scholarly journals Interactions of FliJ with the Salmonella Type III Flagellar Export Apparatus

2003 ◽  
Vol 185 (18) ◽  
pp. 5546-5554 ◽  
Author(s):  
Gillian M. Fraser ◽  
Bertha González-Pedrajo ◽  
Jeremy R. H. Tame ◽  
Robert M. Macnab
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Analytical Ultracentrifugation ◽  
Terminal Region ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Size Exclusion ◽  
Dominant Negative Effect ◽  
Type Iii ◽  
Self Association

ABSTRACT FliJ, a 17-kDa protein, is a soluble component of the Salmonella type III flagellar protein export system that has antiaggregation properties and several other characteristics that suggest it may have a chaperone-like function. We have now examined this protein in detail. Ten-amino-acid scanning deletions covering the entire 147-amino-acid sequence were tested for complementation of a fliJ null strain; only the first and last deletions complemented. A few of the deletions, especially towards the C terminus, exerted a dominant negative effect on wild-type cells, indicating that they were actively interfering with function. Two truncated versions of FliJ, representing its N- and C-terminal halves, failed to complement and were not dominant. We tested for FliJ self-association by several techniques. Size-exclusion chromatography (Superdex 200) indicated an apparent molecular mass of around 50 kDa, which could reflect either multimerization or an elongated shape or both. Multiangle light scattering gave a peak value of 20 kDa, close to the molecular mass of the monomer. Analytical ultracentrifugation gave evidence for weak self-association as a trimer or tetramer. It was known from previous studies that FliJ interacts with the N-terminal region of FliH, a negative regulator of the ATPase FliI. Using both truncation and deletion versions of FliJ, we now show that it is its C-terminal region that is responsible for this interaction. We also show that FliJ interacts with the soluble cytoplasmic domain of the largest membrane component of the export apparatus, FlhA; although small deletions in FliJ did not interfere with the association, both truncated versions failed to associate, indicating that a substantial amount of the central region of the FliJ sequence participates in the association. We present a model summarizing these multiple interactions.

scholarly journals A Dimerization Site at SCR-17/18 in Factor H Clarifies a New Mechanism for Complement Regulatory Control

2021 ◽  
Vol 11 ◽  
Author(s):  
Orla M. Dunne ◽  
Xin Gao ◽  
Ruodan Nan ◽  
Jayesh Gor ◽  
Penelope J. Adamson ◽  
...  
Keyword(s):  
Host Cell ◽  
Analytical Ultracentrifugation ◽  
Dissociation Constants ◽  
Alternative Pathway ◽  
Factor H ◽  
Regulatory Control ◽  
Size Exclusion ◽  
Complement Factor ◽  
Dimer Formation ◽  
Self Association

Complement Factor H (CFH), with 20 short complement regulator (SCR) domains, regulates the alternative pathway of complement in part through the interaction of its C-terminal SCR-19 and SCR-20 domains with host cell-bound C3b and anionic oligosaccharides. In solution, CFH forms small amounts of oligomers, with one of its self-association sites being in the SCR-16/20 domains. In order to correlate CFH function with dimer formation and the occurrence of rare disease-associated variants in SCR-16/20, we identified the dimerization site in SCR-16/20. For this, we expressed, in Pichia pastoris, the five domains in SCR-16/20 and six fragments of this with one-three domains (SCR-19/20, SCR-18/20, SCR-17/18, SCR-16/18, SCR-17 and SCR-18). Size-exclusion chromatography suggested that SCR dimer formation occurred in several fragments. Dimer formation was clarified using analytical ultracentrifugation, where quantitative c(s) size distribution analyses showed that SCR-19/20 was monomeric, SCR-18/20 was slightly dimeric, SCR-16/20, SCR-16/18 and SCR-18 showed more dimer formation, and SCR-17 and SCR-17/18 were primarily dimeric with dissociation constants of ~5 µM. The combination of these results located the SCR-16/20 dimerization site at SCR-17 and SCR-18. X-ray solution scattering experiments and molecular modelling fits confirmed the dimer site to be at SCR-17/18, this dimer being a side-by-side association of the two domains. We propose that the self-association of CFH at SCR-17/18 enables higher concentrations of CFH to be achieved when SCR-19/20 are bound to host cell surfaces in order to protect these better during inflammation. Dimer formation at SCR-17/18 clarified the association of genetic variants throughout SCR-16/20 with renal disease.

