scholarly journals Functional analyses ofSTIM1mutations reveal a common pathomechanism for tubular aggregate myopathy and Stormorken syndrome

2019 ◽  
Author(s):  
Georges Arielle Peche ◽  
Coralie Spiegelhalter ◽  
Roberto Silva-Rojas ◽  
Jocelyn Laporte ◽  
Johann Böhm
Keyword(s):  
Nuclear Import ◽  
Protein Function ◽  
Circular Membrane ◽  
Wild Type ◽  
Dominant Effect ◽  
Downstream Effects ◽  
Similarities And Differences ◽  
Functional Analyses ◽  
Progressive Disorder ◽  
Transcription Factor Nfat

ABSTRACTTubular aggregate myopathy (TAM) is a progressive disorder essentially involving muscle weakness, cramps, and myalgia. TAM clinically overlaps with Stormorken syndrome (STRMK), associating TAM with miosis, thrombocytopenia, hyposplenism, ichthyosis, short stature, and dyslexia. TAM and Stormorken syndrome arise from gain-of-function mutations inSTIM1orORAI1, both encoding key regulators of Ca2+homeostasis, and mutations in either gene results in excessive Ca2+entry. The pathomechanistic similarities and differences of TAM and Stormorken syndrome are only partially understood. Here we provide functionalin celluloexperiments demonstrating that STIM1 harboring the TAM D84G or the STRMK R304W mutation similarly cluster and exert a dominant effect on the wild-type protein. Both mutants recruit ORAI1 to the clusters, induce major nuclear import of the Ca2+-dependent transcription factor NFAT, and trigger the formation of circular membrane stacks. In conclusion, the analyzed TAM and STRMK mutations have a comparable impact on STIM1 protein function and downstream effects of excessive Ca2+entry, highlighting that TAM and Stormorken syndrome involve a common pathomechanism.

scholarly journals Novel FGFR1 Variants Are Associated with Congenital Scoliosis

Genes ◽  
2021 ◽  
Vol 12 (8) ◽  
pp. 1126
Author(s):  
Shengru Wang ◽  
Xiran Chai ◽  
Zihui Yan ◽  
Sen Zhao ◽  
Yang Yang ◽  
...  
Keyword(s):  
Protein Function ◽  
Hypogonadotropic Hypogonadism ◽  
Congenital Scoliosis ◽  
Wild Type ◽  
Conservation Analysis ◽  
Gene Assay ◽  
Protein Expressions ◽  
Patient Series ◽  
Reduced Activity

FGFR1 encodes a transmembrane cytokine receptor, which is involved in the early development of the human embryo and plays an important role in gastrulation, organ specification and patterning of various tissues. Pathogenic FGFR1 variants have been associated with Kallmann syndrome and hypogonadotropic hypogonadism. In our congenital scoliosis (CS) patient series of 424 sporadic CS patients under the framework of the Deciphering disorders Involving Scoliosis and COmorbidities (DISCO) study, we identified four unrelated patients harboring FGFR1 variants, including one frameshift and three missense variants. These variants were predicted to be deleterious by in silico prediction and conservation analysis. Signaling activities and expression levels of the mutated protein were evaluated in vitro and compared to that of the wild type (WT) FGFR1. As a result, the overall protein expressions of c.2334dupC, c.2339T>C and c.1261A>G were reduced to 43.9%, 63.4% and 77.4%, respectively. By the reporter gene assay, we observed significantly reduced activity for c.2334dupC, c.2339T>C and c.1261A>G, indicating the diminished FGFR1 signaling pathway. In conclusion, FGFR1 variants identified in our patients led to only mild disruption to protein function, caused milder skeletal and cardiac phenotypes than those reported previously.

scholarly journals Baculovirus Transregulator IE1 Requires a Dimeric Nuclear Localization Element for Nuclear Import and Promoter Activation

