MEF2D Deficiency in Neonatal Cardiomyocytes Triggers Cell Cycle Re-entry and Programmed Cell Death in vitro [Cell Biology]

August 20th, 2015 by

The cardiomyocyte cell cycle is a poorly understood process. Mammalian cardiomyocytes permanently withdraw from the cell cycle shortly after birth but can re-enter the cell cycle and proliferate when subjected to injury within a brief temporal window in the neonatal period. Thus, investigating the mechanisms of cell cycle regulation in neonatal cardiomyocytes may provide critical insight into the molecular events that prevent adult myocytes from proliferating in response to injury or stress. MEF2D is a key transcriptional mediator of pathological remodeling in the adult heart downstream of various stress-promoting insults. However, the specific gene programs regulated by MEF2D in cardiomyocytes are unknown. By performing genome-wide transcriptome analysis using MEF2D-depleted neonatal cardiomyocytes we found a significant impairment in the cell cycle, characterized by the upregulation of numerous positive cell cycle regulators. Expression of PTEN, the primary negative regulator of PI3K/Akt, was significantly reduced in MEF2D-deficient cardiomyocytes and found to be a direct target gene of MEF2D. Consistent with these findings mutant cardiomyocytes showed activation of the PI3K/Akt survival pathway. Paradoxically, prolonged deficiency of MEF2D in neonatal cardiomyocytes did not trigger proliferation but instead resulted in programmed cell death, which is likely mediated by the E2F transcription factor. These results demonstrate a critical role for MEF2D in cell cycle regulation of post-mitotic, neonatal cardiomyocytes in vitro.

Two-step Ligand Binding in a ({beta}{alpha})8 Barrel Enzyme – Substrate-bound Structures Shed New Light on the Catalytic Cycle of HisA [Enzymology]

August 20th, 2015 by

HisA is a (βα)8 barrel enzyme that catalyzes the Amadori rearrangement of ProFAR to PRFAR in the histidine biosynthesis pathway and it is a paradigm for the study of enzyme evolution. Still, its exact catalytic mechanism has remained unclear. Here, we present crystal structures of wild type Salmonella enterica HisA (SeHisA) in its apo state and of mutants D7N and D7N/D176A in complex with two different conformations of the labile substrate ProFAR, which was structurally visualized for the first time. Site-directed mutagenesis and kinetics demonstrated that Asp7 acts as the catalytic base and Asp176 as the catalytic acid. The SeHisA structures with ProFAR display two different states of the long loops on the catalytic face of the structure, and demonstrate that initial binding of ProFAR to the active site is independent of loop interactions. When the long loops enclose the substrate, ProFAR adopts an extended conformation where its non-reacting half is in a product-like conformation. This change is associated with shifts in a hydrogen-bond network including His47, Asp129, Thr171 and Ser202, all shown to be functionally important. The closed-conformation structure is highly similar to the bi-functional HisA homologue PriA in complex with PRFAR, thus proving that structure and mechanism are conserved between HisA and PriA. This study clarifies the mechanistic cycle of HisA and provides a striking example of how an enzyme and its substrate can undergo coordinated conformational changes before catalysis.
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Recapitulating the Structural Evolution of Redox-Regulation in Adenosine-5′-Phosphosulfate Kinase from Cyanobacteria to Plants [Protein Structure and Folding]

August 20th, 2015 by Herrmann, J., Nathin, D., Lee, S. G., Sun, T., Jez, J. M.

In plants, adenosine 5'-phosphosulfate (APS) kinase (APSK) is required for reproductive viability and the production of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as a sulfur-donor in specialized metabolism. Previous studies of the APSK from Arabidopsis thaliana (AtAPSK) identified a regulatory disulfide bond formed between the N-terminal domain (NTD) and a cysteine on the core scaffold. This thiol-switch is unique to mosses, gymnosperms, and angiosperms. To understand the structural evolution of redox-control of APSK, we investigated the redox-insensitive APSK from the cyanobacterium Synechocystis sp. PCC 6803 (SynAPSK). Crystallographic analysis of SynAPSK in complex with either APS and a non-hydrolyzable ATP analog or APS and sulfate reveals the overall structure of the enzyme, which lacks the NTD found in homologs from mosses and plants. A series of engineered SynAPSK variants reconstruct the structural evolution of the plant APSK. Biochemical analyses of SynAPSK, SynAPSK H23C mutant, SynAPSK fused to the AtAPSK NTD, and the fusion protein with the H23C mutation show that addition of the NTD and cysteines recapitulates thiol-based regulation. These results reveal the molecular basis for structural changes leading to the evolution of redox-control in APSK of the green lineage from cyanobacteria to plants.
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Impaired dNTPase Activity of SAMHD1 by Phosphomimetic Mutation of T592 [Protein Structure and Folding]

August 20th, 2015 by Tang, C., Ji, X., Wu, L., Xiong, Y.

