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C-terminal Truncation (c-terminal + truncation)
Selected AbstractsCanine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-,2(I) and Severe Osteogenesis ImperfectaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Bonnie G. Campbell Abstract RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-,2(I) suggested comigration with the similarly sized pro-,2(I) derived from the mutant allele. Furthermore, ,-chains were overhydroxylated and the ratio of ,1(I):,2(I) was 3.2:1, consistent with the presence of ,1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-,2(I) C-propeptide and confirmed a diagnosis of OI. [source] Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanismDEVELOPMENTAL DYNAMICS, Issue 4 2004Gary R. Hime Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source] Co-expression of C-terminal truncated alpha-synuclein enhances full-length alpha-synuclein-induced pathologyEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2010Ayse Ulusoy Abstract Lewy bodies, which are a pathological hallmark of Parkinson's disease, contain insoluble polymers of alpha-synuclein (,syn). Among the different modifications that can promote the formation of toxic ,syn species, C-terminal truncation is among the most abundant alterations in patients with Parkinson's disease. In vitro, C-terminal truncated ,syn aggregates faster and sub-stoichiometric amounts of C-terminal truncated ,syn promote aggregation of the full-length ,syn (,synFL) and induce neuronal toxicity. To address in vivo the putative stimulation of ,syn-induced pathology by the presence of truncated ,syn, we used recombinant adeno-associated virus to express either ,synFL or a C-terminal truncated ,syn (1-110) in rats. We adjusted the recombinant adeno-associated virus vector concentrations so that either protein alone led to only mild to moderate axonal pathology in the terminals of nigrostriatal dopamine neurons without frank cell loss. When these two forms of ,syn were co-expressed at these pre-determined levels, it resulted in a more aggressive pathology in fiber terminals as well as dopaminergic cell loss in the substantia nigra. Using an antibody that did not detect the C-terminal truncated ,syn (1-110) but only ,synFL, we demonstrated that the co-expressed truncated protein promoted the progressive accumulation of ,synFL and formation of larger pathological accumulations. Moreover, in the co-expression group, three of the eight animals showed apomorphine-induced turning, suggesting prominent post-synaptic alterations due to impairments in the dopamine release, whereas the mild pathology induced by either form alone did not cause motor abnormalities. Taken together these data suggest that C-terminal truncated ,syn can interact with and exacerbate the formation of pathological accumulations containing ,synFL in vivo. [source] The porcine trophoblastic interferon-,, secreted by a polarized epithelium, has specific structural and biochemical propertiesFEBS JOURNAL, Issue 11 2002Avrelija Cenci At the time of implantation in the maternal uterus, the trophectoderm of the pig blastocyst is the source of a massive secretion of interferon-gamma (IFN-,), together with lesser amounts of IFN-,, a unique species of type I IFN. This trophoblastic IFN-, (TrIFN-,) is an unprecedented example of IFN-, being produced spontaneously by an epithelium. We therefore studied some of its structural and biochemical properties, by comparison with pig IFN-, from other sources, either natural LeIFN-, (from adult leucocytes), or recombinant. Biologically active TrIFN-, is a dimeric molecule, of which monomers are mainly composed of a truncated polypeptide chain with two glycotypes, unlike LeIFN-, which is formed of at least two polypeptide chains and four glycotypes. TrIFN-, collected in the uterus lumen was enzymatically deglycosylated and analysed by mass spectrometry (MALDI-TOF). The data revealed that the more abundant polypeptide has a mass of 14.74 kDa, corresponding to a C-terminal cleavage of 17 residues from the expected 143-residue long mature sequence. A minor polypeptide, with a mass of 12.63 kDa, corresponds to a C-terminal truncation of 36 amino acids. MALDI-TOF analysis of tryptic peptides from the glycosylated molecule(s) identifies a single branched carbohydrate motif, with six N -acetylgalactosamines, and no sialic acid. The only glycan microheterogeneity seems to reside in the number of l -fucose residues (one to three). The lack of the C-terminal cluster of basic residues, and the presence of nonsialylated glycans, result in a very low net charge of TrIFN-, molecule. However, the 17-residue truncation does not affect the antiproliferative activity of TrIFN-, on different cells, among which is a porcine uterine epithelial cell line. It is suggested that these specific properties might confer on TrIFN-, a particular ability to invade the uterine mucosa and exert biological functions beyond the endometrial epithelium. [source] Cloning and comparison of phylogenetically related chitinases from Listeria monocytogenes EGD and Enterococcus faecalis V583JOURNAL OF APPLIED MICROBIOLOGY, Issue 6 2009J.J. Leisner Abstract Aims:, To compare enzymatic activities of two related chitinases, ChiA and EF0361, encoded by Listeria monocytogenes and Enterococcus faecalis, respectively. Methods and Results:, The chiA and EF0361 genes were amplified by PCR, cloned and expressed with histidine tags, allowing easy purification of the gene products. ChiA had a molecular weight as predicted from the amino acid sequence, whereas EF0361 was 1840 Da lower than expected because of C-terminal truncation. The ChiA and EF0361 enzymes showed activity towards 4-nitrophenyl N,N,-diacetyl-,- d -chitobioside with Km values of 1·6 and 2·1 mmol l,1, respectively, and kcat values of 21·6 and 6·5 s,1. The enzymes also showed activity towards 4-nitrophenyl ,- d - N, N,, N,-triacetylchitotriose and carboxy-methyl-chitin-Remazol Brilliant Violet but not towards 4-nitrophenyl N- acetyl-,- d -glucosaminide. Chitinolytic specificities of the enzymes were supported by their inactivity towards the substrates 4-nitrophenyl ,- d -cellobioside and peptidoglycan. The pH and temperature profiles for catalytic activities were relatively similar for both the enzymes. Conclusion:, The ChiA and EF0361 enzymes show a high degree of similarity in their catalytic activities although their hosts share environmental