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Biochemistry
Mapping Genes for Calcium Signaling and Their Associated Human Genetic Disorders
Apr 21, 2017   Bioinformatics (Oxford, England)
Hörtenhuber M, Toledo EM, Smedler E, Arenas E, Malmersjö S, Louhivuori L, Uhlén P
Mapping Genes for Calcium Signaling and Their Associated Human Genetic Disorders
Apr 21, 2017
Bioinformatics (Oxford, England)
Signal transduction via calcium ions (Ca 2+ ) represents a fundamental signaling pathway in all eukaryotic cells. A large portion of the human genome encodes proteins used to assemble signaling systems that can transduce signals with diverse spatial and temporal dynamics. Here, we provide a map of all of the genes involved in Ca 2+ signaling and link these genes to human genetic disorders. Using Gene Ontology terms and genome databases, 1,805 genes were identified as regulators or targets of intracellular Ca 2+ signals. Associating these 1,805 genes with human genetic disorders uncovered 1,470 diseases with mutated "Ca 2+ genes". A network with scale-free properties appeared when the Ca 2+ genes were mapped to their associated genetic disorders. The Ca 2+ genome database is freely available at http://cagedb.uhlenlab.org and will foster studies of gene functions and genetic disorders associated with Ca 2+ signaling. per.uhlen@ki.se. Supplementary data are available at Bioinformatics online.
Bio-catalytic performance and dye-based industrial pollutants degradation potential of agarose-immobilized MnP using a Packed Bed Reactor System
Apr 22, 2017   International Journal Of Biological Macromolecules
Bilal M, Asgher M, Iqbal HMN, Hu H, Wang W, Zhang X
Bio-catalytic performance and dye-based industrial pollutants degradation potential of agarose-immobilized MnP using a Packed Bed Reactor System
Apr 22, 2017
International Journal Of Biological Macromolecules
In this study, the matrix-entrapment technique was adopted to immobilize a novel manganese peroxidase (MnP). Agarose beads developed from 3.0% agarose concentration furnished the preeminent immobilization yield (92.76%). The immobilized MnP exhibited better resistance to changes in the pH and temperature as compared to the free counterpart, with optimal conditions being pH 6.0 and 45°C. Thermal and storage stability characteristics were significantly improved after immobilization, and the immobilized-MnP displayed higher tolerance against different temperatures than free MnP state. After 72h, the insolubilized MnP retained its activity up to 41.2±1.7% and 33.6±1.4% at 55°C and 60°C, respectively, and 34.3±1.9% and 22.0±1.1% activities at 65°C and 70°C, respectively, after 48h of the incubation period. A considerable reusability profile was recorded with ten consecutive cycles. Moreover, to explore the industrial applicability, the agarose-immobilized-MnP was tested for bioremediation of textile industry effluent purposes. After six consecutive cycles, the tested effluents were decolorized to different extents (with a maximum of 98.4% decolorization). In conclusion, the remarkable bioremediation potential along with catalytic, thermo-stability, reusability, as well as storage stability features of the agarose-immobilized-MnP reflect its prospects as a biocatalyst for bioremediation and other industrial applications. Copyright © 2017 Elsevier B.V. All rights reserved.
Single-molecule visualization of fast polymerase turnover in the bacterial replisome
Apr 22, 2017   ELife
Lewis JS, Spenkelink LM, Jergic S, Wood EA, Monachino E, Horan NP, Duderstadt KE, Cox MM, Robinson A, Dixon NE, van Oijen AM
Single-molecule visualization of fast polymerase turnover in the bacterial replisome
Apr 22, 2017
ELife
The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the β2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.
Structure of Fam20A reveals a pseudokinase featuring unique disulfide pattern and inverted ATP-binding
Apr 22, 2017   ELife
Cui J, Zhu Q, Zhang H, Cianfrocco MA, Leschziner AE, Dixon JE, Xiao J
Structure of Fam20A reveals a pseudokinase featuring unique disulfide pattern and inverted ATP-binding
Apr 22, 2017
ELife
Mutations in FAM20A cause tooth enamel defects known as Amelogenesis Imperfecta (AI) and renal calcification. We previously showed that Fam20A is a secretory pathway pseudokinase and allosterically activates the physiological casein kinase Fam20C to phosphorylate secreted proteins important for biomineralization (Cui et al., 2015). Here we report the nucleotide-free and ATP-bound structures of Fam20A. Fam20A exhibits a distinct disulfide bond pattern mediated by a unique insertion region. Loss of this insertion due to abnormal mRNA splicing interferes with the structure and function of Fam20A, resulting in AI. Fam20A binds ATP in the absence of divalent cations, and strikingly, ATP is bound in an inverted orientation compared to other kinases. Fam20A forms a dimer in the crystal, and residues in the dimer interface are critical for Fam20C activation. Together, these results provide structural insights into the function of Fam20A and shed light on the mechanism by which Fam20A mutations cause disease.
Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling
Apr 22, 2017   ELife
Grieve AG, Xu H, Künzel U, Bambrough P, Sieber B, Freeman M
Phosphorylation of iRhom2 at the plasma membrane controls mammalian TACE-dependent inflammatory and growth factor signalling
Apr 22, 2017
ELife
Proteolytic cleavage and release from the cell surface of membrane-tethered ligands is an important mechanism of regulating intercellular signalling. TACE is a major shedding protease, responsible for the liberation of the inflammatory cytokine TNFα and ligands of the epidermal growth factor receptor. iRhoms, catalytically inactive members of the rhomboid-like superfamily, have been shown to control the ER-to-Golgi transport and maturation of TACE. Here, we reveal that iRhom2 remains associated with TACE throughout the secretory pathway, and is stabilised at the cell surface by this interaction. At the plasma membrane, ERK1/2-mediated phosphorylation and 14-3-3 protein binding of the cytoplasmic amino-terminus of iRhom2 alter its interaction with mature TACE, thereby licensing its proteolytic activity. We show that this molecular mechanism is responsible for triggering inflammatory responses in primary mouse macrophages. Overall, iRhom2 binds to TACE throughout its lifecycle, implying that iRhom2 is a primary regulator of stimulated cytokine and growth factor signalling.
Role of the Ion Channel Extracellular Collar in AMPA Receptor Gating
Apr 22, 2017   Scientific Reports
Yelshanskaya MV, Mesbahi-Vasey S, Kurnikova MG, Sobolevsky AI
Role of the Ion Channel Extracellular Collar in AMPA Receptor Gating
Apr 22, 2017
Scientific Reports
AMPA subtype ionotropic glutamate receptors mediate fast excitatory neurotransmission and are implicated in numerous neurological diseases. Ionic currents through AMPA receptor channels can be allosterically regulated via different sites on the receptor protein. We used site-directed mutagenesis and patch-clamp recordings to probe the ion channel extracellular collar, the binding region for noncompetitive allosteric inhibitors. We found position and substitution-dependent effects for introduced mutations at this region on AMPA receptor gating. The results of mutagenesis suggested that the transmembrane domains M1, M3 and M4, which contribute to the ion channel extracellular collar, undergo significant relative displacement during gating. We used molecular dynamics simulations to predict an AMPA receptor open state structure and rationalize the results of mutagenesis. We conclude that the ion channel extracellular collar plays a distinct role in gating and represents a hub for powerful allosteric modulation of AMPA receptor function that can be used for developing novel therapeutics.
Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
Apr 22, 2017   Scientific Reports
Islam MF, Watanabe A, Wong L, Lazarou C, Vizeacoumar FS, Abuhussein O, Hill W, Uppalapati M, Geyer CR, Vizeacoumar FJ
Enhancing the throughput and multiplexing capabilities of next generation sequencing for efficient implementation of pooled shRNA and CRISPR screens
Apr 22, 2017
Scientific Reports
Next generation sequencing is becoming the method of choice for functional genomic studies that use pooled shRNA or CRISPR libraries. A key challenge in sequencing these mixed-oligo libraries is that they are highly susceptible to hairpin and/or heteroduplex formation. This results in polyclonal, low quality, and incomplete reads and reduces sequencing throughput. Unfortunately, this challenge is significantly magnified in low-to-medium throughput bench-top sequencers as failed reads significantly perturb the maximization of sequence coverage and multiplexing capabilities. Here, we report a methodology that can be adapted to maximize the coverage on a bench-top, Ion PGM System for smaller shRNA libraries with high efficiency. This ligation-based, half-shRNA sequencing strategy minimizes failed sequences and is also equally amenable to high-throughput sequencers for increased multiplexing. Towards this, we also demonstrate that our strategy to reduce heteroduplex formation improves multiplexing capabilities of pooled CRISPR screens using Illumina NextSeq 500. Overall, our method will facilitate sequencing of pooled shRNA or CRISPR libraries from genomic DNA and maximize sequence coverage.
