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Rosentreter A Hofmann A Xavier CP Stumpf M Noegel AA Clemen CS 《Experimental cell research》2007,313(5):878-895
The actin interaction of coronin 3 has been mainly documented by in vitro experiments. Here, we discuss coronin 3 properties in the light of new structural information and focus on assays that reflect in vivo roles of coronin 3 and its impact on F-actin-associated functions. Using GFP-tagged coronin 3 fusion proteins and RNAi silencing we show that coronin 3 has roles in wound healing, protrusion formation, cell proliferation, cytokinesis, endocytosis, axonal growth, and secretion. During formation of cell protrusions actin accumulation precedes the focal enrichment of coronin 3 suggesting a role for coronin 3 in events that follow the initial F-actin assembly. Moreover, we show that coronin 3 similar to other coronins interacts with the Arp2/3-complex and cofilin indicating that this family in general is involved in regulating Arp2/3-mediated events. 相似文献
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Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes 总被引:2,自引:0,他引:2
Ward DM Shiflett SL Huynh D Vaughn MB Prestwich G Kaplan J 《Traffic (Copenhagen, Denmark)》2003,4(6):403-415
The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size. 相似文献
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Gupta A Chattopadhyay I Dey S Nasipuri P Das SK Gangopadhyay PK Ray K 《Cellular and molecular neurobiology》2007,27(8):1023-1033
Aims We aim to identify the molecular defects in the ATP7B, the causal gene for Wilson disease (WD), in eastern Indian patients and attempt to assess the overall mutation spectrum
in India for detection of mutant allele for diagnostic purposes. Methods Patients from 109 unrelated families and their first-degree relatives comprising 400 individuals were enrolled in this study
as part of an ongoing project. Genomic DNA was prepared from the peripheral blood of Indian WD patients. PCR was done to amplify
the exons and flanking regions of the WD gene followed by sequencing, to identify the nucleotide variants. Results In addition to previous reports, we recently identified eight mutations including three novel (c.3412 + 1G > A, c.1771 G > A,
c.3091 A > G) variants, and identified patients with variable phenotype despite similar mutation background suggesting potential
role of modifier locus. Conclusions So far we have identified 17 mutations in eastern India including five common mutations that account for 44% of patients.
Comparative study on WD mutations between different regions of India suggests high genetic heterogeneity and the absence of
a single or a limited number of common founder mutations. Genotype–phenotype correlation revealed that no particular phenotype
could be assigned to a particular mutation and even same set of mutations in different patients showed different phenotypes. 相似文献
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Emmanuel S. Burgos Carola Wilczek Takashi Onikubo Jeffrey B. Bonanno Janina Jansong Ulf Reimer David Shechter 《The Journal of biological chemistry》2015,290(15):9674-9689
The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate Km, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site. 相似文献
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Molecular cloning and characterization of AAAS-V2, a novel splice variant of human AAAS 总被引:4,自引:0,他引:4
Li X Ji C Gu J Xu J Jin Z Sun L Zou X Lin Y Sun R Wang P Gu S Mao Y 《Molecular biology reports》2005,32(2):127-131
Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Much initial molecular analysis supported that Triple-A syndrome was caused by mutations in AAAS, a WD-repeat protein gene. Here we report cloning and characterization of a novel splice variant of human AAAS, which we named AAAS-v2, which is located on the human chromosome 12p13. The cDNA is 1703bp, encoding a 513-amino acid polypeptide, which contains three WD40 domains, one less than the original which we called AAAS-v1 (Gen Bank: NM_015665.3). RT-PCR analysis in our work revealed that AAAS-v2 and AAAS-v1 were ubiquitously detected in human multiple tissue cDNA (MTC) panels (CLONTECH).The nucleotide sequence reported in this paper has been submitted to GenBank under accession number AY237818.Xin Li: These two authors contributed equally to this paper.Chaoneng Ji: These two authors contributed equally to this paper. 相似文献
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WD (tryptophan/aspartic acid) repeat proteins perform a wide variety of functions in eukaryotic cells. They are characterised by the presence of a number of conserved repeat motifs that contribute to the beta-propeller structures which are the common feature of this large group of proteins. We report here the properties of the first characterised member of this family in the American trypanosome, Trypanosoma cruzi (TcBPP1). In the CL Brener clone the protein is 482 amino acids long and is predicted to contain four WD repeat motifs, flanked by amino and carboxyl terminal extensions. TcBPP1 is a single copy gene present on a 1.0/1.6 Mb pair of homologous chromosomes in a locus that is syntenic with the corresponding regions of Trypanosoma brucei and Leishmania major chromosomes. Consistent with the proposed hybrid nature of the CL Brener clone, the proteins encoded by the two different alleles share only 97% identity at the amino acid level. To determine subcellular location, we examined transfected parasites for the distribution of green fluorescent protein (GFP) fused with different regions of TcBPP1. These studies demonstrated that a 115 amino acid peptide derived from the amino terminal domain of TcBPP1 is able to target GFP to the mitochondrion. Interestingly this region lacks a typical amino terminal presequence suggesting that mitochondrial import is mediated by an alternative targeting signal. 相似文献
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Hames RS Hames R Prosser SL Euteneuer U Lopes CA Moore W Woodland HR Fry AM 《Experimental cell research》2008,314(3):574-589
In many animals, the germ line develops from a distinct mitochondria-rich region of embryonic cytoplasm called the germ plasm. However, the protein composition of germ plasm and its formation remain poorly understood, except in Drosophila. Here, we show that Xpat, a recently identified protein component of Xenopus germ plasm, interacts via its C-terminal domain with a novel protein, xPix1. Xpat and xPix1 are co-expressed in ovaries, eggs and early embryos and colocalize to the mitochondrial cloud and germ plasm in stage I and stage VI oocytes, respectively. Although Xpat appears unique to Xenopus, Pix proteins, which contain an N-terminal WD40 domain and C-terminal coiled-coil, are widely conserved. In humans, two proteins, Pix1 and Pix2, are expressed at varying levels in different cancer cell lines. Importantly, as well as localizing to mitochondria, human Pix proteins localize to centrosomes and associate with microtubules in vitro and in vivo. Although, Pix proteins are stably expressed through the cell cycle, Pix2 concentrates on microtubule structures in mitosis and microinjection of Pix antibodies interferes with cell division. Based on these data, we propose that Pix1 and Pix2 are microtubule-associated adaptor proteins that likely contribute to a range of developmental and cell division processes. 相似文献