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https://www.readbyqxmd.com/read/28095497/replication-coupled-recruitment-of-viral-and-cellular-factors-to-herpes-simplex-virus-type-1-replication-forks-for-the-maintenance-and-expression-of-viral-genomes
#1
Jill A Dembowski, Sarah E Dremel, Neal A DeLuca
Herpes simplex virus type 1 (HSV-1) infects over half the human population. Much of the infectious cycle occurs in the nucleus of cells where the virus has evolved mechanisms to manipulate host processes for the production of virus. The genome of HSV-1 is coordinately expressed, maintained, and replicated such that progeny virions are produced within 4-6 hours post infection. In this study, we selectively purify HSV-1 replication forks and associated proteins from virus-infected cells and identify select viral and cellular replication, repair, and transcription factors that associate with viral replication forks...
January 17, 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28088608/gene-analysis-of-six-cases-of-congenital-protein-s-deficiency-and-functional-analysis-of-protein-s-mutations-a139v-c449f-r451q-c475f-a525v-and-d599tfster13
#2
Fumina Taniguchi, Eriko Morishita, Akiko Sekiya, Haruka Nomoto, Shiori Katsu, Shounosuke Kaneko, Hidesaku Asakura, Shigeki Ohtake
Congenital deficiency of protein S (PS), an anticoagulant factor, leads to venous thrombosis, with onset predominantly beginning in adolescence. In the present study, gene analysis of six unrelated Japanese families diagnosed with congenital PS deficiency identified five missense mutations in the PROS1 gene - c.757C>T (Ala139Val; A139V), c.1346 G>T (Cys449Phe; C449F), c.1352G>A (Arg451Gln; R451Q), c.1424G>T (Cys475Phe; C475F) and c.1574C>T (Ala525Val; A525V) - and one frameshift mutation, c.2135delA (Asp599ThrfsTer13; D599TfsTer13)...
December 24, 2016: Thrombosis Research
https://www.readbyqxmd.com/read/28079880/nonradioactive-quantification-of-autophagic-protein-degradation-with-l-azidohomoalanine-labeling
#3
Jigang Wang, Jianbin Zhang, Yew Mun Lee, Shukie Ng, Yin Shi, Zi-Chun Hua, Qingsong Lin, Han-Ming Shen
At present, several assays that use radioisotope labeling to quantify the degradation of long-lived proteins have been developed to measure autophagic flux. Here, we describe a nonradioactive pulse-chase protocol using L-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation during autophagy. AHA is used as a surrogate for L-methionine, and, when added to cultured cells grown in methionine-free medium, AHA is incorporated into proteins during de novo protein synthesis. After a chase period to remove short-lived proteins, autophagy is induced by starvation or other stimuli...
December 2017: Nature Protocols
https://www.readbyqxmd.com/read/28076784/coupled-proliferation-and-apoptosis-maintain-the-rapid-turnover-of-microglia-in-the-adult-brain
#4
Katharine Askew, Kaizhen Li, Adrian Olmos-Alonso, Fernando Garcia-Moreno, Yajie Liang, Philippa Richardson, Tom Tipton, Mark A Chapman, Kristoffer Riecken, Sol Beccari, Amanda Sierra, Zoltán Molnár, Mark S Cragg, Olga Garaschuk, V Hugh Perry, Diego Gomez-Nicola
Microglia play key roles in brain development, homeostasis, and function, and it is widely assumed that the adult population is long lived and maintained by self-renewal. However, the precise temporal and spatial dynamics of the microglial population are unknown. We show in mice and humans that the turnover of microglia is remarkably fast, allowing the whole population to be renewed several times during a lifetime. The number of microglial cells remains steady from late postnatal stages until aging and is maintained by the spatial and temporal coupling of proliferation and apoptosis, as shown by pulse-chase studies, chronic in vivo imaging of microglia, and the use of mouse models of dysregulated apoptosis...
January 10, 2017: Cell Reports
https://www.readbyqxmd.com/read/28069747/a-targeted-rnai-screen-identifies-factors-affecting-diverse-stages-of-receptor-mediated-transcytosis
#5
Bradlee Nelms, Natasha Furtado Dalomba, Wayne Lencer
Endosome transport by transcytosis is the primary mechanism by which proteins and other large cargo traverse epithelial barriers in normal tissue. Transcytosis is also essential for establishing and maintaining membrane polarity in epithelia and other polarized cells. To identify novel components of this pathway, we conducted a high-throughput RNA interference screen for factors necessary for the bidirectional transcytosis of IgG by the Fcγ receptor FcRn. This screen identified 23 genes whose suppression resulted in a reproducible decrease in FcRn-mediated transcytosis...