Modèle topologique de la structure d’un antiport vacuolaire de type NHX chez la vigne cultivée (Vitis vinifera)

Botany ◽  
2009 ◽  
Vol 87 (3) ◽  
pp. 339-347 ◽  
Author(s):  
Mohsen Hanana ◽  
Olivier Cagnac ◽  
Ahmed Mliki ◽  
Eduardo Blumwald
Keyword(s):  
Amino Acid ◽  
Vitis Vinifera ◽  
Molecular Mass ◽  
Electrostatic Interactions ◽  
Functional Characterization ◽  
Terminal Region ◽  
Vitis Vinifera L ◽  
Amino Acid Residues ◽  
Transmembrane Segments ◽  
Α Helix

After identifying and isolating a grapevine ( Vitis vinifera L.) NHX vacuolar antiporter and before initializing functional genomic studies, we juged necessary to acquire a minimum of knowledge about the VvNHX1 protein. Thus, we realized a bioinformatic analysis to determine its basic characteristics and to get structural informations that could guide us through the functional characterization. We have determined important physico-chemical parameters (molecular mass, isoelectric point, hydrophobic regions, etc.) and obtained interesting structural data (primary, secondary, and tertiary structures; conserved domains and interaction motives; etc.). The VvNHX1 gene, which encodes this 541 amino-acid protein with a predicted molecular mass of 60 kDa, is made of 14 exons and measures 6.5 kb. The amino-acidic composition of this protein is very important, in particular, for the establishment of the α-helix structure, which represents more than 50% of the protein, but also for charge distribution, which generates critical electrostatic interactions for the ionic flux. The secondary structure of VvNHX1 contains multiple transmembrane α-helix segments that are made of hydrophobic amino-acid residues, thus facilitating its insertion in the membrane. Globally, VvNHX1 has one hydrophobic N-terminal region, made of 10 transmembrane segments with 440 amino-acid residues, and one hydrophilic C-terminal region, made of 100 residues. The region located between the fourth and fifth transmembrane segments represents, with its structure mainly helicoidal and the presence of a favourable electrostatic environment, the pore where cation flux is performed across the membrane. VvNHX1 contains various interaction domains as well as several putative posttranslational modification sites, mainly at the C-terminus but also at the N-terminus, that play an important part in regulating protein activities, influence protein structural stability, or interact with other proteins or signalling molecules.

scholarly journals Molecular Determinants for Targeting Heterochromatin Protein 1-Mediated Gene Silencing: Direct Chromoshadow Domain–KAP-1 Corepressor Interaction Is Essential

2000 ◽  
Vol 20 (17) ◽  
pp. 6449-6465 ◽  
Author(s):  
Mark S. Lechner ◽  
Gillian E. Begg ◽  
David W. Speicher ◽  
Frank J. Rauscher
Keyword(s):  
Amino Acid ◽  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Optical Biosensor ◽  
Dominant Negative ◽  
Heterochromatin Protein 1 ◽  
Heterochromatin Protein ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Assembly Factor

ABSTRACT The KRAB domain is a highly conserved transcription repression module commonly found in eukaryotic zinc finger proteins. KRAB-mediated repression requires binding to the KAP-1 corepressor, which in turn recruits members of the heterochromatin protein 1 (HP1) family. The HP1 proteins are nonhistone chromosomal proteins, although it is unclear how they are targeted to unique chromosomal domains or promoters. In this report, we have reconstituted and characterized the HP1–KAP-1 interaction using purified proteins and have compared KAP-1 to three other known HP1 binding proteins: SP100, lamin B receptor (LBR), and the p150 subunit from chromatin assembly factor (CAF-1 p150). We show that the chromoshadow domain (CSD) of HP1 is a potent repression domain that binds directly to all four previously described proteins. For KAP-1, we have mapped the CSD interaction region to a 15-amino-acid segment, termed the HP1BD, which is also present in CAF-1 p150 but not SP100 or LBR. The region of KAP-1 harboring the HP1BD binds as a monomer to a dimer of the CSD, as revealed by gel filtration, analytical ultracentrifugation, and optical biosensor analyses. The use of a spectrum of amino acid substitutions in the human HP1α CSD revealed a strong correlation between CSD-mediated repression and binding to KAP-1, CAF-1 p150, and SP100 but not LBR. Differences among the HP1 binding partners could also be discerned by fusion to a heterologous DNA binding domain and by the potential to act as dominant negative molecules. Together, these results strongly suggest that KAP-1 is a physiologically relevant target for HP1 function.

