scholarly journals Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽  
2003 ◽  
Vol 163 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Erin N Asleson ◽  
Dennis M Livingston
Keyword(s):  
Half Life ◽  
Translational Regulation ◽  
Cellular Level ◽  
Missense Mutations ◽  
Wild Type ◽  
Mutant Proteins ◽  
Gal1 Promoter ◽  
Rad52 Protein ◽  
The Stability ◽  
Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

scholarly journals Prediction of Functional Consequences of Missense Mutations in ANO4 Gene

2021 ◽  
Vol 22 (5) ◽  
pp. 2732
Author(s):  
Nadine Reichhart ◽  
Vladimir M. Milenkovic ◽  
Christian H. Wetzel ◽  
Olaf Strauß
Keyword(s):  
Transmembrane Protein ◽  
Functional Characterization ◽  
Missense Mutations ◽  
Mutant Proteins ◽  
Functional Consequences ◽  
New Perspective ◽  
The Stability ◽  
Dynamics Simulations ◽  
And Function

The anoctamin (TMEM16) family of transmembrane protein consists of ten members in vertebrates, which act as Ca2+-dependent ion channels and/or Ca2+-dependent scramblases. ANO4 which is primarily expressed in the CNS and certain endocrine glands, has been associated with various neuronal disorders. Therefore, we focused our study on prioritizing missense mutations that are assumed to alter the structure and stability of ANO4 protein. We employed a wide array of evolution and structure based in silico prediction methods to identify potentially deleterious missense mutations in the ANO4 gene. Identified pathogenic mutations were then mapped to the modeled human ANO4 structure and the effects of missense mutations were studied on the atomic level using molecular dynamics simulations. Our data show that the G80A and A500T mutations significantly alter the stability of the mutant proteins, thus providing new perspective on the role of missense mutations in ANO4 gene. Results obtained in this study may help to identify disease associated mutations which affect ANO4 protein structure and function and might facilitate future functional characterization of ANO4.

The Bβ-sheet in the PAI-1 Molecule Plays an Important Role for Its Stability

2000 ◽  
Vol 83 (06) ◽  
pp. 896-901 ◽  
Author(s):  
Guang-Chao Sui ◽  
Björn Wiman
Keyword(s):  
Amino Acids ◽  
Half Life ◽  
Ph Dependence ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Reactive Center ◽  
The Stability ◽  
Β Sheet ◽  
Pai 1

SummaryWe have investigated the B β-sheet in PAI-1 regarding its role for the stability of the molecule. The residues from His219 to Tyr241 (except for Gly230 and Pro240), covering the s2B and s3B strands, and in addition His185 and His190 were substituted by amino acids with opposite properties. The 23 generated single-site changed mutants and also wild type PAI-1 (wtPAI-1) were expressed in E. coli. Subsequently they were purified by heparin-Sepharose and anhydrotrypsin agarose affinity chromatographies. The stability of the purified PAI-1 variants was analyzed at 37° C and at different pHs (5.5, 6.5 or 7.5). At pH 7.5 and 37° C, single substitutions of the residues in the central portions of both strands 2 and 3 in the B β-sheet (Ile223 to Leu226 on s2B and Met235 to Ile237 on s3B), caused a significant decrease in stability, yielding half-lives of about 10–25% as compared to wtPAI-1. On the other hand, mutations at both sides of the central portion of the B β-sheet (Tyr221, Asp222, Tyr228 and Thr232) frequently resulted in an increased PAI-1 stability (up to 7-fold). While wtPAI-1 exhibited prolonged half-lives at pH 6.5 and 5.5, the PAI-1 variant Y228S was more stable at neutral pH (half-life of 9.6 h at pH 7.5) as compared to its half-life at pH 5.5 (1.1 h). One of the 4 modified histidine residues (His229) resulted in a variant with a clearly affected stability as a function of pH, suggesting that it may, at least in part, be of importance for the pH dependence of the PAI-1 stability. Thus, our data demonstrate that the B β-sheet is of great importance for the stability of the molecule. Modifications in this part causes decreased or increased stability in a certain pattern, suggesting effects on the insertion rate of the reactive center loop into the A β-sheet of the molecule.

