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Variant Proteins (variant + protein)

Distribution by Scientific Domains

Life Sciences	66%
Chemistry	33%

Selected Abstracts

A new ferrous iron-uptake transporter, EfeU (YcdN), from Escherichia coli

MOLECULAR MICROBIOLOGY, Issue 1 2006
Cornelia Große
Summary Escherichia coli possesses multiple routes for iron uptake. Here we present EfeU (YcdN), a novel iron acquisition system of E. coli strain Nissle 1917. Laboratory strains of E. coli such as K12 lack a functional (efeU) ycdN gene caused by a frameshift mutation. EfeU, a member of the oxidase-dependent iron transporters (OFeT), is a homologue of the iron permease Ftr1p from yeast. The ycdN gene is part of the ycdNOB tricistronic operon which is expressed in response to iron deprivation in a Fur-dependent manner. Expression of efeU resulted in improved growth of an E. coli mutant lacking all known iron-uptake systems and mediated increased iron uptake into cells. Furthermore, the presence of other divalent metal cations did not impair growth of strains expressing efeU. The EfeU protein functioned as ferrous iron permease in proteoliposomes in vitro. Topology analysis indicated that EfeU is an integral cytoplasmic membrane protein exhibiting seven transmembrane helices. Two REXXE motifs within transmembrane helices of OFeT family members are implicated in iron translocation. Site-directed mutagenesis of each REGLE motif of EfeU diminished iron uptake in vivo and growth yield. In vitro the EfeU variant protein with an altered first REGLE motif was impaired in iron permeation, whereas activity of the EfeU variant with a mutation in the second motif was similar to the wild-type protein. [source]

Detection and localization of an estrogen receptor beta splice variant protein (ER,2) in the adult female rat forebrain and midbrain regions

THE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 3 2007
Wilson C.J. Chung
Abstract Estrogens regulate neural processes such as neuronal development, reproductive behavior, and hormone secretion, and signal through estrogen receptor (ER) , and ER, (here called ER,1). Recent studies have found variations in ER, and ER,1 mRNA splicing in rodents and humans. Functional reporter gene assays suggest that these splicing variations alter ER-mediated transcriptional regulation. Estrogen receptor beta 2 (ER,2), an ER,1 splice variant containing an 18 amino acid (AA) insert in the ligand binding domain, binds estradiol with ,10-fold lower affinity than ER,1, suggesting that it may serve as a low-affinity ER. Moreover, ER,2 reportedly acts in a dominant-negative fashion when heterodimerized with ER,1 or ER,. To explore the function of ER,2 in brain, an antiserum (Two,ER.1) targeting the 18 AA insert was developed and characterized. Western blot analysis and transient expression of ER,2 in cell lines demonstrated that Two,ER.1 recognizes ER,2. In the adult female rat brain, ER,2 immunoreactivity is localized in the cell nucleus and is expressed with a distribution similar to that of ER,1 mRNA. ER,2 immunoreactive cell numbers were high in, for example, piriform cortex, paraventricular nucleus, supraoptic nucleus, arcuate nucleus, and hippocampal CA regions, whereas it was low in the dentate gyrus. Moreover, ER,2 is coexpressed in gonadotropin-releasing hormone and oxytocin neurons. These studies demonstrate ER, splice variant proteins in brain and support the hypothesis that ER signaling diversity depends not only on ligand or coregulatory proteins, but also on regional and phenotypic selectivity of ER splice variant proteins. J. Comp. Neurol. 505:249,267, 2007. © 2007 Wiley-Liss, Inc. [source]

Better library design: data-driven protein engineering

BIOTECHNOLOGY JOURNAL, Issue 2 2007
Javier F. Chaparro-Riggers
Abstract Data-driven protein engineering is increasingly used as an alternative to rational design and combinatorial engineering because it uses available knowledge to limit library size, while still allowing for the identification of unpredictable substitutions that lead to large effects. Recent advances in computational modeling and bioinformatics, as well as an increasing databank of experiments on functional variants, have led to new strategies to choose particular amino acid residues to vary in order to increase the chances of obtaining a variant protein with the desired property. Strategies for limiting diversity at each position, design of small sub-libraries, and the performance of scouting experiments, have also been developed or even automated, further reducing the library size. [source]