scholarly journals CHMP7, a novel ESCRT-III-related protein, associates with CHMP4b and functions in the endosomal sorting pathway

2006 ◽  
Vol 400 (1) ◽  
pp. 23-32 ◽  
Author(s):  
Mio Horii ◽  
Hideki Shibata ◽  
Ryota Kobayashi ◽  
Keiichi Katoh ◽  
Chiharu Yorikawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Amino Acid Residues ◽  
Related Protein ◽  
Positive Interaction ◽  
Leukaemia Virus ◽  
Gag Protein ◽  
Endosomal Sorting ◽  
Aaa Atpase

All CHMPs (charged multivesicular body proteins) reported to date have common features: they all contain approx. 200 amino acid residues, have coiled-coil regions and have a biased distribution of charged residues (basic N-terminal and acidic C-terminal halves). Yeast orthologues of CHMPs, including an ESCRT-III component Snf7, are required for the sorting of cargo proteins to intraluminal vesicles of multivesicular bodies. We have characterized a novel human ESCRT-III-related protein, designated CHMP7, which consists of 453 amino acid residues. CHMP7 contains an SNF7 domain and a distantly SNF7-related domain in its C-terminal half and N-terminal half respectively. Among the ten CHMP proteins classified previously in six subfamilies (CHMP1–CHMP6), the C-terminal SNF7 domain of CHMP7 is most similar to the SNF7 domain of CHMP6, which associates with CHMP4 proteins and EAP20, a component of ESCRT-II. Pull-down assays using lysates of HEK-293T (human embryonic kidney) cells that overexpressed Strep-tagged CHMP7 and GFP (green fluorescent protein)-fused CHMP4b (also named Shax1) revealed a positive interaction between the C-terminal half of CHMP7 and CHMP4b. However, interaction was not observed between CHMP7 and EAP20. Confocal fluorescence microscopic analyses revealed that FLAG–CHMP7 is distributed in HeLa cells diffusely throughout the cytoplasm, but with some accumulation, especially in the perinuclear area. The distribution of FLAG–CHMP7 was altered to a cytoplasmic punctate pattern by overexpression of either CHMP4b–GFP or GFP–Vps4BE235Q, a dominant-negative mutant of the AAA (ATPase associated with various cellular activities) Vps4B, and partially co-localized with them. Ubiquitinated proteins and endocytosed EGF accumulated in GFP–CHMP7-expressing cells. A dominant-negative effect of overexpressed GFP–CHMP7 was also observed in the release of virus-like particles from HEK-293T cells that transiently expressed the MLV (murine leukaemia virus) Gag protein. These results suggest that CHMP7, a novel CHMP4-associated ESCRT-III-related protein, functions in the endosomal sorting pathway.

scholarly journals Different Roles of Mitochondrial Calcium Uniporter Complex Subunits in Growth and Infectivity of Trypanosoma cruzi

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Miguel A. Chiurillo ◽  
Noelia Lander ◽  
Mayara S. Bertolini ◽  
Melissa Storey ◽  
Anibal E. Vercesi ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Human Cells ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Host Cells ◽  
Dominant Negative Effect ◽  
Mitochondrial Calcium ◽  
Mitochondrial Calcium Uniporter ◽  
Calcium Uniporter ◽  
Molecular Nature