Systematic Synergy of Glucose and GLP-1 to Stimulate Insulin Secretion Revealed by Quantitative Phosphoproteomics
Apr 22, 2017   Scientific Reports
Tang JS, Li QR, Li JM, Wu JR, Zeng R
Systematic Synergy of Glucose and GLP-1 to Stimulate Insulin Secretion Revealed by Quantitative Phosphoproteomics
Apr 22, 2017
Scientific Reports
GLP-1 synergizes with glucose in regulating pancreatic β-cell function, including facilitating β-cell survival and insulin secretion. Though it has been widely accepted that phosphorylation is extremely important in regulating β-cell functions, our knowledge to the global mechanism is still limited. Here we performed a quantitative phosphoproteomics study to systematically present the synergistic regulation of INS-1E cell phosphoproteome mediated by glucose and GLP-1. We generated the largest pancreatic β-cell phosphoproteome by identifying 25,327 accurately localized phosphorylation sites on 5,389 proteins. Our results discovered several novel kinases regulated by glucose, GLP-1 or their synergism, and some of these kinases might act as downstream molecules of GLP-1 mediated PKA signaling cascade. A few phosphosites were regulated by both GLP-1 and glucose alone, and these target proteins were highly related to their biological function on pancreatic β-cells. Finally, we found glucose and GLP-1 executed their synergistic effect at multiple levels, especially at pathway level. Both GLP-1 and glucose participated in regulating every single step of the secretion pathway, and systematically synergized their effects in inducing insulin secretion.
Quiescence of adult oligodendrocyte precursor cells requires thyroid hormone and hypoxia to activate Runx1
Apr 22, 2017   Scientific Reports
Tokumoto Y, Tamaki S, Kabe Y, Takubo K, Suematsu M
Quiescence of adult oligodendrocyte precursor cells requires thyroid hormone and hypoxia to activate Runx1
Apr 22, 2017
Scientific Reports
The adult mammalian central nervous system (CNS) contains a population of slowly dividing oligodendrocyte precursor cells (OPCs), i.e., adult OPCs, which supply new oligodendrocytes throughout the life of animal. While adult OPCs develop from rapidly dividing perinatal OPCs, the mechanisms underlying their quiescence remain unknown. Here, we show that perinatal rodent OPCs cultured with thyroid hormone (TH) under hypoxia become quiescent and acquire adult OPCs-like characteristics. The cyclin-dependent kinase inhibitor p15/INK4b plays crucial roles in the TH-dependent cell cycle deceleration in OPCs under hypoxia. Klf9 is a direct target of TH-dependent signaling. Under hypoxic conditions, hypoxia-inducible factors mediates runt-related transcription factor 1 activity to induce G1 arrest in OPCs through enhancing TH-dependent p15/INK4b expression. As adult OPCs display phenotypes of adult somatic stem cells in the CNS, the current results shed light on environmental requirements for the quiescence of adult somatic stem cells during their development from actively proliferating stem/progenitor cells.
IL-35 induces N2 phenotype of neutrophils to promote tumor growth
Apr 22, 2017   Oncotarget
Zou JM, Qin J, Li YC, Wang Y, Li D, Shu Y, Luo C, Wang SS, Chi G, Guo F, Zhang GM, Feng ZH
IL-35 induces N2 phenotype of neutrophils to promote tumor growth
Apr 22, 2017
Oncotarget
IL-35 is an immunosuppressive cytokine and exerts regulatory effects on T cells, B cells, macrophages and dendritic cells. Neutrophils are important innate immune cells that play key roles in tumor development. The effect of IL-35 on neutrophils remains unknown. Here, we report that IL-35 can induce N2 neutrophil polarization (protumor phenotype) by increasing G-CSF and IL-6 production, and promote neutrophil infiltration into tumor microenvironment. The sustained expression of IL-35 could promote chronic inflammation to augment the proangiogenic and immunosuppressive function of neutrophils. IL-35 stimulated macrophages to secrete proinflammatory cytokines IL-1β and IL-6. IL-1β stimulated γδ T cells to produce IL-17, which in turn increased the production of G-CSF. By increasing the expression of G-CSF and IL-6, IL-35 could up-regulate the expression of MMP-9 and Bv8, and down-regulate TRAIL expression in neutrophils, thus augmenting the proangiogenic function of neutrophils. Moreover, G-CSF/IL-6 induced the enhanced activation of STAT3 and ERK pathways in neutrophils, thus increasing the expression of iNOS to suppress T cell activation. Our findings suggest that IL-35 can promote tumor progression by functioning as an up-stream cytokine to promote cancer-associated inflammation and control neutrophil polarization. Targeting IL-35 might be an important approach for designing new strategy of tumor therapy.