2002 ◽  
Vol 76 (18) ◽  
pp. 9505-9515 ◽  
Author(s):  
Victoria A. Olson ◽  
Justin A. Wetter ◽  
Paul D. Friesen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Direct Interaction ◽  
Productive Infection ◽  
Homologous Region ◽  
Wild Type ◽  
Nuclear Entry ◽  
Early Protein ◽  
Biochemical Fractionation ◽  
Localization Element

ABSTRACT Immediate-early protein IE1 is a principal regulator of viral transcription and a contributor to origin-specific DNA replication of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Since these viral functions involve interaction of dimeric IE1 with palindromic homologous region (hr) enhancer-origin elements of the AcMNPV genome within the nucleus, it is presumed that proper nuclear transport of IE1 is essential for productive infection. To investigate the mechanisms of IE1 nuclear import, we analyzed the effect of site-directed mutations on IE1 subcellular distribution. As demonstrated by fluorescence microscopy and biochemical fractionation of plasmid-transfected cells, wild-type IE1 localized predominantly to the nucleus. Substitution or deletion of amino acid residues within a positively charged domain (residues 534 to 538) adjacent to IE1's oligomerization motif impaired nuclear import and caused loss of transactivation. Moreover, upon coexpression, these import-defective mutations prevented nuclear entry of wild-type IE1. In contrast, double-mutated IE1 defective for both nuclear import and dimerization failed to block nuclear entry or transactivation by wild-type IE1. Thus, import-defective IE1 dominantly interfered with wild-type IE1 by direct interaction and cytosolic trapping. Collectively, our data indicate that the small basic domain encompassing residues R537 and R538 constitutes a novel nuclear localization element that functions only upon IE1 dimerization. These findings support a model wherein IE1 oligomerizes within the cytosol as a prerequisite for nuclear entry and subsequent high-affinity interaction with the symmetrical binding sites comprising AcMNPV hr enhancer-origin elements.

scholarly journals Protease-Dependent Uncoating of a Complex Retrovirus

2005 ◽  
Vol 79 (14) ◽  
pp. 9244-9253 ◽  
Author(s):  
Jacqueline Lehmann-Che ◽  
Marie-Lou Giron ◽  
Olivier Delelis ◽  
Martin Löchelt ◽  
Patricia Bittoun ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Viral Genome ◽  
Productive Infection ◽  
Wild Type Virus ◽  
Target Cells ◽  
Wild Type ◽  
Microtubule Organizing Center ◽  
Viral Protease ◽  
Virus Life Cycle ◽  
Organizing Center

ABSTRACT Although retrovirus egress and budding have been partly unraveled, little is known about early stages of the replication cycle. In particular, retroviral uncoating, a process during which incoming retroviral cores are altered to allow the integration of the viral genome into host chromosomes, is poorly understood. To get insights into these early events of the retroviral cycle, we have used foamy complex retroviruses as a model. In this report, we show that a protease-defective foamy retrovirus is noninfectious, although it is still able to bud and enter target cells efficiently. Similarly, a retrovirus mutated in an essential viral protease-dependent cleavage site in the central part of Gag is noninfectious. Following entry, wild-type and mutant retroviruses are able to traffic along microtubules towards the microtubule-organizing center (MTOC). However, whereas nuclear import of Gag and of the viral genome was observed for the wild-type virus as early as 8 hours postinfection, incoming capsids and genome from mutant viruses remained at the MTOC. Interestingly, a specific viral protease-dependent Gag cleavage product was detected only for the wild-type retrovirus early after infection, demonstrating that cleavage of Gag by the viral protease at this stage of the virus life cycle is absolutely required for productive infection, an unprecedented observation among retroviruses.

scholarly journals Similarities and differences between IL11 and IL11RA1 knockout mice for lung fibro-inflammation, fertility and craniosynostosis