January 9, 2017: Journal of Cell Biology
https://www.readbyqxmd.com/read/28062662/disease-causing-mutations-affecting-surface-residues-of-mitochondrial-glutaryl-coa-dehydrogenase-impair-stability-heteromeric-complex-formation-and-mitochondria-architecture
#6
Jessica Schmiesing, Benjamin Lohmöller, Michaela Schweizer, Henning Tidow, Søren W Gersting, Ania C Muntau, Thomas Braulke, Chris Mühlhausen
The neurometabolic disorder glutaric aciduria type 1 (GA1) is caused by mutations in the GCDH gene encoding the mitochondrial matrix protein glutaryl-CoA dehydrogenase (GCDH), which forms homo- and heteromeric complexes. Twenty percent of all pathogenic mutations affect single amino acid residues on the surface of GCDH resulting in a severe clinical phenotype. We report here on heterologous expression studies of 18 missense mutations identified in GA1 patients affecting surface amino acids. Western blot and pulse chase experiments revealed that the stability of half of the GCDH mutants was significantly reduced...
January 5, 2017: Human Molecular Genetics
https://www.readbyqxmd.com/read/28002502/ehnpc1-and-ehnpc2-proteins-participate-in-trafficking-of-exogenous-cholesterol-in-entamoeba-histolytica-trophozoites-relevance-for-phagocytosis
#7
Jeni Bolaños, Abigail Betanzos, Rosario Javier-Reyna, Guillermina García-Rivera, Miriam Huerta, Jonnatan Pais-Morales, Arturo González-Robles, Mario A Rodríguez, Michael Schnoor, Esther Orozco
Entamoeba histolytica, the highly phagocytic protozoan causative of human amoebiasis lacks the machinery to synthesize cholesterol. Here, we investigated the presence of NPC1 and NPC2 proteins in this parasite, which are involved in cholesterol trafficking in mammals. Bioinformatics analysis revealed one Ehnpc1 and two Ehnpc2 genes. EhNPC1 appeared as a transmembrane protein and both EhNPC2 as peripheral membrane proteins. Molecular docking predicted that EhNPC1 and EhNPC2 bind cholesterol and interact with each other...
December 2016: PLoS Pathogens
https://www.readbyqxmd.com/read/27974639/visualizing-the-dynamic-coupling-of-claudin-strands-to-the-actin-cytoskeleton-through-zo-1
#8
Christina M Van Itallie, Amber Jean Tietgens, James M Anderson
The organization and integrity of epithelial tight junctions depend on interactions between claudins, ZO scaffolding proteins and the cytoskeleton. However, although binding between claudins and ZO-1/2/3 and between ZO-1/2/3 and numerous cytoskeletal proteins has been demonstrated in vitro, FRAP (Fluorescence Recovery After Photobleaching) analysis suggests interactions in vivo are likely highly dynamic. Here we use super resolution live cell imaging in a model fibroblast system to examine relationships between claudins, ZO-1, occludin and actin...
December 14, 2016: Molecular Biology of the Cell
https://www.readbyqxmd.com/read/27932476/high-fat-diet-induced-lysosomal-dysfunction-and-impaired-autophagic-flux-contribute-to-lipotoxicity-in-the-kidney
#9
Takeshi Yamamoto, Yoshitsugu Takabatake, Atsushi Takahashi, Tomonori Kimura, Tomoko Namba, Jun Matsuda, Satoshi Minami, Jun-Ya Kaimori, Isao Matsui, Taiji Matsusaka, Fumio Niimura, Motoko Yanagita, Yoshitaka Isaka
Excessive fat intake contributes to the progression of metabolic diseases via cellular injury and inflammation, a process termed lipotoxicity. Here, we investigated the role of lysosomal dysfunction and impaired autophagic flux in the pathogenesis of lipotoxicity in the kidney. In mice, a high-fat diet (HFD) resulted in an accumulation of phospholipids in enlarged lysosomes within kidney proximal tubular cells (PTCs). In isolated PTCs treated with palmitic acid, autophagic degradation activity progressively stagnated in association with impaired lysosomal acidification and excessive lipid accumulation...