ABSTRACT Trypanosoma cruzi is the agent of Chagas disease, and the finding that this parasite possesses a mitochondrial calcium uniporter (TcMCU) with characteristics similar to that of mammalian mitochondria was fundamental for the discovery of the molecular nature of MCU in eukaryotes. We report here that ablation of TcMCU , or its paralog TcMCUb , by clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 led to a marked decrease in mitochondrial Ca 2+ uptake without affecting the membrane potential of these cells, whereas overexpression of each gene caused a significant increase in the ability of mitochondria to accumulate Ca 2+ . While TcMCU- knockout (KO) epimastigotes were viable and able to differentiate into trypomastigotes, infect host cells, and replicate normally, ablation of TcMCUb resulted in epimastigotes having an important growth defect, lower rates of respiration and metacyclogenesis, more pronounced autophagy changes under starvation, and significantly reduced infectivity. Overexpression of TcMCUb , in contrast to what was proposed for its mammalian ortholog, did not result in a dominant negative effect on TcMCU. IMPORTANCE The finding of a mitochondrial calcium uniporter (MCU) in Trypanosoma cruzi was essential for the discovery of the molecular nature of this transporter in mammals. In this work, we used the CRISPR/Cas9 technique that we recently developed for T. cruzi to knock out two components of the uniporter: MCU, the pore subunit, and MCUb, which was proposed as a negative regulator of MCU in human cells. In contrast to what occurs in human cells, MCU is not essential, while MCUb is essential for growth, differentiation, and infectivity; has a bioenergetic role; and does not act as a dominant negative subunit of MCU.

Novel Germline CEBPA Sequence Variations in Familial AML and Cytogenetically Normal AML.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2566-2566 ◽  
Author(s):  
Rupa A. Udani ◽  
Mary Parlow ◽  
Lihui Yin ◽  
Daniel B. Bellissimo
Keyword(s):  
Conflicts Of Interest ◽  
Terminal Region ◽  
Dominant Negative ◽  
Negative Regulator ◽  
Buccal Swab ◽  
Truncated Form ◽  
Frame Shift ◽  
Sequence Variations ◽  
Cebpa Mutations ◽  
Cytogenetically Normal Aml

Abstract Abstract 2566 The CCAAT enhancer binding protein alpha encoded by CEBPA gene is a transcription factors involved in myeloid lineage differentiation. Somatic CEBPA mutations are associated with a favorable prognosis in patients with normal to intermediate karyotype and lacking the internal tandem duplication in the fms-like tyrosine kinase -3 gene (FLT3/ITD) or mutation in the nucleophosmin gene (NPM1). N-terminal CEBPA mutations typically result in a frame shift encoding a truncated form of the 42-kD CEBPα protein and potentially increase translation of the alternative 30-kD isoform. The 30-kD isoform functions as a dominant negative regulator of the full-length 42-kD isoform of the CEBPα protein. C-terminal mutations are generally in-frame insertion/deletions in the DNA binding or the leucine zipper domains that cause alteration of the dimerization domain (bZIP). Germline mutations in CEBPA gene are recognized as the major cause of familial acute myeloid leukemia (AML). Familial AML is inherited in an autosomal dominant manner with complete penetrance. In contrast to somatic AML, the age onset of familial AML is earlier ranging from 4 to 39 years. The overall survival of the familial AML patients with germline CEBPA mutation is ∼50%–65% better than ∼34%–50% observed with the somatic CEBPA mutations (FLT3/NPM1 negative). In all of the familial AML pedigrees reported, the mutations were frame-shift insertions/deletions in the N-terminal domain resulting in translation of a truncated form of the CEBPα protein. We have identified four novel germline sequence variants in patients with history of familial AML and unknown karyotypes (cases 1–4). The patients' age ranges from 3 to 32 years. Of the four novel variants, one variant was identified in the C-terminal region of the gene. This is the first reported C-terminal variant detected in the proband of a familial AML pedigree. The variant is out of frame insertion/deletion of ∼401 bp in the C-terminal region of the CEBPA gene (there is a deletion ‘∼171bp then an insertion of 401bp). Another novel C-terminal variation was identified in a patient with cytogenetically normal AML undergoing testing for somatically acquired CEBPA mutations (case 5). The detection of this C-terminal variation in a buccal swab sample confirmed the germline origin of the variation. In two additional cases (cases 6 and 7), we observed that one of the two sequence variations identified was no longer present after treatment, suggesting the remaining variation was germline in origin. Our results demonstrate that germline C-terminal CEBPA mutations can cause familial AML and that germline CEBPA mutations may be identified in cytogenetically normal AML patients. The possibility of germline variants should be considered in AML patients since other family members, who may be possible transplant donors for the patient, may have also inherited the same germline variant. Case AML Type Sequence variation CEBPA gene region 1 Familial AML c.142delG; p.Ala48fs N terminal 2 c.168C>A; p.Cys56* N terminal 3 c.175G>T; p.Glu59* N terminal 4 c.643_814delins401; p.Thr216fs C terminal 5 Somatic AML c.1073delC; p.Ala358fs C terminal 6 c.68_78del; p.Pro23fs N terminal 7 c. 938T>G; p. Val328G C terminal Disclosures: No relevant conflicts of interest to declare.