2020 ◽  
Author(s):  
Benjamin Ng ◽  
Anissa A. Widjaja ◽  
Sivakumar Viswanathan ◽  
Jinrui Dong ◽  
Sonia P. Chothani ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Lung Fibroblasts ◽  
Loss Of Function ◽  
Wild Type ◽  
Reduced Body ◽  
Body Weights ◽  
Show Evidence ◽  
Similarities And Differences ◽  
The Impact

AbstractGenetic loss of function (LOF) in IL11RA infers IL11 signaling as important for fertility, fibrosis, inflammation and craniosynostosis. The impact of genetic LOF in IL11 has not been characterized. We generated IL11-knockout (Il11-/-) mice, which are born in normal Mendelian ratios, have normal hematological profiles and are protected from bleomycin-induced lung fibro-inflammation. Noticeably, baseline IL6 levels in the lungs of Il11-/- mice are lower than those of wild-type mice and are not induced by bleomycin damage, placing IL11 upstream of IL6. Lung fibroblasts from Il11-/- mice are resistant to pro-fibrotic stimulation and show evidence of reduced autocrine IL11 activity. Il11-/- female mice are infertile. Unlike Il11ra1-/- mice, Il11-/- mice do not have a craniosynostosis-like phenotype and exhibit mildly reduced body weights. These data highlight similarities and differences between LOF in IL11 or IL11RA while establishing further the role of IL11 signaling in fibrosis and stromal inflammation.

scholarly journals METABOLIC CONSEQUENCES OF METHIONINE REDOX IN METHIONINE RESTRICTION

2019 ◽  
Vol 3 (Supplement_1) ◽  
pp. S106-S107
Author(s):  
Kevin Thyne ◽  
Yuhong Liu ◽  
Adam B Salmon
Keyword(s):  
Protein Function ◽  
Methionine Sulfoxide ◽  
Redox Status ◽  
Significant Loss ◽  
Methionine Sulfoxide Reductase ◽  
Wild Type ◽  
Entire Family ◽  
Methionine Sulfoxide Reductase A ◽  
Methionine Restriction

Abstract While caloric restriction (CR) provides highly robust improvements to longevity and health, dietary restriction of the essential amino acid methionine can provide similar benefits including improved metabolic function and increased longevity. Despite these similarities between CR and methionine restriction (MR), there is growing evidence to suggest they may be mediated by different mechanisms that require further elucidation. The sulfur side-chain of methionine is highly prone to oxidation, even in vivo, with redox changes of these residues potentially altering protein function and interfering with its use as a substrate. An entire family of enzymes, methionine sulfoxide reductases, have evolved in aerobic organisms to regulate the redox status of methionine. We tested the role of methionine sulfoxide reductase A (MsrA) in the physiological and metabolic benefits of MR. After three months of MR, mice lacking MsrA (MsrA KO) showed significant loss of weight, including both fat and lean mass, in comparison to wild-type mice under MR. Both MsrA KO and wild-type mice responded to MR with improvements to both glucose and insulin tolerance. However, MR MsrA KO mice showed lower HbA1c and reduced leptin compared to MR wild-type mice. Overall, our results show mice lacking MsrA have a stronger response to MR suggesting that methionine redox may play an important role in some of the mechanisms responsible for these metabolic outcomes. Further studies clarify whether MsrA could also be a potential regulator of the longevity benefits of MR.

scholarly journals Novel Salmonella enterica Serovar Typhimurium Protein That Is Indispensable for Virulence and Intracellular Replication

2001 ◽  
Vol 69 (12) ◽  
pp. 7413-7418 ◽  
Author(s):  
Tahar van der Straaten ◽  
Angela van Diepen ◽  
Kitty Kwappenberg ◽  
Sjaak van Voorden ◽  
Kees Franken ◽  
...  
Keyword(s):  
Amino Acid ◽  
Protein Function ◽  
Salmonella Enterica ◽  
Host Cells ◽  
Wild Type ◽  
Intracellular Replication ◽  
Oxygen Intermediates ◽  
Serovar Typhimurium

