scholarly journals A Hairpin-like Structure within an AU-rich mRNA-destabilizing Element Regulates trans-Factor Binding Selectivity and mRNA Decay Kinetics

2005 ◽  
Vol 280 (23) ◽  
pp. 22406-22417 ◽  
Author(s):  
Elizabeth J. Fialcowitz ◽  
Brandy Y. Brewer ◽  
Bridget P. Keenan ◽  
Gerald M. Wilson
Keyword(s):  
Mammalian Cells ◽  
Binding Proteins ◽  
Phylogenetic Analyses ◽  
Higher Order ◽  
Mrna Turnover ◽  
Decay Kinetics ◽  
Binding Selectivity ◽  
Factor Binding ◽  
Factor Α

In mammals, rapid mRNA turnover directed by AU-rich elements (AREs) is mediated by selective association of cellular ARE-binding proteins. These trans-acting factors display overlapping RNA substrate specificities and may act to either stabilize or destabilize targeted transcripts; however, the mechanistic features of AREs that promote preferential binding of one trans-factor over another are not well understood. Here, we describe a hairpin-like structure adopted by the ARE from tumor necrosis factor α (TNFα) mRNA that modulates its affinity for selected ARE-binding proteins. In particular, association of the mRNA-destabilizing factor p37AUF1 was strongly inhibited by adoption of the higher order ARE structure, whereas binding of the inducible heat shock protein Hsp70 was less severely compromised. By contrast, association of the mRNA-stabilizing protein HuR was only minimally affected by changes in ARE folding. Consistent with the inverse relationship between p37AUF1 binding affinity and the stability of ARE folding, mutations that stabilized the ARE hairpin also inhibited its ability to direct rapid mRNA turnover in transfected cells. Finally, phylogenetic analyses and structural modeling indicate that TNFα mRNA sequences flanking the ARE are highly conserved and may stabilize the hairpin fold in vivo. Taken together, these data suggest that local higher order structures involving AREs may function as potent regulators of mRNA turnover in mammalian cells by modulating trans-factor binding selectivity.

scholarly journals Identification of Novel Human Cdt1-binding Proteins by a Proteomics Approach: Proteolytic Regulation by APC/CCdh1

2008 ◽  
Vol 19 (3) ◽  
pp. 1007-1021 ◽  
Author(s):  
Nozomi Sugimoto ◽  
Issay Kitabayashi ◽  
Satoko Osano ◽  
Yasutoshi Tatsumi ◽  
Takashi Yugawa ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Binding Proteins ◽  
Mass Spectrometry Analysis ◽  
Anaphase Promoting Complex ◽  
Chromosomal Damage ◽  
Spectrometry Analysis

In mammalian cells, Cdt1 activity is strictly controlled by multiple independent mechanisms, implying that it is central to the regulation of DNA replication during the cell cycle. In fact, unscheduled Cdt1 hyperfunction results in rereplication and/or chromosomal damage. Thus, it is important to understand its function and regulations precisely. We sought to comprehensively identify human Cdt1-binding proteins by a combination of Cdt1 affinity chromatography and liquid chromatography and tandem mass spectrometry analysis. Through this approach, we could newly identify 11 proteins, including subunits of anaphase-promoting complex/cyclosome (APC/C), SNF2H and WSTF, topoisomerase I and IIα, GRWD1/WDR28, nucleophosmin/nucleoplasmin, and importins. In vivo interactions of Cdt1 with APC/CCdh1, SNF2H, topoisomerase I and IIα, and GRWD1/WDR28 were confirmed by coimmunoprecipitation assays. A further focus on APC/CCdh1 indicated that this ubiquitin ligase controls the levels of Cdt1 during the cell cycle via three destruction boxes in the Cdt1 N-terminus. Notably, elimination of these destruction boxes resulted in induction of strong rereplication and chromosomal damage. Thus, in addition to SCFSkp2 and cullin4-based ubiquitin ligases, APC/CCdh1 is a third ubiquitin ligase that plays a crucial role in proteolytic regulation of Cdt1 in mammalian cells.

Surprising roles for phospholipid binding proteins revealed by high throughput geneticsThis paper is one of a selection of papers published in this special issue entitled “Second International Symposium on Recent Advances in Basic, Clinical, and Social Medicine” and has undergone the Journal's usual peer review process.