Design and Validation of Genes Encoding Hyperstable β-Globin mRNAs.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4059-4059
Author(s):  
Osheiza Abdulmalik ◽  
J. Eric Russell
Keyword(s):  
Sickle Cell Disease ◽  
Mrna Stability ◽  
Half Life ◽  
Fold Increase ◽  
Globin Genes ◽  
Wild Type ◽  
Cell Disease ◽  
Globin Mrna ◽  
Stem Loop ◽  
The Stability

Abstract 4059 Poster Board III-994 Transgenic approaches to β thalassemia and sickle cell disease require viral vectors that express high levels of therapeutic β-like globin proteins. We recently proposed that the overall expression of these transgenes would likely be improved by structural modifications that prolong the cytoplasmic half-lives of their encoded mRNAs. Relevant experiments from our laboratory have previously linked the constitutively high stability of β-globin mRNA to a region of its 3'UTR that appears to interact with at least two distinct cytoplasmic mRNA-stabilizing factors, and is predicted to form an imperfect stem-loop (SL) structure. Based upon these findings, we conducted enzymatic secondary-structure mapping studies of the β-globin 3'UTR, unequivocally validating the existence of the predicted functional stem-loop element. We subsequently reasoned that the constitutive half-life of β-globin mRNA might be prolonged by the insertion of multiple SL motifs into its 3'UTR, resulting in increased levels of the mRNA–and its encoded β-globin product–in terminally differentiating erythroid cells. To test this hypothesis, we constructed full-length β-globin genes containing either wild-type 3'UTRs, or variant 3'UTRs that had been modified to contain either two or three tandem SL motifs. Each gene was identically linked to a tetracycline-suppressible promoter, permitting pulse-chase mRNA stability analyses to be conducted in vivo in intact cultured cells. Erythroid-phenotype K562 cells were transiently transfected with SL-variant and control wild-type β-globin genes, exposed to tetracycline, and levels of β-globin mRNA determined by qRT-PCR at defined intervals using tet-indifferent β-actin mRNA as internal control. Relative to wild-type β-globin mRNA, SL-duplicate β-globin mRNAs displayed a position-dependent two-fold increase in cytoplasmic half-life; SL-triplicate β-globin mRNAs did not exhibit any additional stability. These experiments confirm the existence of a defined SL structure within the β-globin 3'UTR, and demonstrate that duplication of this motif can substantially increase the stability of β-globin mRNA. We subsequently designed a series of experiments to elucidate post-transcriptional processes involved in mRNA hyperstability. These studies required the construction of HeLa cells that stably express either wild-type β-globin mRNA (11 subclones) or SL-duplicate β-globin mRNAs (10 subclones). Preliminary analyses indicate an approximate 1.5-fold increase in the median steady-state expression of SL-duplicate genes, consistent with a prolongation in the half-life of its encoded mRNA. While formal mRNA stability studies are not yet complete, early data appear to replicate results from experiments conducted in transiently transfected cells. We have also initiated structural studies to link differences in the stability of SL-variant β-globin mRNA to alterations in its poly(A) tail. Using an RNase H-based strategy, we identified a previously unknown poly(A)-site heterogeneity–of undetermined significance–affecting both wild-type and SL-duplicate β-globin mRNAs. Finally, we recently isolated fifty-four K562 subclones expressing SL-duplicate or control β-globin mRNAs; parallel analyses of these cells will permit the cell-specificity of β-globin SL-directed mRNA stabilization to be investigated in detail. Results from each of these studies will be immediately applicable to the design of high-efficiency therapeutic transgenes for β thalassemia and sickle-cell disease. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss-of-Function Piezo1 Mutations Display Altered Stability Driven by Ubiquitination and Proteasomal Degradation

2021 ◽  
Vol 12 ◽  
Author(s):  
Zijing Zhou ◽  
Jinyuan Vero Li ◽  
Boris Martinac ◽  
Charles D. Cox
Keyword(s):  
Membrane Trafficking ◽  
Proteasome Inhibition ◽  
Proteasomal Degradation ◽  
Missense Mutations ◽  
Turnover Rates ◽  
Loss Of Function ◽  
Wild Type ◽  
Mutant Proteins ◽  
Lymphatic Dysplasia ◽  
Therapeutic Avenue

Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.

Effect of Stabilizing versus Destabilizing Interactions on Plasminogen Activator Inhibitor-1

2000 ◽  
Vol 84 (11) ◽  
pp. 871-875 ◽  
Author(s):  
Nele Vleugels ◽  
John Leys ◽  
Isabelle Knockaert ◽  
Paul Declerck
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Plasminogen Activator Inhibitor ◽  
Reactive Site ◽  
Wild Type ◽  
Plasminogen Activator Inhibitor 1 ◽  
The Stability ◽  
Gate Region ◽  
Activator Inhibitor ◽  
Pai 1