Sensitive detection of viable circulating tumor cells using a novel conditionally telomerase-selective replicating adenovirus in non-small cell lung cancer patients
Apr 22, 2017   Oncotarget
Togo S, Katagiri N, Namba Y, Tulafu M, Nagahama K, Kadoya K, Takamochi K, Oh S, Suzuki K, Sakurai F, Mizuguchi H, Urata Y, Takahashi K
Sensitive detection of viable circulating tumor cells using a novel conditionally telomerase-selective replicating adenovirus in non-small cell lung cancer patients
Apr 22, 2017
Oncotarget
Circulating tumor cells (CTCs) have a crucial role in the clinical outcome of cancer patients. Detection of non-small cell lung cancer (NSCLC) using an antibody against epithelial cell adhesion molecule (EpCAM) in captured CTCs has low sensitivity; the loss of epithelial markers leads to underestimation of CTCs with mesenchymal phenotype. We propose a new approach for detection of viable CTCs, including those with epithelial-mesenchymal transition status (EMT-CTCs), using the new telomerase-specific replication-selective adenovirus (OBP-1101), TelomeScan F35. Peripheral venous blood samples and clinicopathological data were collected from 123 NSCLC patients. The sensitivity of CTC detection was 69.1%, and for patients with stage I, II, III and IV, it was 59.6%, 40.0%, 85.7%, and 75.0%, respectively. Among the EMT-CTC samples, 46% were vimentin positive and 39.0% of non-EMT-CTC samples were EpCAM positive. Patients testing positive for EMT-CTCs at baseline had poor response to chemotherapy (P = 0.025) and decreased progression-free survival (EMT-CTC positive vs. negative: 193 ± 47 days vs. 388 ± 47. days, P = 0.040) in comparison to those testing negative. TelomeScan F35 is a highly sensitive CTC detection system and will be a useful screening tool for early diagnosis of NSCLC patients. Mesenchymal-phenotype CTCs are crucial indicators of chemotherapeutic efficacy in NSCLC patients.
Acute Hepatopancreatic Necrosis Disease (AHPND)-causing Vibrio parahaemolyticus strains maintain an antibacterial Type VI Secretion System with versatile effector repertoires
Apr 22, 2017   Applied And Environmental Microbiology
Li P, Kinch LN, Ray A, Dalia AB, Cong Q, Nunan LM, Camilli A, Grishin NV, Salomon D, Orth K
Acute Hepatopancreatic Necrosis Disease (AHPND)-causing Vibrio parahaemolyticus strains maintain an antibacterial Type VI Secretion System with versatile effector repertoires
Apr 22, 2017
Applied And Environmental Microbiology
Acute hepatopancreatic necrosis disease (AHPND) is a newly emerging shrimp disease that has severely damaged the global shrimp industry. AHPND is caused by toxic strains of Vibrio parahaemolyticus (V. parahaemolyticus) that have acquired a "selfish plasmid" encoding the deadly binary toxins PirAvp/PirBvp To better understand the repertoire of virulence factors in AHPND-causing V. parahaemolyticus, we conducted a comparative analysis using genome sequences of the clinical strain RIMD2210633, environmental non-AHPND and toxic AHPND isolates of V. parahaemolyticus Interestingly, we found that all of the AHPND strains, but none of the non-AHPND strains, harbor the antibacterial type VI secretion system 1 (T6SS1) which we previously identified and characterized in the clinical isolate RIMD2210633. This finding suggests that the acquisition of this T6SS might confer AHPND-causing V. parahaemolyticus a fitness advantage over competing bacteria and facilitate shrimp infection. Additionally, we find highly dynamic effector loci in the T6SS1 of AHPND-causing strains, leading to diverse effector repertoires. Our discovery provides novel insights into AHPND-causing pathogens and reveals a potential target for disease control.Importance Acute hepatopancreatic necrosis disease (AHPND) is a serious disease that has caused severe damage and significant financial losses to the global shrimp industry. To better understand and prevent this shrimp disease, it is essential to thoroughly characterize its causative agent, Vibrio paraehaemolyticus (V. parahaemolyticus). Although the plasmid-encoded binary toxins PirAvp/PirBvp have been shown to be the primary cause of AHPND, it remains unknown whether other virulent factors are commonly present in V. parahaemolyticus and might play important roles during shrimp infection. Here, we analyzed the genome sequences of clinical, non-AHPND and AHPND strains to characterize their repertoires of key virulence determinants. Our studies reveal that an antibacterial type VI secretion system is associated with the AHPND strains and differentiates them from non-AHPND strains, similar to the PirA/PirB toxins. We propose that T6SS1 provides a selective advantage during shrimp infections. Copyright © 2017 American Society for Microbiology.
Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1)
Apr 22, 2017   Developmental And Comparative Immunology
Li H, Jin H, Li Y, Liu D, Foda MF, Jiang Y, Luo R
Molecular cloning and functional characterization of duck nucleotide-binding oligomerization domain 1 (NOD1)
Apr 22, 2017
Developmental And Comparative Immunology
Nucleotide-binding oligomerization domain 1 (NOD1) is an imperative cytoplasmic pattern recognition receptor (PRR) and considered as a key member of the NOD-like receptor (NLR) family which plays a critical role in innate immunity through sensing microbial components derived from bacterial peptidoglycan. In the current study, the full-length of duck NOD1 (duNOD1) cDNA from duck embryo fibroblasts (DEFs) was cloned. Multiple sequence alignment and phylogenetic analysis demonstrated that duNOD1 exhibited a strong evolutionary relationship with chicken and rock pigeon NOD1. Tissue-specific expression analysis showed that duNOD1 was widely distributed in various organs, with the highest expression observed in the liver. Furthermore, duNOD1 overexpression induced NF-κB activation in DEFs and the CARD domain is crucial for duNOD1-mediated NF-κB activation. In addition, silencing the duNOD1 decreased the activity of NF-κB in DEFs stimulated by iE-DAP. Overexpression of duNOD1 significantly increased the expression of TNF-α, IL-6, and RANTES in DEFs. These findings highlight the crucial role of duNOD1 as an intracellular sensor in duck innate immune system. Copyright © 2017. Published by Elsevier Ltd.