December 8, 2016: Journal of the American Society of Nephrology: JASN
https://www.readbyqxmd.com/read/27929926/comparing-lamin-proteins-post-translational-relative-stability-using-a-2a-peptide-based-system-reveals-elevated-resistance-of-progerin-to-cellular-degradation
#10
Di Wu, Phillip A Yates, Haoyue Zhang, Kan Cao
Nuclear lamins are the major components of the nuclear lamina at the periphery of the nucleus, supporting the nuclear envelope and participating in many nuclear processes, including DNA replication, transcription and chromatin organization. A group of diseases, the laminopathies, is associated with mutations in lamin genes. One of the most striking cases is Hutchinson-Gilford progeria syndrome (HGPS) which is the consequence of a lamin A dominant negative mutant named progerin. Due to the abnormal presence of a permanent C-terminal farnesyl tail, progerin gradually accumulates on the nuclear membrane, perturbing a diversity of signalings and transcriptional events...
November 2016: Nucleus
https://www.readbyqxmd.com/read/27896768/two-dimensional-gel-electrophoresis-of-insulin-secretory-granule-proteins-from-biosynthetically-labeled-pancreatic-islets
#11
Paul C Guest
Pulse-chase radiolabeling of cells with radioactive amino acids is a common method for tracking the biosynthesis of proteins. Radiolabeled newly synthesized proteins can be analyzed by a number of techniques such as two dimensional gel electrophoresis (2DE). This chapter presents a protocol for the biosynthetic labeling of pancreatic islets with (35)S-methionine in the presence of basal and stimulatory concentrations of glucose, followed by subcellular fractionation to produce a secretory granule fraction and analysis of the granule protein contents by 2DE...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27815386/pten-signaling-in-the-postnatal-perivascular-progenitor-niche-drives-medulloblastoma-formation
#12
Guo Zhu, Sherri L Rankin, Jon D Larson, Xiaoyan Zhu, Lionel M L Chow, Chunxu Qu, Jinghui Zhang, David W Ellison, Suzanne J Baker
Loss of the tumor suppressor gene PTEN exerts diverse outcomes on cancer in different developmental contexts. To gain insight into the effect of its loss on outcomes in the brain, we conditionally inactivated the murine Pten gene in neonatal neural stem/progenitor cells. Pten inactivation created an abnormal perivascular proliferative niche in the cerebellum that persisted in adult animals but did not progress to malignancy. Proliferating cells showed undifferentiated morphology and expressed the progenitor marker Nestin but not Math1, a marker of committed granule neuron progenitors...
January 1, 2017: Cancer Research
https://www.readbyqxmd.com/read/27799163/to-b1a-or-not-to-b1a-do-hematopoietic-stem-cells-contribute-to-tissue-resident-immune-cells
#13
REVIEW
Anna E Beaudin, E Camilla Forsberg
Hematopoietic stem cells (HSCs) have long been considered the continuous source of all hematopoietic cells for the life of an individual. Recent findings have questioned multiple aspects of this view, including the ability of lifelong HSCs to contribute to tissue-resident immune cells. Here we discuss the most recent findings on the source of B1a cells, innatelike lymphocytes that primarily reside in serous cavities. Powerful experimental approaches including bar coding, single cell transplantation, in vivo lineage tracing, and HSC-specific pulse-chase labeling have provided novel insights on B1a-cell generation during ontogeny...
December 15, 2016: Blood
https://www.readbyqxmd.com/read/27738101/hyperactivation-of-the-insulin-signaling-pathway-improves-intracellular-proteostasis-by-coordinately-up-regulating-the-proteostatic-machinery-in-adipocytes
#14
Annabel Y Minard, Martin K L Wong, Rima Chaudhuri, Shi-Xiong Tan, Sean J Humphrey, Benjamin L Parker, Jean Y Yang, D Ross Laybutt, Gregory J Cooney, Adelle C F Coster, Jacqueline Stöckli, David E James
Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy...