SummaryPlasminogen activator inhibitor-1 (PAI-1) is a unique member of the serpin family, as it spontaneously converts into a latent conformation. However, the exact mechanism of this conversion is not known. Previous studies reported that neutralizing monoclonal antibodies as well as reversal or removal of charges on the s3C-s4C turn results in a destabilization of PAI-1 leading to an accelerated conversion to its latent form.In this study the effect of the reversal or removal of charges in this “gate region” (R186E/R187E, H190E/K191E, H190L/K191L and R356E) on a stable PAI-1-variant (PAI-1-stab) was investigated. Whereas PAI-1-stab has a half-life of 150 ± 66 h, PAI-1-stab-R186ER187E, PAI-1-stab-H190E-K191E, PAI-1-stab-H190L-K191L and PAI-1-stab-R356E have a strongly decreased half-life (p< 0.005 versus PAI-1-stab) of 175 ± 48 min, 75 ± 34 min, 68 ± 38 min and 79 ± 16 min, respectively. Wild-type PAI-1 (wtPAI-1) had a half-life of 55 ± 19 min. These data indicate that the stabilization induced by the mutated residues in PAI-1-stab is counteracted by the additional mutations, resulting in half-lives similar to that of wtPAI-1, thereby suggesting that the stabilizing and destabilizing forces act mainly independently in these mutants. Extrapolation of these data to other (stable) serpins leads to the hypothesis that the s3C-s4C turn and the distal hinge region of the reactive site loop plays a role for the stability of serpins in general.

scholarly journals Altering the stability of the Cdc8 overlap region modulates the ability of this tropomyosin to bind co-operatively to actin and regulate myosin

2011 ◽  
Vol 438 (2) ◽  
pp. 265-273 ◽  
Author(s):  
Daniel A. East ◽  
Duncan Sousa ◽  
Stephen R. Martin ◽  
Thomas A. Edwards ◽  
William Lehman ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Coiled Coil ◽  
Regulatory Function ◽  
Wild Type ◽  
Mutant Proteins ◽  
Methionine Residue ◽  
Protein Dimers ◽  
End To End ◽  
The Stability ◽  
Overlap Region

Tm (tropomyosin) is an evolutionarily conserved α-helical coiled-coil protein, dimers of which form end-to-end polymers capable of associating with and stabilizing actin filaments, and regulating myosin function. The fission yeast Schizosaccharomyces pombe possesses a single essential Tm, Cdc8, which can be acetylated on its N-terminal methionine residue to increase its affinity for actin and enhance its ability to regulate myosin function. We have designed and generated a number of novel Cdc8 mutant proteins with N-terminal substitutions to explore how stability of the Cdc8 overlap region affects the regulatory function of this Tm. By correlating the stability of each protein, its propensity to form stable polymers, its ability to associate with actin and to regulate myosin, we have shown that the stability of the N-terminal of the Cdc8 α-helix is crucial for Tm function. In addition we have identified a novel Cdc8 mutant with increased N-terminal stability, dimers of which are capable of forming Tm polymers significantly longer than the wild-type protein. This protein had a reduced affinity for actin with respect to wild-type, and was unable to regulate actomyosin interactions. The results of the present paper are consistent with acetylation providing a mechanism for modulating the formation and stability of Cdc8 polymers within the fission yeast cell. The data also provide evidence for a mechanism in which Tm dimers form end-to-end polymers on the actin filament, consistent with a co-operative model for Tm binding to actin.

Properties of lamin A mutants found in Emery-Dreifuss muscular dystrophy, cardiomyopathy and Dunnigan-type partial lipodystrophy

2001 ◽  
Vol 114 (24) ◽  
pp. 4435-4445 ◽  
Author(s):  
Cecilia Östlund ◽  
Gisèle Bonne ◽  
Ketty Schwartz ◽  
Howard J. Worman
Keyword(s):  
Muscular Dystrophy ◽  
Dilated Cardiomyopathy ◽  
Lamin A ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Partial Lipodystrophy ◽  
Lamin B1 ◽  
The Stability ◽  
Mutant Forms ◽  
Two Hybrid

Autosomal dominant Emery-Dreifuss muscular dystrophy is caused by mutations in the LMNA gene, which encodes lamin A and lamin C. Mutations in this gene also give rise to limb girdle muscular dystrophy type 1B, dilated cardiomyopathy with atrioventricular conduction defect and Dunnigan-type partial lipodystrophy. The properties of the mutant lamins that cause muscular dystrophy, lipodystrophy and dilated cardiomyopathy are not known. We transfected C2C12 myoblasts with cDNA encoding wild-type lamin A and 15 mutant forms found in patients affected by these diseases. Immunofluorescence microscopy showed that four mutants, N195K, E358K, M371K and R386K, could have a dramatically aberrant localization, with decreased nuclear rim staining and formation of intranuclear foci. The distributions of endogenous lamin A/C, lamin B1 and lamin B2 were also altered in cells expressing these four mutants and three of them caused a loss of emerin from the nuclear envelope. In the yeast two-hybrid assay, the 15 lamin A mutants studied interacted with themselves and with wild-type lamin A and lamin B1. Pulse-chase experiments showed no decrease in the stability of several representative lamin A mutants compared with wild-type. These results indicate that some lamin A mutants causing disease can be aberrantly localized, partially disrupt the endogenous lamina and alter emerin localization, whereas others localize normally in transfected cells.