2010 ◽  
Vol 88 (4) ◽  
pp. 565-574 ◽  
Author(s):  
Marissa A. LeBlanc ◽  
Christopher R. McMaster
Keyword(s):  
High Throughput ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Essential Gene ◽  
Peer Review Process ◽  
Vesicular Transport ◽  
Lipid Binding ◽  
And Function ◽  
Lipid Binding Proteins

Saccharomyces cerevisiae remains an ideal organism for studying the cell biological roles of lipids in vivo, as yeast has phospholipid metabolic pathways similar to mammalian cells, is easy and economical to manipulate, and is genetically tractable. The availability of isogenic strains containing specific genetic inactivation of each non-essential gene allowed for the development of a high-throughput method, called synthetic genetic analysis (SGA), to identify and describe precise pathways or functions associated with specific genes. This review describes the use of SGA to aid in elucidating the function of two lipid-binding proteins that regulate vesicular transport, Sec14 and Kes1. Sec14 was first identified as a phosphatidylcholine (PC) – phosphatidylinositol (PI) transfer protein required for viability, with reduced Sec14 function resulting in diminished vesicular transport out of the trans-Golgi. Although Sec14 is required for cell viability, inactivating the KES1 gene that encodes for a member of the oxysterol binding protein family in cells lacking Sec14 function results in restoration of vesicular transport and cell growth. SGA analysis identified a role for Kes1 and Sec14 in regulating the level and function of Golgi PI-4-phosphate (PI-4-P). SGA also determined that Sec14 not only regulates vesicular transport out of the trans-Golgi, but also transport from endosomes to the trans-Golgi. Comparing SGA screens in databases, coupled with genetic and cell biological analyses, further determined that the PI-4-P pool affected by Kes1 is generated by the PI 4-kinase Pik1. An important biological role for Sec14 and Kes1 revealed by SGA is coordinate regulation of the Pik1-generated Golgi PI-4-P pool that in turn is essential for vesicular transport into and out of the trans-Golgi.

In vivo effects of 17α-ethinylestradiol, 17β-estradiol and 4-nonylphenol on insulin-like growth-factor binding proteins (igfbps) in Atlantic salmon

2018 ◽  
Vol 203 ◽  
pp. 28-39 ◽  
Author(s):  
Jason P. Breves ◽  
Tara A. Duffy ◽  
Ingibjörg E. Einarsdottir ◽  
Björn Thrandur Björnsson ◽  
Stephen D. McCormick
Keyword(s):  
Growth Factor ◽  
Atlantic Salmon ◽  
Binding Proteins ◽  
Insulin Like Growth Factor ◽  
Factor Binding ◽  
17Β Estradiol ◽  
In Vivo Effects

scholarly journals Versatile Role for hnRNP D Isoforms in the Differential Regulation of Cytoplasmic mRNA Turnover

2001 ◽  
Vol 21 (20) ◽  
pp. 6960-6971 ◽  
Author(s):  
Nianhua Xu ◽  
Chyi-Ying A. Chen ◽  
Ann-Bin Shyu
Keyword(s):  
Mammalian Cells ◽  
Mrna Decay ◽  
Sequence Similarity ◽  
Class Ii ◽  
Negative Regulator ◽  
Mrna Turnover ◽  
Dependent Manner ◽  
Coding Region ◽  
Hnrnp D

ABSTRACT An important emerging theme is that heterogeneous nuclear ribonucleoproteins (hnRNPs) not only function in the nucleus but also control the fates of mRNAs in the cytoplasm. Here, we show that hnRNP D plays a versatile role in cytoplasmic mRNA turnover by functioning as a negative regulator in an isoform-specific and cell-type-dependent manner. We found that hnRNP D discriminates among the three classes of AU-rich elements (AREs), most effectively blocking rapid decay directed by class II AREs found in mRNAs encoding cytokines. Our experiments identified the overlapping AUUUA motifs, one critical characteristic of class II AREs, to be the key feature recognized in vivo by hnRNP D for its negative effect on ARE-mediated mRNA decay. The four hnRNP D isoforms, while differing in their ability to block decay of ARE-containing mRNAs, all potently inhibited mRNA decay directed by another mRNA cis element that shares no sequence similarity with AREs, the purine-rich c-fosprotein-coding region determinant of instability. Further experiments indicated that different mechanisms underlie the inhibitory effect of hnRNP D on the two distinct mRNA decay pathways. Our study identifies a potential mechanism by which cytoplasmic mRNA turnover can be differentially and selectively regulated by hnRNP D isoforms in mammalian cells. Our results support the notion that hnRNP D serves as a key factor broadly involved in general mRNA decay.

scholarly journals What Can Mushroom Proteins Teach Us about Lipid Rafts?