December 2, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27723268/polymorphisms-in-the-f-pocket-of-hla-b27-subtypes-strongly-impact-on-assembly-chaperone-interactions-and-heavy-chain-misfolding
#15
David B Guiliano, Helen North, Eleni Panayoitou, Elaine C Campbell, Kirsty McHugh, Fiona G M Cooke, Marine Silvestre, Paul Bowness, Simon J Powis, Antony N Antoniou
Objective - HLA-B27 is associated with the inflammatory spondyloarthropathies (SpAs). Of significance, subtypes HLA-B*27:06 and HLA-B*27:09 are not associated with the SpAs. These subtypes primarily differ from the HLA-B*27:05 disease associated allele at residues 114 and 116 of the heavy chain, part of the F pocket of the antigen-binding groove. Dimerisation of HLA-B27 during assembly has been implicated in disease onset. This study investigated the factors influencing differences in dimerisation between disease associated and non-associated HLA-B27 alleles...
October 9, 2016: Arthritis & Rheumatology
https://www.readbyqxmd.com/read/27720452/kinetic-analysis-of-protein-stability-reveals-age-dependent-degradation
#16
Erik McShane, Celine Sin, Henrik Zauber, Jonathan N Wells, Neysan Donnelly, Xi Wang, Jingyi Hou, Wei Chen, Zuzana Storchova, Joseph A Marsh, Angelo Valleriani, Matthias Selbach
Do young and old protein molecules have the same probability to be degraded? We addressed this question using metabolic pulse-chase labeling and quantitative mass spectrometry to obtain degradation profiles for thousands of proteins. We find that >10% of proteins are degraded non-exponentially. Specifically, proteins are less stable in the first few hours of their life and stabilize with age. Degradation profiles are conserved and similar in two cell types. Many non-exponentially degraded (NED) proteins are subunits of complexes that are produced in super-stoichiometric amounts relative to their exponentially degraded (ED) counterparts...
October 20, 2016: Cell
https://www.readbyqxmd.com/read/27704601/comparing-single-feeding-and-multi-feeding-approach-for-experimentally-assessing-trophic-transfer-of-metals-in-fish
#17
Simon Pouil, Michel Warnau, François Oberhänsli, Jean-Louis Teyssié, Paco Bustamante, Marc Metian
Diet is an important pathway for metal uptake in marine organisms, and assimilation efficiency (AE) is one of the most relevant parameter to quantify trophic transfer of metals along aquatic food webs. The most commonly used method to estimate this parameter is pulse-chase feeding using radiolabelled food. This approach is, however, based on several assumptions that are not always tested in experimental context. The present work aimed at validating the approach by assessing single-feeding and multiple-feeding approaches, using a model species (the turbot Scophthalmus maximus)...
October 5, 2016: Environmental Toxicology and Chemistry
https://www.readbyqxmd.com/read/27681595/loss-of-ift122-a-retrograde-intraflagellar-transport-ift-complex-component-leads-to-slow-progressive-photoreceptor-degeneration-due-to-inefficient-opsin-transport
#18
Meriam Boubakri, Taro Chaya, Hiromi Hirata, Naoko Kajimura, Ryusuke Kuwahara, Akiko Ueno, Jarema Malicki, Takahisa Furukawa, Yoshihiro Omori
In the retina, aberrant opsin transport from cell bodies to outer segments leads to retinal degenerative diseases such as retinitis pigmentosa. Opsin transport is facilitated by the intraflagellar transport (IFT) system that mediates the bidirectional movement of proteins within cilia. In contrast to functions of the anterograde transport executed by IFT complex B (IFT-B), the precise functions of the retrograde transport mediated by IFT complex A (IFT-A) have not been well studied in photoreceptor cilia. Here, we analyzed developing zebrafish larvae carrying a null mutation in ift122 encoding a component of IFT-A...
November 18, 2016: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/27665551/chemical-secretory-pathway-modulation-in-plant-protoplasts
#19
Francesca De Marchis, Andrea Pompa, Michele Bellucci
The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence...
2016: Methods in Molecular Biology
https://www.readbyqxmd.com/read/27614019/dpp9-is-a-novel-component-of-the-n-end-rule-pathway-targeting-the-tyrosine-kinase-syk
#20
Daniela Justa-Schuch, Maria Silva-Garcia, Esther Pilla, Michael Engelke, Markus Kilisch, Christof Lenz, Ulrike Möller, Fumihiko Nakamura, Henning Urlaub, Ruth Geiss-Friedlander
The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1...
September 10, 2016: ELife
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