SAMHD1 is a cellular protein that plays key roles in HIV-1 restriction and regulation of cellular dNTP levels. Mutations in SAMHD1 are also implicated in the pathogenesis of chronic lymphocytic leukemia and Aicardi-Goutieres syndrome. The anti-HIV-1 activity of SAMHD1 is negatively modulated by phosphorylation at residue T592. The mechanism underlying the effect of phosphorylation on anti-HIV-1 activity remains unclear. SAMHD1 forms tetramers that possess deoxyribonucleotide triphosphate triphosphohydrolase (dNTPase) activity, which is allosterically controlled by the combined action of GTP and all four dNTPs. Here we demonstrate that the phosphomimetic mutation T592E reduces the stability of the SAMHD1 tetramer and the dNTPase activity of the enzyme. To better understand the underlying mechanisms, we determined the crystal structures of SAMHD1 variants T592E and T592V. While the neutral substitution T592V does not perturb the structure, the charged T592E induces large conformational changes, likely triggered by electrostatic repulsion from a distinct negatively charged environment surrounding T592. The phosphomimetic mutation results in a significant decrease in the population of active SAMHD1 tetramers and hence the dNTPase activity is substantially decreased. These results provide a mechanistic understanding of how SAMHD1 phosphorylation at residue T592 may modulate its cellular and antiviral functions.

Structure of the Legionella Effector, lpg1496, Suggests a Role in Nucleotide Metabolism [Protein Structure and Folding]

August 20th, 2015 by Wong, K., Kozlov, G., Zhang, Y., Gehring, K.

Pathogenic gram-negative bacteria use specialized secretion systems that translocate bacterial proteins, termed effectors, directly into host cells where they interact with host proteins and biochemical processes for the benefit of the pathogen. Lpg1496 is a previously uncharacterized effector of Legionella pneumophila, the causative agent of Legionnaire's disease. Here, we crystallized three nucleotide-binding domains from lpg1496. The C-terminal domain, which is conserved among the SidE family of effectors, is formed of two largely α-helical lobes with a nucleotide-binding cleft. A structural homology search has shown similarity to phosphodiesterases involved in cleavage of cyclic nucleotides. We have also crystallized a novel domain that occurs twice in the N-terminal half of the protein that we term KLAMP domains due to the presence of homologous domains in bacterial histidine kinase-like ATP-binding region-containing proteins and S-adenosylmethionine-dependent methyltransferase proteins. Both KLAMP structures are very similar but selectively bind 3′,5′-cAMP and ADP. A co-crystal of the KLAMP1 domain with 3′,5′-cAMP reveals the contribution of Tyr61 and Tyr69 that produce π-stacking interactions with the adenine ring of the nucleotide. Our study provides the first structural insights into two novel nucleotide-binding domains associated with bacterial virulence.

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP [Signal Transduction]

August 20th, 2015 by

Evidence suggests that the plasma membrane Ca2+ ATPase (PMCA), which is critical for maintaining a low intracellular Ca2+ concentration ([Ca2+]i), utilises glycolytically-derived ATP in pancreatic ductal adenocarcinoma (PDAC), and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition and an irreversible [Ca2+]i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype towards mitochondrial metabolism and assessing the effects of mitochondrial vs glycolytic inhibitors on ATP depletion, PMCA inhibition and [Ca2+]i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate (KIC) or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitising cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca2+]i in KIC and galactose-cultured cells, suggesting that the glycolytic dependency of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.

Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing [Immunology]

August 20th, 2015 by

Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high-throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions, and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, Pertussis Toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day.