ABSTRACT Upon contact with host cells, the intracellular pathogenSalmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribedSalmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonellachromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 104 to 107bacteria in C3H/HeN and 101 to 104 bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

scholarly journals The Karyopherin Kap142p/Msn5p Mediates Nuclear Import and Nuclear Export of Different Cargo Proteins

2001 ◽  
Vol 152 (4) ◽  
pp. 729-740 ◽  
Author(s):  
Kimihisa Yoshida ◽  
Günter Blobel
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Protein Import ◽  
Protein A ◽  
Double Strand Breaks ◽  
Wild Type ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
Highly Sensitive ◽  
Cargo Proteins

We have identified a novel pathway for protein import into the nucleus. Although the product of Saccharomyces cerevisiae gene MSN5 was previously shown to function as a karyopherin (Kap) for nuclear export of various proteins, we discovered a nuclear import pathway mediated by Msn5p (also referred to as Kap142p). We have purified from yeast cytosol a complex containing Kap142p and the trimeric replication protein A (RPA), which is required for multiple aspects of DNA metabolism, including DNA replication, DNA repair, and recombination. In wild-type cells, RPA was localized primarily to the nucleus but, in a KAP142 deletion strain, RPA was mislocalized to the cytoplasm and the strain was highly sensitive to bleomycin (BLM). BLM causes DNA double-strand breaks and, in S. cerevisiae, the DNA damage is repaired predominantly by RPA-dependent homologous recombination. Therefore, our results indicate that in wild-type cells a critical portion of RPA was imported into the nucleus by Kap142p. Like several other import-related Kap–substrate complexes, the endogenous RPA–Kap142p complex was dissociated by RanGTP, but not by RanGDP. All three RPA genes are essential for viability, whereas KAP142 is not. Perhaps explaining this disparity, we observed an interaction between RPA and Kap95p in a strain lacking Kap142p. This interaction could provide a mechanism for import of RPA into the nucleus and cell viability in the absence of Kap142p. Together with published results (Kaffman, A., N.M. Rank, E.M. O'Neill, L.S. Huang, and E.K. O'Shea. 1998. Nature. 396:482–486; Blondel, M., P.M. Alepuz, L.S. Huang, S. Shaham, G. Ammerer, and M. Peter. 1999. Genes Dev. 13:2284–2300; DeVit, M.J., and M. Johnston. 1999. Curr. Biol. 9:1231–1241; Mahanty, S.K., Y. Wang, F.W. Farley, and E.A. Elion. 1999. Cell. 98:501–512) our data indicate that the karyopherin Kap142p is able to mediate nuclear import of one set of proteins and nuclear export of a different set of proteins.

scholarly journals A Nuclear Import Pathway for a Protein Involved in tRNA Maturation

1997 ◽  
Vol 139 (7) ◽  
pp. 1655-1661 ◽  
Author(s):  
Jonathan S. Rosenblum ◽  
Lucy F. Pemberton ◽  
Günter Blobel
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nuclear Import ◽  
Ribosomal Proteins ◽  
Ribosome Biogenesis ◽  
Sequence Similarity ◽  
Wild Type ◽  
Major Pathway ◽  
Trna Processing ◽  
Transport Factors ◽  
Limited Sequence

A limited number of transport factors, or karyopherins, ferry particular substrates between the cytoplasm and nucleoplasm. We identified the Saccharomyces cerevisiae gene YDR395w/SXM1 as a potential karyopherin on the basis of limited sequence similarity to known karyopherins. From yeast cytosol, we isolated Sxm1p in complex with several potential import substrates. These substrates included Lhp1p, the yeast homologue of the human autoantigen La that has recently been shown to facilitate maturation of pre-tRNA, and three distinct ribosomal proteins, Rpl16p, Rpl25p, and Rpl34p. Further, we demonstrate that Lhp1p is specifically imported by Sxm1p. In the absence of Sxm1p, Lhp1p was mislocalized to the cytoplasm. Sxm1p and Lhp1p represent the karyopherin and a cognate substrate of a unique nuclear import pathway, one that operates upstream of a major pathway of pre-tRNA maturation, which itself is upstream of tRNA export in wild-type cells. In addition, through its association with ribosomal proteins, Sxm1p may have a role in coordinating ribosome biogenesis with tRNA processing.

