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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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.

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.

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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Human telomerase reverse transcriptase (hTERT) transcription requires Sp1/Sp3 binding to the promoter and a permissive chromatin environment [DNA and Chromosomes]

October 20th, 2015 by Cheng, D., Zhao, Y., Wang, S., Jia, W., Kang, J., Zhu, J.

The transcription of human telomerase gene hTERT is regulated by transcription factors (TFs), including Sp1 family proteins, and its chromatin environment. To understand its regulation in a relevant chromatin context, we employed BAC reporters containing 160-kb of human genomic sequence containing the hTERT gene. Upon chromosomal integration, the BACs recapitulated endogenous hTERT expression, contrary to transient reporters. Sp1/Sp3 expression did not correlate with hTERT promoter activity and these TFs bound to the hTERT promoters in both telomerase-positive and -negative cells. Mutation of the proximal GC-box resulted in a dramatic decrease of hTERT promoter activity and mutations of all five GC-boxes eliminated its transcriptional activity. Neither mutations of GC-boxes nor knockdown of endogenous Sp1 impacted promoter binding by other TFs, including E-box binding proteins, and histone acetylation and trimethylation of histone H3K9 at the hTERT promoter in telomerase-positive and -negative cells. The result indicated that promoter binding by Sp1/Sp3 was essential, but not a limiting step, for hTERT transcription. hTERT transcription required a permissive chromatin environment. Importantly, our data also revealed different functions of GC-boxes and E-boxes in hTERT regulation: while GC-boxes were essential for promoter activity, factors bound to the E-boxes functioned to de-repress hTERT promoter.
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A Nucleolar PUF RNA-binding Protein with Specificity for a Unique RNA Sequence [Plant Biology]

October 20th, 2015 by Zhang, C., Muench, D. G.

PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins, and these show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for non-classical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was ten nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, as all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5[prime] end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of three of the ten predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred ten-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA processing. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.

C1q Deficiency Promotes Pulmonary Vascular Inflammation and Enhances the Susceptibility of the Lung Endothelium to Injury. [Cell Biology]

October 20th, 2015 by

The collectin proteins are innate immune molecules found in high concentrations on the epithelial and endothelial surfaces of the lung. While these proteins are known to have important anti-inflammatory actions in the airways of the lung little is known of their functional importance in the pulmonary circulation. We recently demonstrated that the circulating collectin protein adiponectin has potent anti-inflammatory effects on the lung endothelium, leading us to reason that other structurally-related proteins might have similar effects. To test this hypothesis, we investigated the anti-inflammatory actions of C1q in lung endothelial homeostasis and the pulmonary vascular response to LPS or HCl injury. We show that lung endothelium from C1q deficient (C1q-/-) mice expresses higher baseline levels of the vascular adhesion markers ICAM-1, VCAM-1 and E-selectin when compared to wild-type mice. Further, we demonstrate that these changes are associated with enhanced susceptibility of the lung to injury as evident by increased expression of adhesion markers, enhanced production of pro-inflammatory cytokines, and augmented neutrophil recruitment. Additionally, we found that C1q-/- mice also exhibited enhanced endothelial barrier dysfunction after injury as manifested by decreased expression of junctional adherens proteins and enhanced vascular leakage. Mechanistically, C1q appears to mediate its effects by inhibiting phosphorylation of p38 mitogen-activated protein kinase (MAPK) and blocking nuclear translocation of the P65 subunit of nuclear factor (NF)-kB. In summary, our findings indicate a previously unrecognized role for C1q in pulmonary vascular homeostasis and provide added support for the hypothesis that circulating collectin proteins have protective effects on the lung endothelium.

Normal Fertility Requires Expression of Carbonic Anhydrases II and IV in Sperm [Cell Biology]

October 20th, 2015 by

HCO3- is a key factor in the regulation of sperm motility. High concentrations of HCO3- in the female genital tract induce an increase in sperm beat frequency which speeds their progress through the female reproductive tract. Carbonic anhydrases (CA) which catalyze the reversible hydration of CO2 to HCO3-, represent potential candidates in the regulation of the HCO3- homeostasis in sperm and the composition of the male and female genital tract fluids. We show that two CA isoforms - CAII and CAIV - are distributed along the epididymal epithelium and appear with the onset of puberty. Expression analyses reveal an up-regulation of CAII and CAIV in the different epididymal sections of the knockout lines. In sperm, we find CAII is located in the principal piece whereas CAIV is present in the plasma membrane of the entire sperm tail. CAII and CAIV single knockout animals display an imbalanced HCO3- homeostasis, resulting in substantially reduced sperm motility, swimming speed and HCO3--enhanced beat frequency. The CA activity remaining in sperm of CAII and CAIV-null mutants is 35% and 68% of that found for WT mice. Sperm of the double knockout mutant mice showed responses to stimulus by HCO3- or CO2 that were delayed in onset and reduced in magnitude. In contrast to sperm from double knockout animals, pharmacological loss of CAIV in sperm from CAII knockout animals, show an even lower response to HCO3-. These results suggest that CAII and CAIV are required for optimal fertilization.