Emerging evidence for the role of differential tumor microenvironment in breast cancer racial disparity: a closer look at the surroundings
Apr 21, 2017   Carcinogenesis
Deshmukh SK, Srivastava SK, Tyagi N, Ahmad A, Singh AP, Ghadhban AA, Dyess DL, Carter JE, Dugger K, Singh S
Emerging evidence for the role of differential tumor microenvironment in breast cancer racial disparity: a closer look at the surroundings
Apr 21, 2017
Carcinogenesis
Although increased awareness leading to early detection and prevention, as well as advancements in treatment strategies, have resulted in superior clinical outcomes, African American women with breast cancer continue to have greater mortality rates, compared to Caucasian American counterparts. Moreover, African American women are more likely to have breast cancer at a younger age and be diagnosed with aggressive tumor sub-types. Such racial disparities can be attributed to socioeconomic differences, but it is increasingly being recognized that these disparities may indeed be due to certain genetic and other non-genetic biological differences. Tumor microenvironment, which provides a favorable niche for the growth of tumor cells, is comprised of several types of stromal cells and the various proteins secreted as a consequence of bi-directional tumor-stromal cross-talk. Emerging evidence suggests inherent biological differences in the tumor microenvironment of breast cancer patients from different racial backgrounds. Tumor microenvironment components, affected by the genetic make-up of the tumor cells as well as other non-tumor-associated factors, may also render patients more susceptible to the development of aggressive tumors and faster progression of disease resulting in early onset, thus adversely affecting patients' survival. This review provides an overview of breast cancer racial disparity and discusses the existence of race-associated differential tumor microenvironment and its underlying genetic and non-genetic causal factors. A better understanding of these aspects would help further research on effective cancer management and improved approaches for reducing the racial disparities gaps in breast cancer patients. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
Heterogeneous ribonuclear protein A3 (hnRNP A3) is present in dipeptide repeat protein containing inclusions in Frontotemporal Lobar Degeneration and Motor Neurone disease associated with expansions in C9orf72 gene
Apr 22, 2017   Acta Neuropathologica Communications
Davidson YS, Flood L, Robinson AC, Nihei Y, Mori K, Rollinson S, Richardson A, Benson BC, Jones M, Snowden JS, Pickering-Brown S, Haass C, Lashley T, Mann DMA
Heterogeneous ribonuclear protein A3 (hnRNP A3) is present in dipeptide repeat protein containing inclusions in Frontotemporal Lobar Degeneration and Motor Neurone disease associated with expansions in C9orf72 gene
Apr 22, 2017
Acta Neuropathologica Communications
Frontotemporal Lobar Degeneration (FTLD) encompasses certain related neurodegenerative disorders which alter behaviour, personality and language. Heterogeneous ribonuclear proteins (hnRNPs) maintain RNA metabolism and changes in their function may underpin the pathogenesis of FTLD. Immunostaining for hnRNP A1, A2/B1 and A3 was performed on sections of temporal cortex with hippocampus from 61 patients with FTLD, stratified by pathological hallmarks into FTLD-tau and FTLD-TDP type A, B and C subtypes, and by genetics into patients with C9orf72 expansions, MAPT or GRN mutations, or those without known mutation. Four patients with Motor Neurone Disease (MND) with C9orf72 expansions and 10 healthy controls were also studied. Semi-quantitative analysis assessed hnRNP staining intensity in dentate gyrus (DG) and CA4 region of hippocampus, and temporal cortex (Tcx) in the different pathological and genetic groups.Immunostaining for hnRNP A1, A2/B1 and A3 revealed no consistent changes in pattern or amount of physiological staining across any of the pathological or genetic groups. No immunostaining of any inclusions resembling TDP-43 immunoreactive neuronal cytoplasmic inclusions or dystrophic neurites, was seen in either Tcx or DG of the hippocampus in any of the FTLD cases investigated for hnRNP A1, A2/B1 and A3. However, immunostaining for hnRNP A3 showed that inclusion bodies, resembling those TDP-43 negative, p62-immunopositive structures containing dipeptide repeat proteins (DPR) were variably observed in hippocampus and cerebellum. The proportion of cases showing hnRNP A3-immunoreactive DPR, and the number of hnRNP A3-positive inclusions within cases, was significantly greater in DG than in cells of CA4 region and cerebellum, but the latter was significantly less in all three regions compared to that detected by p62 immunostaining.