scholarly journals Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

2010 ◽  
Vol 21 (22) ◽  
pp. 3973-3984 ◽  
Author(s):  
Yukinao Shibukawa ◽  
Natsuko Yamazaki ◽  
Keiichi Kumasawa ◽  
Etsuko Daimon ◽  
Michiko Tajiri ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Cell Fusion ◽  
Syncytium Formation ◽  
Terminal Region ◽  
Negative Regulator ◽  
Dominant Negative Effect ◽  
Bewo Cell ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Cytoskeleton Rearrangement

Cell–cell fusion is an intriguing differentiation process, essential for placental development and maturation. A proteomic approach identified a cytoplasmic protein, calponin 3 (CNN3), related to the fusion of BeWo choriocarcinoma cells. CNN3 was expressed in cytotrophoblasts in human placenta. CNN3 gene knockdown promoted actin cytoskeletal rearrangement and syncytium formation in BeWo cells, suggesting CNN3 to be a negative regulator of trophoblast fusion. Indeed, CNN3 depletion promoted BeWo cell fusion. CNN3 at the cytoplasmic face of cytoskeleton was dislocated from F-actin with forskolin treatment and diffused into the cytoplasm in a phosphorylation-dependent manner. Phosphorylation sites were located at Ser293/296 in the C-terminal region, and deletion of this region or site-specific disruption of Ser293/296 suppressed syncytium formation. These CNN3 mutants were colocalized with F-actin and remained there after forskolin treatment, suggesting that dissociation of CNN3 from F-actin is modulated by the phosphorylation status of the C-terminal region unique to CNN3 in the CNN family proteins. The mutant missing these phosphorylation sites displayed a dominant negative effect on cell fusion, while replacement of Ser293/296 with aspartic acid enhanced syncytium formation. These results indicated that CNN3 regulates actin cytoskeleton rearrangement which is required for the plasma membranes of trophoblasts to become fusion competent.

scholarly journals Type III CRISPR complexes from Thermus thermophilus.

2016 ◽  
Vol 63 (2) ◽  
Author(s):  
Marta Szychowska ◽  
Wojciech Siwek ◽  
Damian Pawolski ◽  
Asgar Abbas Kazrani ◽  
Krzysztof Pyrc ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Size Exclusion Chromatography ◽  
Mass Spectroscopy ◽  
Thermus Thermophilus ◽  
Size Exclusion ◽  
Acquired Immunity ◽  
Type I ◽  
Type Iii ◽  
Exclusion Chromatography

Pathogen-specific acquired immunity in bacteria is mediated by the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems. Thermus thermophilus strain HB8 contains CRISPR systems of several major subtypes (type I, IIIA and IIIB), and has become a widely studied model for CRISPR biology. We have selected two highly expressed CRISPR spacers, crRNA 2.1 and crRNA 2.2, and have enriched endogenous T. thermophilus proteins that co-purify with these crRNAs. Mass spectroscopy indicates that the chromatography protocol enriches predominantly Csm complex subunits, but also Cmr subunits. After several chromatographic steps, size exclusion chromatography indicated a molecular mass of the crRNA associated complex of 265±69 kDa. In agreement with earlier work, crRNAs of different lengths (containing the selected spacers) were observed. Most of these were completely lost when several T. thermophilus csm genes were ablated.

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