preferences only to some extent. Significance and Impact of the Study:, This study contributes to an understanding of the chitinolytic activities by L. monocytogenes and Ent. faecalis. Detailed information on their chitinolytic systems will help define potential reservoirs in the natural environment and possible transmission routes into food-manufacturing plants. [source] Analysis of the requirements for pilus biogenesis at the outer membrane usher and the function of the usher C-terminusMOLECULAR MICROBIOLOGY, Issue 2 2006Stephane Shu Kin So Summary Uropathogenic strains of Escherichia coli assemble type 1 and P pili to colonize the bladder and kidney respectively. These pili are prototype structures assembled by the chaperone/usher secretion pathway. In this pathway, a periplasmic chaperone works together with an outer membrane (OM) usher to control the folding of pilus subunits, their assembly into a pilus fibre and secretion of the fibre to the cell surface. The usher serves as the assembly and secretion platform in the OM. The usher has distinct functional domains, with the N-terminus providing the initial targeting site for chaperone,subunit complexes and the C-terminus required for subsequent stages of pilus biogenesis. In this study, we investigated the molecular interactions occurring at the usher during pilus biogenesis and the function of the usher C-terminus. We provide genetic and biochemical evidence that the usher functions as a complex in the OM and that interaction of the pilus adhesin with the usher is critical to prime the usher for pilus biogenesis. Analysis of C-terminal truncation and substitution mutants of the P pilus usher PapC demonstrated that the C-terminus is required for proper binding of chaperone,subunit complexes to the usher and plays an important role in assembly of complete pili. [source] The human ACC2 CT-domain C-terminus is required for full functionality and has a novel twistACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2009Kevin P. Madauss Inhibition of acetyl-CoA carboxylase (ACC) may prevent lipid-induced insulin resistance and type 2 diabetes, making the enzyme an attractive pharmaceutical target. Although the enzyme is highly conserved amongst animals, only the yeast enzyme structure is available for rational drug design. The use of biophysical assays has permitted the identification of a specific C-terminal truncation of the 826-residue human ACC2 carboxyl transferase (CT) domain that is both functionally competent to bind inhibitors and crystallizes in their presence. This C-terminal truncation led to the determination of the human ACC2 CT domain,CP-640186 complex crystal structure, which revealed distinctions from the yeast-enzyme complex. The human ACC2 CT-domain C-terminus is comprised of three intertwined ,-helices that extend outwards from the enzyme on the opposite side to the ligand-binding site. Differences in the observed inhibitor conformation between the yeast and human structures are caused by differing residues in the binding pocket. [source] Epidermal growth factor receptor lacking C-terminal autophosphorylation sites retains signal transduction and high sensitivity to epidermal growth factor receptor tyrosine kinase inhibitorCANCER SCIENCE, Issue 3 2009Mari Maegawa Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR. (Cancer Sci 2009; 100: 552,557) [source] Identification of a novel region of the GABAB2 C-terminus that regulates surface expression and neuronal targeting of the GABAB receptorEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 5 2009A. M. Pooler Abstract GABAB is a G protein-coupled receptor composed of two subunits, GABAB1 and GABAB2. GABAB1 contains an endoplasmic reticulum-retention sequence and is trafficked to the cell surface only in association with GABAB2. To determine whether the C-terminus of GABAB2 regulates GABAB trafficking, we constructed forms of GABAB2 with various C-terminal truncations and examined their surface expression. Truncation of GABAB2 after residue 841 significantly reduced surface expression of both the subunit and the heterodimerized receptor. Turnover of the ,841 construct, however, did not differ from that of full-length GABAB2. To determine whether the C-terminus of GABAB2 might target GABAB to neurites, cultured hippocampal neurons were transfected with the truncated GABAB2 constructs. Truncation of GABAB2 at residue 841 resulted in primarily somatic localization; furthermore, axonal trafficking of this construct was significantly more restricted than dendritic trafficking. Finally, to biochemically assess trafficking of the truncated GABAB2 constructs, we digested transfected HEK293 cell lysates with endoglycosidase H. When GABAB2 was truncated at residue 841, it became sensitive to digestion by this enzyme, indicating incomplete trafficking. Taken together, these data show that the region of the GABAB2 C-terminus between residues 841 and 862 is important for regulating forward trafficking and neuronal targeting of the GABAB receptor. [source] The solution structure of ZNF593 from Homo sapiens reveals a zinc finger in a predominately unstructured proteinPROTEIN SCIENCE, Issue 3 2008Paulette L. Hayes Abstract Here, we report the solution structure of ZNF593, a protein identified in a functional study as a negative modulator of the DNA-binding activity of the Oct-2 transcription factor. ZNF593 contains a classic C2H2 zinc finger domain flanked by about 40 disordered residues on each terminus. Although the protein contains a high degree of intrinsic disorder, the structure of the zinc finger domain was resolved by NMR spectroscopy without a need for N- or C-terminal truncations. The tertiary structure of the zinc finger domain is composed of a ,-hairpin that positions the cysteine side chains for zinc coordination, followed by an atypical kinked ,-helix containing the two histidine side chain ligands. The structural topology of ZNF593 is similar to a fragment of the double-stranded RNA-binding protein Zfa and the C-terminal zinc finger of MBP-1, a human enhancer binding protein. The structure presented here will provide a guide for future functional studies of how ZNF593 negatively modulates the DNA-binding activity of Oct-2, a POU domain-containing transcription factor. Our work illustrates the unique capacity of NMR spectroscopy for structural analysis of folded domains in a predominantly disordered protein. [source] | ||||||||||||||||