scholarly journals Structural insight into DNA binding and oligomerization of the multifunctional Cox protein of bacteriophage P2

2013 ◽  
Vol 42 (4) ◽  
pp. 2725-2735 ◽  
Author(s):  
Ronnie P.-A. Berntsson ◽  
Richard Odegrip ◽  
Wilhelmina Sehlén ◽  
Karin Skaar ◽  
Linda M. Svensson ◽  
...  
Keyword(s):  
Dna Binding ◽  
Protein Function ◽  
Specific Binding ◽  
Gene Products ◽  
Structural Determinants ◽  
Bacteriophage P2 ◽  
Defective Prophage ◽  
Key Residues ◽  
Functional Analyses ◽  
Insight Into

Abstract The Cox protein from bacteriophage P2 is a small multifunctional DNA-binding protein. It is involved in site-specific recombination leading to P2 prophage excision and functions as a transcriptional repressor of the P2 Pc promoter. Furthermore, it transcriptionally activates the unrelated, defective prophage P4 that depends on phage P2 late gene products for lytic growth. In this article, we have investigated the structural determinants to understand how P2 Cox performs these different functions. We have solved the structure of P2 Cox to 2.4 Å resolution. Interestingly, P2 Cox crystallized in a continuous oligomeric spiral with its DNA-binding helix and wing positioned outwards. The extended C-terminal part of P2 Cox is largely responsible for the oligomerization in the structure. The spacing between the repeating DNA-binding elements along the helical P2 Cox filament is consistent with DNA binding along the filament. Functional analyses of alanine mutants in P2 Cox argue for the importance of key residues for protein function. We here present the first structure from the Cox protein family and, together with previous biochemical observations, propose that P2 Cox achieves its various functions by specific binding of DNA while wrapping the DNA around its helical oligomer.

scholarly journals sir2 mutants of Kluyveromyces lactis are hypersensitive to DNA-targeting drugs.

1994 ◽  
Vol 14 (7) ◽  
pp. 4501-4508 ◽  
Author(s):  
X J Chen ◽  
G D Clark-Walker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Function ◽  
Kluyveromyces Lactis ◽  
Functional Equivalence ◽  
Striking Difference ◽  
Multicopy Plasmid ◽  
Wild Type ◽  
Dna Targeting ◽  
Mating Efficiency ◽  
Type Gene

A Kluyveromyces lactis mutant, hypersensitive to the DNA-targeting drugs ethidium bromide (EtBr), berenil, and HOE15030, can be complemented by a wild-type gene with homology to SIR2 of Saccharomyces cerevisiae (ScSIR2). The deduced amino acid sequence of the K. lactis Sir2 protein has 53% identity with ScSir2 protein but is 108 residues longer. K. lactis sir2 mutants show decreased mating efficiency, deficiency in sporulation, an increase in recombination at the ribosomal DNA locus, and EtBr-induced death. Some functional equivalence between the Sir2 proteins of K. lactis and S. cerevisiae has been demonstrated by introduction of ScSIR2 into a sir2 mutant of K. lactis. Expression of ScSIR2 on a multicopy plasmid restores resistance to EtBr and complements sporulation deficiency. Similarly, mating efficiency of a sir2 mutant of S. cerevisiae is partially restored by K. lactis SIR2 on a multicopy plasmid. Although these observations suggest that there has been some conservation of Sir2 protein function, a striking difference is that sir2 mutants of S. cerevisiae, unlike their K. lactis counterparts, are not hypersensitive to DNA-targeting drugs.

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