exposure. These results suggest that a major source of toxicity of trivalent arsenic derives from misfolding of newly synthesized proteins, and identifies ribosome reduction as a rapid, effective, and reversible proteotoxic stress response.

Histone deacetylase inhibitors target the leukemic microenvironment by enhancing a Nherf1-Protein Phosphatase 1{alpha}-TAZ signaling pathway in osteoblasts [Molecular Bases of Disease]

October 21st, 2015 by

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage which reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts. We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated in order to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein Phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis.
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