Serum levels of lead, mercury and cadmium in relation to coronary artery disease in the elderly: A cross-sectional study
Apr 21, 2017   Chemosphere
Asgary S, Movahedian A, Keshvari M, Taleghani M, Sahebkar A, Sarrafzadegan N
Serum levels of lead, mercury and cadmium in relation to coronary artery disease in the elderly: A cross-sectional study
Apr 21, 2017
Chemosphere
There is a continuing attempt to identify novel factors that can predict the risk of cardiovascular disease beyond the established coronary risk factors. It has been suggested that serum levels of lead, mercury and cadmium are associated with the risk of coronary artery disease (CAD). In the present study, we aimed to evaluate serum concentrations of lead (s-Pb), mercury (s-Hg) and cadmium (s-Cd) in patients with CAD in comparison with those of healthy individuals. The correlation between serum levels of these heavy metals and lipid profile parameters was also investigated. In this case-control study, we included 65 patients (35 females) aged 50-70 years with angiographically-documented CAD and 65 healthy controls (43 female) matched for sex, age and place of residence. Serum concentrations of heavy metals were determined using graphite furnace atomic absorption (GFAA). Serum lipids were measured using routine enzymatic methods. It was observed that the mean concentration of s-Pb (12.54 ± 8.41 vs. 5.89 ± 4.44 μg/L, p 
Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5
Apr 21, 2017   Cell
Komolov KE, Du Y, Duc NM, Betz RM, Rodrigues JPGLM, Leib RD, Patra D, Skiniotis G, Adams CM, Dror RO, Chung KY, Kobilka BK, Benovic JL
Structural and Functional Analysis of a β2-Adrenergic Receptor Complex with GRK5
Apr 21, 2017
Cell
The phosphorylation of agonist-occupied G-protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) functions to turn off G-protein signaling and turn on arrestin-mediated signaling. While a structural understanding of GPCR/G-protein and GPCR/arrestin complexes has emerged in recent years, the molecular architecture of a GPCR/GRK complex remains poorly defined. We used a comprehensive integrated approach of cross-linking, hydrogen-deuterium exchange mass spectrometry (MS), electron microscopy, mutagenesis, molecular dynamics simulations, and computational docking to analyze GRK5 interaction with the β2-adrenergic receptor (β2AR). These studies revealed a dynamic mechanism of complex formation that involves large conformational changes in the GRK5 RH/catalytic domain interface upon receptor binding. These changes facilitate contacts between intracellular loops 2 and 3 and the C terminus of the β2AR with the GRK5 RH bundle subdomain, membrane-binding surface, and kinase catalytic cleft, respectively. These studies significantly contribute to our understanding of the mechanism by which GRKs regulate the function of activated GPCRs. PAPERCLIP. Copyright © 2017 Elsevier Inc. All rights reserved.
A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis
Apr 21, 2017   Molecular Cell
Treiber T, Treiber N, Plessmann U, Harlander S, Daiß JL, Eichner N, Lehmann G, Schall K, Urlaub H, Meister G
A Compendium of RNA-Binding Proteins that Regulate MicroRNA Biogenesis
Apr 21, 2017
Molecular Cell
During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.
Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a
Apr 21, 2017   Molecular Cell
Swarts DC, van der Oost J, Jinek M
Structural Basis for Guide RNA Processing and Seed-Dependent DNA Targeting by CRISPR-Cas12a
Apr 21, 2017
Molecular Cell
The CRISPR-associated protein Cas12a (Cpf1), which has been repurposed for genome editing, possesses two distinct nuclease activities: endoribonuclease activity for processing its own guide RNAs and RNA-guided DNase activity for target DNA cleavage. To elucidate the molecular basis of both activities, we determined crystal structures of Francisella novicida Cas12a bound to guide RNA and in complex with an R-loop formed by a non-cleavable guide RNA precursor and a full-length target DNA. Corroborated by biochemical experiments, these structures reveal the mechanisms of guide RNA processing and pre-ordering of the seed sequence in the guide RNA that primes Cas12a for target DNA binding. Furthermore, the R-loop complex structure reveals the strand displacement mechanism that facilitates guide-target hybridization and suggests a mechanism for double-stranded DNA cleavage involving a single active site. Together, these insights advance our mechanistic understanding of Cas12a enzymes and may contribute to further development of genome editing technologies. Copyright © 2017 Elsevier Inc. All rights reserved.
Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster
Apr 21, 2017   ELife
Park HB, Perez CE, Barber KW, Rinehart J, Crawford JM
Genome mining unearths a hybrid nonribosomal peptide synthetase-like-pteridine synthase biosynthetic gene cluster
Apr 21, 2017
ELife
Nonribosomal peptides represent a large class of metabolites with pharmaceutical relevance. Pteridines, such as pterins, folates, and flavins, are heterocyclic metabolites that often serve as redox-active cofactors. The biosynthetic machineries for construction of these distinct classes of small molecules operate independently in the cell. Here, we discovered an unprecedented nonribosomal peptide synthetase-like-pteridine synthase hybrid biosynthetic gene cluster in Photorhabdus luminescens using genome synteny analysis. P. luminescens is a Gammaproteobacterium that undergoes phenotypic variation and can have both pathogenic and mutualistic roles. Through extensive gene deletion, pathway-targeted molecular networking, quantitative proteomic analysis, and NMR, we show that the genetic locus affects the regulation of quorum sensing and secondary metabolic enzymes and encodes new pteridine metabolites functionalized with cis-amide acyl-side chains, termed pepteridine A (1) and B (2). The pepteridines are produced in the pathogenic phenotypic variant and represent the first reported metabolites to be synthesized by a hybrid NRPS-pteridine pathway. These studies expand our view of the combinatorial biosynthetic potential available in bacteria.
Respiratory chain enzyme deficiency induces mitochondrial location of actin-binding Gelsolin to modulate the oligomerization of VDAC complexes and cell survival
Apr 21, 2017   Human Molecular Genetics
García-Bartolomé A, Peñas A, Marín-Buera L, Lobo-Jarne T, Pérez-Pérez R, Morán M, Arenas J, Martín MA, Ugalde C
Respiratory chain enzyme deficiency induces mitochondrial location of actin-binding Gelsolin to modulate the oligomerization of VDAC complexes and cell survival
Apr 21, 2017
Human Molecular Genetics
Despite considerable knowledge on the genetic basis of mitochondrial disorders, their pathophysiological consequences remain poorly understood. We previously used 2D-DIGE analyses to define a protein profile characteristic for respiratory chain complex III-deficiency that included a significant overexpression of cytosolic Gelsolin (GSN), a cytoskeletal protein that regulates the severing and capping of the actin filaments. Biochemical and immunofluorescence assays confirmed a specific increase of GSN levels in the mitochondria from patientś fibroblasts and from transmitochondrial cybrids with complex III assembly defects. A similar effect was obtained in control cells upon treatment with antimycin A in a dose-dependent manner, showing that the enzymatic inhibition of complex III is sufficient to promote the mitochondrial localization of GSN. Mitochondrial subfractionation showed the localization of GSN to the mitochondrial outer membrane, where it interacts with the voltage-dependent anion channel protein 1 (VDAC1). In control cells, VDAC1 was present in five stable oligomeric complexes, which showed increased levels and a modified distribution pattern in the complex III-deficient cybrids. Downregulation of GSN expression induced cell death in both cell types, in parallel with the specific accumulation of VDAC1 dimers and the release of mitochondrial cytochrome c into the cytosol, indicating a role for GSN in the oligomerization of VDAC complexes and in the prevention of apoptosis. Our results demonstrate that respiratory chain complex III dysfunction induces the physiological upregulation and mitochondrial location of GSN, probably to promote cell survival responses through the modulation of the oligomeric state of the VDAC complexes. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.
DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination
Apr 21, 2017   DNA Research : An International Journal For Rapid Publication Of Reports On Genes And Genomes
Srivastava S, Dahal S, Naidu SJ, Anand D, Gopalakrishnan V, Kooloth Valappil R, Raghavan SC
DNA double-strand break repair in Penaeus monodon is predominantly dependent on homologous recombination
Apr 21, 2017
DNA Research : An International Journal For Rapid Publication Of Reports On Genes And Genomes
DNA double-strand breaks (DSBs) are mostly repaired by nonhomologous end joining (NHEJ) and homologous recombination (HR) in higher eukaryotes. In contrast, HR-mediated DSB repair is the major double-strand break repair pathway in lower order organisms such as bacteria and yeast. Penaeus monodon, commonly known as black tiger shrimp, is one of the economically important crustaceans facing large-scale mortality due to exposure to infectious diseases. The animals can also get exposed to chemical mutagens under the culture conditions as well as in wild. Although DSB repair mechanisms have been described in mammals and some invertebrates, its mechanism is unknown in the shrimp species. In the present study, we show that HR-mediated DSB repair is the predominant mode of repair in P. monodon. Robust repair was observed at a temperature of 30 °C, when 2 µg of cell-free extract derived from hepatopancreas was used for the study. Although HR occurred through both reciprocal recombination and gene conversion, the latter was predominant when the bacterial colonies containing recombinants were evaluated. Unlike mammals, NHEJ-mediated DSB repair was undetectable in P. monodon. However, we could detect evidence for an alternative mode of NHEJ that uses microhomology, termed as microhomology-mediated end joining (MMEJ). Interestingly, unlike HR, MMEJ was predominant at lower temperatures. Therefore, the results suggest that, while HR is major DSB repair pathway in shrimp, MMEJ also plays a role in ensuring the continuity and stability of the genome. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.