Actin mutations in hypertrophic and dilated cardiomyopathy cause inefficient protein folding and perturbed filament formation

FEBS JOURNAL, Issue 8 2005
Søren Vang
Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) are the most common hereditary cardiac conditions. Both are frequent causes of sudden death and are often associated with an adverse disease course. Alpha-cardiac actin is one of the disease genes where different missense mutations have been found to cause either HCM or DCM. We have tested the hypothesis that the protein-folding pathway plays a role in disease development for two actin variants associated with DCM and six associated with HCM. Based on a cell-free coupled translation assay the actin variants could be graded by their tendency to associate with the chaperonin TCP-1 ring complex/chaperonin containing TCP-1 (TRiC/CCT) as well as their propensity to acquire their native conformation. Some variant proteins are completely stalled in a complex with TRiC and fail to fold into mature globular actin and some appear to fold as efficiently as the wild-type protein. A fraction of the translated polypeptide became ubiquitinated and detergent insoluble. Variant actin proteins overexpressed in mammalian cell lines fail to incorporate into actin filaments in a manner correlating with the degree of misfolding observed in the cell-free assay; ranging from incorporation comparable to wild-type actin to little or no incorporation. We propose that effects of mutations on folding and fiber assembly may play a role in the molecular disease mechanism. [source]

The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive

FEBS JOURNAL, Issue 20 2004
Linda O'Reilly
Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the ,-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures. [source]

In silico analysis of missense substitutions using sequence-alignment based methods,

HUMAN MUTATION, Issue 11 2008
Sean V. Tavtigian
Abstract Genetic testing for mutations in high-risk cancer susceptibility genes often reveals missense substitutions that are not easily classified as pathogenic or neutral. Among the methods that can help in their classification are computational analyses. Predictions of pathogenic vs. neutral, or the probability that a variant is pathogenic, can be made based on: 1) inferences from evolutionary conservation using protein multiple sequence alignments (PMSAs) of the gene of interest for almost any missense sequence variant; and 2) for many variants, structural features of wild-type and variant proteins. These in silico methods have improved considerably in recent years. In this work, we review and/or make suggestions with respect to: 1) the rationale for using in silico methods to help predict the consequences of missense variants; 2) important aspects of creating PMSAs that are informative for classification; 3) specific features of algorithms that have been used for classification of clinically-observed variants; 4) validation studies demonstrating that computational analyses can have predictive values (PVs) of ,75 to 95%; 5) current limitations of data sets and algorithms that need to be addressed to improve the computational classifiers; and 6) how in silico algorithms can be a part of the "integrated analysis" of multiple lines of evidence to help classify variants. We conclude that carefully validated computational algorithms, in the context of other evidence, can be an important tool for classification of missense variants. Hum Mutat 29(11), 1327,1336, 2008. © 2008 Wiley-Liss, Inc. [source]

Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometry

MASS SPECTROMETRY REVIEWS, Issue 5 2006
Akira Shimizu
Abstract Some variant proteins cause diseases, and some diseases result in increases of proteins with abnormally modified structures. The detection, characterization, and estimation of the relative amounts of protein variants and abnormally modified proteins are important for clinical diagnosis and for elucidation of the mechanisms of the pathogenesis of diseases. Analysis of the covalent structures of proteins using matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS) and liquid chromatography-electrospray ionization MS (LC-ESI-MS), which had been developed by the early 1990s, have largely replaced analyses by conventional protein chemistry. Here, we review the detection and characterization of hemoglobin variants, HbA1c measurement, detection of carbohydrate-deficient transferrin, and identification of variants of transthyretin (TTR) and Cu/Zn-superoxide dismutase (SOD-1) using soft ionization MS. We also propose the diagnostic application of the signals of modified forms of TTR, that is, S-sulfonated TTR and S-homocysteinyl TTR. The relative peak height ratio of the abnormal/normal components gives valuable information about the instability of variants and enables the detection of unstable Hb subunits or thalassemia heterozygotes. We found unique modified structures of TTR that suggested changes in amyloid fibrils. © 2006 Wiley Periodicals, Inc. [source]