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C-terminal Amino Acids (c-terminal + amino_acids)
Selected AbstractsMouse cytosolic sulfotransferase SULT2B1b interacts with cytoskeletal proteins via a proline/serine-rich C-terminusFEBS JOURNAL, Issue 18 2010Katsuhisa Kurogi Cytosolic sulfotransferase (SULT) SULT2B1b had previously been characterized as a cholesterol sulfotransferase. Like human SULT2B1, mouse SULT2B1b contains a unique, 31 amino acid C-terminal sequence with a proline/serine-rich region, which is not found in members of other SULT families. To gain insight into the functional relevance of this proline/serine-rich region, we constructed a truncated mouse SULT2B1b lacking the 31 C-terminal amino acids, and compared it with the wild-type enzyme. Enzymatic characterization indicated that the catalytic activity was not significantly affected by the absence of those C-terminal residues. Glutathione S -transferase pulldown assays showed that several proteins interacted with mouse SULT2B1b specifically through this C-terminal proline/serine-rich region. Peptide mass fingerprinting revealed that of the five SULT2B1b-binding proteins analyzed, three were cytoskeletal proteins and two were cytoskeleton-binding molecular chaperones. Furthermore, wild-type mouse SULT2B1b, but not the truncated enzyme, was associated with the cytoskeleton in experiments with a cytoskeleton-stabilizing buffer. Collectively, these results suggested that the unique, extended proline/serine-rich C-terminus of mouse SULT2B1b is important for its interaction with cytoskeletal proteins. Such an interaction may allow the enzyme to move along microfilaments such as actin filaments, and catalyze the sulfation of hydroxysteroids, such as cholesterol and pregnenolone, at specific intracellular locations. Structured digital abstract ,,MINT-7975854: Sult2B1b (uniprotkb:O35400) physically interacts (MI:0914) with Myosin-Ic (uniprotkb:Q9WTI7), Alpha-actinin-1 (uniprotkb:Q7TPR4), Alpha-actinin-4 (uniprotkb:P57780), HSP 90-beta (uniprotkb:P11499), Hsc70, (uniprotkb:P63017), Beta-actin (uniprotkb:P60710) and Gamma-actin (uniprotkb:P63260) by pull down (MI:0096) [source] Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical formFEBS JOURNAL, Issue 8 2007R. Ndoria Thuku Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy, followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily, enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1,327. [source] Structural requirements for the apical sorting of human multidrug resistance protein 2 (ABCC2)FEBS JOURNAL, Issue 7 2002Anne T. Nies The human multidrug resistance protein 2 (MRP2, symbol ABCC2) is a polytopic membrane glycoprotein of 1545 amino acids which exports anionic conjugates across the apical membrane of polarized cells. A chimeric protein composed of C-proximal MRP2 and N-proximal MRP1 localized to the apical membrane of polarized Madin,Darby canine kidney cells (MDCKII) indicating involvement of the carboxy-proximal part of human MRP2 in apical sorting. When compared to other MRP family members, MRP2 has a seven-amino-acid extension at its C-terminus with the last three amino acids (TKF) comprising a PDZ-interacting motif. In order to analyze whether this extension is required for apical sorting of MRP2, we generated MRP2 constructs mutated and stepwise truncated at their C-termini. These constructs were fused via their N-termini to green fluorescent protein (GFP) and were transiently transfected into polarized, liver-derived human HepG2 cells. Quantitative analysis showed that full-length GFP,MRP2 was localized to the apical membrane in 73% of transfected, polarized cells, whereas it remained on intracellular membranes in 27% of cells. Removal of the C-terminal TKF peptide and stepwise deletion of up to 11 amino acids did not change this predominant apical distribution. However, apical localization was largely impaired when GFP,MRP2 was C-terminally truncated by 15 or more amino acids. Thus, neither the PDZ-interacting TKF motif nor the full seven-amino-acid extension were necessary for apical sorting of MRP2. Instead, our data indicate that a deletion of at least 15 C-terminal amino acids impairs the localization of MRP2 to the apical membrane of polarized cells. [source] Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cellsFEBS JOURNAL, Issue 3 2000Stavit Drori Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3,-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3,-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3,-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating , 65,90 kDa native reduced folate carrier and a , 40,45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed. [source] Influence of "Alternative" C-terminal amino acids on the formation of [b3 + 17 + Cat]+ products from metal cationized synthetic tetrapeptides,JOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 5 2004V. Anbalagan Abstract The aim of this study was to investigate the dissociation patterns, and in particular the relative abundance of [b3 + 17 + Cat]+, for peptides with C-termini designed to allow transfer of the ,OH required to generate the product ion, but not necessarily as the most favored pathway. Working with the hypothesis that formation of a five-membered ring intermediate, including intramolecular nucleophilic attack by a carbonyl oxygen atom, is an important mechanistic step, several model peptides with general sequence AcFGGX were synthesized, metal cationized by electrospray ionization and subjected to collision-induced dissociation (CID). The amino acid at position X was one that either required a larger ring intermediate (,-alanine, ,-aminobutyric acid and ,-amino- n -caproic acid to generate six-, seven- or nine- membered rings, respectively) to transfer ,OH, lacked a structural element required for nucleophilic attack (aminoethanol) or prohibited cyclization because of the inclusion of a rigid ring (p - and m -aminobenzoic acid). For Ag+, Li+ and Na+ cationized peptides, our results show that amino acids requiring the adoption of larger ring intermediates suppressed the formation of [b3 + 17 + Cat]+, while amino acids that prohibit cyclization eliminated the reaction pathway completely. Formation of [b3 , 1 + Cat]+ from the alkali metal cationized versions was not a favorable process upon suppression or elimination of the [b3 + 17 + Cat]+ pathway: the loss of H2O to form [M , H2O + Cat]+ was instead the dominant dissociation reaction observed. Multiple-stage dissociation experiments suggest that [M , H2O + Cat]+ is not [b4 , 1 + Cat]+ arising from the loss of H2O from the C-terminus, but may instead be a species that forms via a mechanism involving the elimination of an oxygen atom from an amide group. Copyright © 2004 John Wiley & Sons, Ltd. [source] Expression, purification and crystallization of human prolylcarboxypeptidaseACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Pravien D. Abeywickrema Prolylcarboxypeptidase (PrCP) is a lysosomal serine carboxypeptidase that cleaves a variety of C-terminal amino acids adjacent to proline and has been implicated in diseases such as hypertension and obesity. Here, the robust production, purification and crystallization of glycosylated human PrCP from stably transformed CHO cells is described. Purified PrCP yielded crystals belonging to space group R32, with unit-cell parameters a = b = 181.14, c = 240.13,Å, that diffracted to better than 2.8,Å resolution. 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