scholarly journals The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

1991 ◽  
Vol 113 (3) ◽  
pp. 605-614 ◽  
Author(s):  
M J Schibler ◽  
B Huang
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Missense Mutations ◽  
Wild Type ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Microtubule Inhibitors ◽  
Microtubule Stability ◽  
The Stability ◽  
Tubulin Mutations

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.

scholarly journals Dual Lipid Modification of the Yeast Gγ Subunit Ste18p Determines Membrane Localization of Gβγ

1999 ◽  
Vol 19 (11) ◽  
pp. 7705-7711 ◽  
Author(s):  
Jodi E. Hirschman ◽  
Duane D. Jenness
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cysteine Residue ◽  
Wild Type ◽  
Molecular Weights ◽  
Sds Page ◽  
Membrane Attachment ◽  
The Stability ◽  
Mutant Forms

ABSTRACT The pheromone response in the yeast Saccharomyces cerevisiae is mediated by a heterotrimeric G protein. The Gβγ subunit (a complex of Ste4p and Ste18p) is associated with both internal and plasma membranes, and a portion is not stably associated with either membrane fraction. Like Ras, Ste18p contains a farnesyl-directing CaaX box motif (C-terminal residues 107 to 110) and a cysteine residue (Cys 106) that is a potential site for palmitoylation. Mutant Ste18p containing serine at position 106 (mutation ste18-C106S) migrated more rapidly than wild-type Ste18p during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The electrophoretic mobility of wild-type Ste18p (but not the mutant Ste18p) was sensitive to hydroxylamine treatment, consistent with palmitoyl modification at Cys 106. Furthermore, immunoprecipitation of the Gβγ complex from cells cultured in the presence of [3H]palmitic acid resulted in two radioactive species on nonreducing SDS-PAGE gels, with molecular weights corresponding to Gγ and Gβγ. Substitution of serine for either Cys 107 or Cys 106 resulted in the failure of Gβγ to associate with membranes. The Cys 107 substitution also resulted in reduced steady-state accumulation of Ste18p, suggesting that the stability of Ste18p requires modification at Cys 107. All of the mutant forms of Ste18p formed complexes with Ste4p, as assessed by coimmunoprecipitation. We conclude that tight membrane attachment of the wild-type Gβγ depends on palmitoylation at Cys 106 and prenylation at Cys 107 of Ste18p.

Platelet Activation and Aggregation Induced by Recombinant von Willebrand Factors Reproducing Four Type 2B von Willebrand Disease Missense Mutations

1998 ◽  
Vol 79 (01) ◽  
pp. 211-216 ◽  
Author(s):  
Lysiane Hilbert ◽  
Claudine Mazurier ◽  
Christophe de Romeuf
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Missense Mutations ◽  
Inhibit Platelet Aggregation ◽  
Wild Type ◽  
A1 Domain ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryType 2B of von Willebrand disease (vWD) refers to qualitative variants with increased affinity of von Willebrand factor (vWF) for platelet glycoprotein Ib (GPIb). All the mutations responsible for type 2B vWD have been located in the A1 domain of vWF. In this study, various recombinant von Willebrand factors (rvWF) reproducing four type 2B vWD missense mutations were compared to wild-type rvWF (WT-rvWF) for their spontaneous binding to platelets and their capacity to induce platelet activation and aggregation. Our data show that the multimeric pattern of each mutated rvWF is similar to that of WT-rvWF but the extent of spontaneous binding and the capacity to induce platelet activation and aggregation are more important for the R543Q and V553M mutations than for the L697V and A698V mutations. Both the binding of mutated rvWFs to platelets and platelet aggregation induced by type 2B rvWFs are inhibited by monoclonal anti-GPIb and anti-vWF antibodies, inhibitors of vWF binding to platelets in the presence of ristocetin, as well as by aurin tricarboxylic acid. On the other hand, EDTA and a monoclonal antibody directed against GPIIb/IIIa only inhibit platelet aggregation. Furthermore, the incubation of type 2B rvWFs with platelets, under stirring conditions, results in the decrease in high molecular weight vWF multimers in solution, the extent of which appears correlated with that of plasma vWF from type 2B vWD patients harboring the corresponding missense mutation. This study supports that the binding of different mutated type 2B vWFs onto platelet GPIb induces various degrees of platelet activation and aggregation and thus suggests that the phenotypic heterogeneity of type 2B vWD may be related to the nature and/or location of the causative point mutation.

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