A multi-systemic mitochondrial disorder due to a dominant p.Y955H disease variant in DNA polymerase gamma
Apr 21, 2017   Human Molecular Genetics
Siibak T, Clemente P, Bratic A, Bruhn H, Kauppila TES,   . . . . . .   , Wedell A, Peter B, Freyer C, Falkenberg M, Wredenberg A
A multi-systemic mitochondrial disorder due to a dominant p.Y955H disease variant in DNA polymerase gamma
Apr 21, 2017
Human Molecular Genetics
Mutations in the mitochondrial DNA polymerase, POLG, are associated with a variety of clinical presentations, ranging from early onset fatal brain disease in Alpers syndrome to chronic progressive external ophthalmoplegia. The majority of mutations are linked with disturbances of mitochondrial DNA (mtDNA) integrity and maintenance. On a molecular level, depending on their location within the enzyme, mutations either lead to mtDNA depletion or the accumulation of multiple mtDNA deletions, and in some cases these molecular changes can be correlated to the clinical presentation. We identified a patient with a dominant p.Y955H mutation in POLG, presenting with a severe, early-onset multi-systemic mitochondrial disease with bilateral sensorineural hearing loss, cataract, myopathy, and liver failure. Using a combination of disease models of Drosophila melanogaster (Dm) and in vitro biochemistry analysis, we compare the molecular consequences of the p.Y955H mutation to the well-documented p.Y955C mutation. We demonstrate that both mutations affect mtDNA replication and display a dominant negative effect, with the p.Y955H allele resulting in a more severe polymerase dysfunction. © The Author 2017. Published by Oxford University Press.
Characterization of YjjJ toxin of Escherichia coli
Apr 21, 2017   FEMS Microbiology Letters
Maeda Y, Lin CY, Ishida Y, Inouye M, Yamaguchi Y, Phadtare S
Characterization of YjjJ toxin of Escherichia coli
Apr 21, 2017
FEMS Microbiology Letters
Reminiscent of eukaryotic apoptotic programmed cell death, bacteria also contain a large number of suicide genes, which are in general co-expressed with their cognate antitoxic genes. These systems called the toxin-antitoxin (TA) systems are associated with cellular dormancy, and play major roles in biofilm formation and persistent multi-drug resistance of many human pathogens. In recent years, the study on TA system toxins has become a hot topic due to the health implications of these toxins by virtue of their role in bacterial pathogenicity. Here we report functional characterization of a hitherto uncharacterized E. coli TA toxin, YjjJ. YjjJ exhibits several uncommon properties such as, (i) unlike the genes encoding most type II TA system toxins, the gene encoding YjjJ is present as a single gene and not in an operon, (ii) despite being a homologue of the well characterized toxin HipA, YjjJ seems to have different cellular target(s), and (iii) HipB, the cognate antitoxin of HipA also acts as an antitoxin for YjjJ. This forms a basis for an interesting next step in the study of TA systems with respect to cross regulation between various TA systems and the evolutionary as well as clinical significance of these observations. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Gates for soluble and membrane proteins, and two trafficking systems (IFT and LIFT), establish a dynamic ciliary signaling compartment
Apr 22, 2017   Current Opinion In Cell Biology
Jensen VL, Leroux MR
Gates for soluble and membrane proteins, and two trafficking systems (IFT and LIFT), establish a dynamic ciliary signaling compartment
Apr 22, 2017
Current Opinion In Cell Biology
Primary cilia are microtubule-based organelles found on most mammalian cell surfaces. They possess a soluble matrix and membrane contiguous with the cell body cytosol and plasma membrane, and yet, have distinct compositions that can be modulated to enable dynamic signal transduction. Here, we discuss how specialized ciliary compartments are established using a coordinated network of gating, trafficking and targeting activities. Cilium homeostasis is maintained by a size-selective molecular mesh that limits soluble protein entry, and by a membrane diffusion barrier localized at the transition zone. Bidirectional protein shuttling between the cell body and cilium uses IntraFlagellar Transport (IFT), and prenylated ciliary protein delivery is achieved through Lipidated protein IntraFlagellar Targeting (LIFT). Elucidating how these gates and transport systems function will help reveal the roles that cilia play in ciliary signaling and the growing spectrum of disorders termed ciliopathies. Copyright © 2017 Elsevier Ltd. All rights reserved.

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