Membranes ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 264
Author(s):  
Maja Grundner ◽  
Anastasija Panevska ◽  
Kristina Sepčić ◽  
Matej Skočaj
Keyword(s):  
Lipid Rafts ◽  
Lipid Raft ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Protein Complexes ◽  
Biological Membranes ◽  
Indirect Evidence ◽  
Lipid Binding ◽  
Liquid Ordered

The lipid raft hypothesis emerged as a need to explain the lateral organization and behavior of lipids in the environment of biological membranes. The idea, that lipids segregate in biological membranes to form liquid-disordered and liquid-ordered states, was faced with a challenge: to show that lipid-ordered domains, enriched in sphingomyelin and cholesterol, actually exist in vivo. A great deal of indirect evidence and the use of lipid-binding probes supported this idea, but there was a lack of tools to demonstrate the existence of such domains in living cells. A whole new toolbox had to be invented to biochemically characterize lipid rafts and to define how they are involved in several cellular functions. A potential solution came from basic biochemical experiments in the late 1970s, showing that some mushroom extracts exert hemolytic activities. These activities were later assigned to aegerolysin-based sphingomyelin/cholesterol-specific cytolytic protein complexes. Recently, six sphingomyelin/cholesterol binding proteins from different mushrooms have been identified and have provided some insight into the nature of sphingomyelin/cholesterol-rich domains in living vertebrate cells. In this review, we dissect the accumulated knowledge and introduce the mushroom lipid raft binding proteins as molecules of choice to study the dynamics and origins of these liquid-ordered domains in mammalian cells.

scholarly journals Septins Arrange F-Actin-Containing Fibers on the Chlamydia trachomatis Inclusion and Are Required for Normal Release of the Inclusion by Extrusion

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Larisa Volceanov ◽  
Katharina Herbst ◽  
Martin Biniossek ◽  
Oliver Schilling ◽  
Dirk Haller ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Higher Order ◽  
Host Cells ◽  
Developmental Cycle ◽  
Content Type ◽  
Gtp Binding Proteins ◽  
Gtp Binding ◽  
Chlamydial Inclusion

ABSTRACTChlamydia trachomatisis an obligate intracellular human pathogen that grows inside a membranous, cytosolic vacuole termed an inclusion. Septins are a group of 13 GTP-binding proteins that assemble into oligomeric complexes and that can form higher-order filaments. We report here that the septins SEPT2, -9, -11, and probably -7 form fibrillar structures around the chlamydial inclusion. Colocalization studies suggest that these septins combine with F actin into fibers that encase the inclusion. Targeting the expression of individual septins by RNA interference (RNAi) prevented the formation of septin fibers as well as the recruitment of actin to the inclusion. At the end of the developmental cycle ofC. trachomatis, newly formed, infectious elementary bodies are released, and this release occurs at least in part through the organized extrusion of intact inclusions. RNAi against SEPT9 or against the combination of SEPT2/7/9 substantially reduced the number of extrusions from a culture of infected HeLa cells. The data suggest that a higher-order structure of four septins is involved in the recruitment or stabilization of the actin coat around the chlamydial inclusion and that this actin recruitment by septins is instrumental for the coordinated egress ofC. trachomatisfrom human cells. The organization of F actin around parasite-containing vacuoles may be a broader response mechanism of mammalian cells to the infection by intracellular, vacuole-dwelling pathogens.IMPORTANCEChlamydia trachomatisis a frequent bacterial pathogen throughout the world, causing mostly eye and genital infections.C. trachomatiscan develop only inside host cells; it multiplies inside a membranous vacuole in the cytosol, termed an inclusion. The inclusion is covered by cytoskeletal “coats” or “cages,” whose organization and function are poorly understood. We here report that a relatively little-characterized group of proteins, septins, is required to organize actin fibers on the inclusion and probably through actin the release of the inclusion. Septins are a group of GTP-binding proteins that can organize into heteromeric complexes and then into large filaments. Septins have previously been found to be involved in the interaction of the cell with bacteria in the cytosol. Our observation that they also organize a reaction to bacteria living in vacuoles suggests that they have a function in the recognition of foreign compartments by a parasitized human cell.