Lipin1 regulates skeletal muscle differentiation through ERK activation and Cyclin D complex regulated cell cycle withdrawal [Molecular Bases of Disease]

August 20th, 2015 by

Lipin1, an intracellular protein, plays critical roles in controlling lipid synthesis and energy metabolism through its enzymatic activity and nuclear transcriptional functions. Several mouse models of skeletal muscle wasting are associated with lipin1 mutation or altered expression. Recent human studies have suggested that children with homozygous null mutations in the LPIN1 gene suffer from rhabdomyolysis. However, the underlying pathophysiologic mechanism is still poorly understood. In the present study, we examined whether lipin1 contributes to regulating muscle regeneration. We characterized the time course of skeletal muscle regeneration in lipin1 deficient fld mice following injury. We found that fld mice exhibited smaller regenerated muscle fiber cross-sectional areas and increased levels of fibrosis compared with wild-type mice in response to injury. Our results from a series of in vitro experiments suggest that lipin1 is upregulated and translocated to the nucleus during myoblast differentiation, and plays a key role in myogenesis by regulating the cytosolic activation of ERK1/2 to form a complex and a downstream effector Cyclin D3-mediated cell cycle withdrawal. Overall, our study reveals a previously unknown role of lipin1 in skeletal muscle regeneration and expands our understanding of the cellular and molecular mechanisms underlying skeletal muscle regeneration.
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Role of Erk1/2 Signaling in the Regulation of Neutrophil versus Monocyte Development in Response to G-CSF and M-CSF [Immunology]

August 20th, 2015 by Hu, N., Qiu, Y., Dong, F.

Lineage specification in the hematopoietic system depends on the expression of lineage specific transcription factors. However, the role of hematopoietic cytokines in this process has been controversial and little is known about the intracellular signaling mechanisms by which cytokines instruct lineage choice. G-CSF and M-CSF are two lineage-specific cytokines that play a dominant role in granulopoiesis and monopoiesis, respectively. We show here that a G-CSFR mutant in which tyrosine 729 had been mutated to phenylalanine (Y729F) promoted monocyte rather than neutrophil development in myeloid precursors, which was associated with prolonged activation of Erk1/2 and augmented activation of downstream targets c-Fos and Egr1. Inhibition of Erk1/2 activation or knockdown of c-Fos or Egr1 largely rescued neutrophil development in cells expressing G-CSFR Y729F. We also show that M-CSF, but not G-CSF, stimulated strong and sustained activation of Erk1/2 in mouse lineage marker negative (Lin-) bone marrow cells. Significantly, inhibition of Erk1/2 signaling in these cells favored neutrophil over monocyte development in response to M-CSF. Thus, prolonged Erk1/2 activation resulted in monocyte development following G-CSF induction whereas inhibition of Erk1/2 signaling promoted neutrophil development at the expense of monocyte formation in response to M-CSF. These results reveal an important mechanism by which G-CSF and M-CSF instruct neutrophil versus monocyte lineage choice, i.e., differential activation of Erk1/2 pathway.

Selective Irreversible Inhibition of Neuronal and Inducible Nitric-Oxide Synthase in the Combined Presence of Hydrogen Sulfide and Nitric Oxide [Enzymology]

August 20th, 2015 by

Citrulline formation by both human neuronal nitric-oxide synthase (nNOS) and mouse macrophage inducible nitric-oxide synthase (iNOS) was inhibited by the hydrogen sulfide (H2S) donor Na2S with IC50-values of ~ 2.4×10−5 M and ~ 7.9×10−5 M, respectively, whereas human endothelial nitric-oxide synthase (eNOS) was hardly affected at all. Inhibition of nNOS was not affected by the concentrations of L-arginine (Arg), NADPH, FAD, FMN, tetrahydrobiopterin (BH4), and calmodulin (CaM), indicating that H2S does not interfere with substrate or cofactor binding. The IC50 decreased to ~ 1.5×10−5 M at pH 6.0 and increased to ~ 8.3×10−5 M at pH 8.0. Preincubation of concentrated nNOS with H2S under turnover conditions decreased activity after dilution by ~ 70 %, suggesting irreversible inhibition. However, when CaM was omitted during preincubation, activity was not affected, suggesting that irreversible inhibition requires both H2S and NO. Likewise, NADPH oxidation was inhibited with IC50 ~ 1.9×10−5 M in the presence of Arg and BH4, but exhibited much higher IC50-values (~ 1.0-6.1×10−4 M) when Arg and/or BH4 were omitted. Moreover, the relatively weak inhibition of nNOS by Na2S in the absence of Arg and/or BH4 was markedly potentiated by the NO-donor PROLI/NO (IC50 ~ 1.3-2.0×10−5 M). These results suggest that nNOS and iNOS, but not eNOS, are irreversibly inhibited by H2S/NO at modest concentrations of H2S in a reaction that may allow feedback inhibition of NO production under conditions of excessive NO/H2S formation.
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