scholarly journals Tetramerizing tGCN4 domain facilitates production of Influenza A H1N1 M2e higher order soluble oligomers that show enhanced immunogenicity in vivo

2020 ◽  
Vol 295 (42) ◽  
pp. 14352-14366
Author(s):  
Sweety Samal ◽  
Tripti Shrivastava ◽  
Praveen Sonkusre ◽  
Zaigham Abbas Rizvi ◽  
Rajesh Kumar ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Influenza A ◽  
Large Scale ◽  
Vaccine Development ◽  
Higher Order ◽  
Amino Terminus ◽  
Th2 Response ◽  
Influenza A H1n1 ◽  
Mammalian System

One strategy for the development of a next generation influenza vaccine centers upon using conserved domains of the virus to induce broader and long-lasting immune responses. The production of artificial proteins by mimicking native-like structures has shown to be a promising approach for vaccine design against diverse enveloped viruses. The amino terminus of influenza A virus matrix 2 ectodomain (M2e) is highly conserved among influenza subtypes, and previous studies have shown M2e-based vaccines are strongly immunogenic, making it an attractive target for further exploration. We hypothesized that stabilizing M2e protein in the mammalian system might influence the immunogenicity of M2e with the added advantage to robustly produce the large scale of proteins with native-like fold and hence can act as an efficient vaccine candidate. In this study, we created an engineered construct in which the amino terminus of M2e is linked to the tetramerizing domain tGCN4, expressed the construct in a mammalian system, and tested for immunogenicity in BALB/c mice. We have also constructed a stand-alone M2e construct (without tGCN4) and compared the protein expressed in mammalian cells and in Escherichia coli using in vitro and in vivo methods. The mammalian-expressed protein was found to be more stable, more antigenic than the E. coli protein, and form higher-order oligomers. In an intramuscular protein priming and boosting regimen in mice, these proteins induced high titers of antibodies and elicited a mixed Th1/Th2 response. These results highlight the mammalian-expressed M2e soluble proteins as a promising vaccine development platform.

scholarly journals A Proteomics Analysis of Calmodulin-Binding Proteins in Dictyostelium discoideum during the Transition from Unicellular Growth to Multicellular Development

2021 ◽  
Vol 22 (4) ◽  
pp. 1722
Author(s):  
William D. Kim ◽  
Shyong Q. Yap ◽  
Robert J. Huber
Keyword(s):  
Dictyostelium Discoideum ◽  
Calcium Binding ◽  
Mammalian Cells ◽  
Binding Proteins ◽  
Model Organism ◽  
Developmental Processes ◽  
Multicellular Development ◽  
Calmodulin Binding ◽  
Search Tool

Calmodulin (CaM) is an essential calcium-binding protein within eukaryotes. CaM binds to calmodulin-binding proteins (CaMBPs) and influences a variety of cellular and developmental processes. In this study, we used immunoprecipitation coupled with mass spectrometry (LC-MS/MS) to reveal over 500 putative CaM interactors in the model organism Dictyostelium discoideum. Our analysis revealed several known CaMBPs in Dictyostelium and mammalian cells (e.g., myosin, calcineurin), as well as many novel interactors (e.g., cathepsin D). Gene ontology (GO) term enrichment and Search Tool for the Retrieval of Interacting proteins (STRING) analyses linked the CaM interactors to several cellular and developmental processes in Dictyostelium including cytokinesis, gene expression, endocytosis, and metabolism. The primary localizations of the CaM interactors include the nucleus, ribosomes, vesicles, mitochondria, cytoskeleton, and extracellular space. These findings are not only consistent with previous work on CaM and CaMBPs in Dictyostelium, but they also provide new insight on their diverse cellular and developmental roles in this model organism. In total, this study provides the first in vivo catalogue of putative CaM interactors in Dictyostelium and sheds additional light on the essential roles of CaM and CaMBPs in eukaryotes.

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