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Joseph Gabriele Giuseppe F. Pontoriero Nancy Thomas Christy A. Thomson Kevin Skoblenick Zdenek B. Pristupa Ram K. Mishra 《Cell stress & chaperones》2009,14(6):555-567
Catecholamine-regulated proteins (CRPs) have been shown to bind dopamine and other structurally related catecholamines; in
particular, the 40-kDa CRP (CRP40) protein has been previously cloned and functionally characterized. To determine putative
human homologs, BLAST analysis using the bovine CRP40 sequence identified a human established sequence tag (EST) with significant
homology (accession #BQ224193). Using this EST, we cloned a recombinant human brain CRP40-like protein, which possessed chaperone
activity. Radiolabeled dopamine binding studies with recombinant human CRP40 protein demonstrated the ability of this protein
to bind dopamine with low affinity and high capacity. The full-length human CRP40 nucleotide sequence was elucidated (accession
#DQ480334) with RNA ligase-mediated rapid amplification of complementary DNA ends polymerase chain reaction, while Northern
blot hybridization suggested that human CRP40 is an alternative splice variant of the 70-kDa mitochondrial heat shock protein,
mortalin. Human SH-SY5Y neuroblastoma cells treated with the antipsychotic drug, haloperidol, exhibited a significant increase
in CRP40 messenger RNA expression compared to untreated control cells, while other dopamine agonists/antagonists also altered
CRP40 expression and immunolocalization. In conclusion, these results show that we have cloned a splice variant of mortalin
with a novel catecholamine binding function and that this chaperone-like protein may be neuroprotective in dopamine-related
central nervous system disorders. 相似文献
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Peng L Oberst MD Huang J Brohawn P Morehouse C Lekstrom K Baeuerle PA Wu H Yao Y Coats SR Dall'Acqua W Damschroder M Hammond SA 《PloS one》2012,7(5):e36412
MEDI-565 (also known as MT111) is a bispecific T-cell engager (BiTE?) antibody in development for the treatment of patients with cancers expressing carcinoembryonic antigen (CEA). MEDI-565 binds CEA on cancer cells and CD3 on T cells to induce T-cell mediated killing of cancer cells. To understand the molecular basis of human CEA recognition by MEDI-565 and how polymorphisms and spliced forms of CEA may affect MEDI-565 activity, we mapped the epitope of MEDI-565 on CEA using mutagenesis and homology modeling approaches. We found that MEDI-565 recognized a conformational epitope in the A2 domain comprised of amino acids 326-349 and 388-410, with critical residues F(326), T(328), N(333), V(388), G(389), P(390), E(392), I(408), and N(410). Two non-synonymous single-nucleotide polymorphisms (SNPs) (rs10407503, rs7249230) were identified in the epitope region, but they are found at low homozygosity rates. Searching the National Center for Biotechnology Information GenBank? database, we further identified a single, previously uncharacterized mRNA splice variant of CEA that lacks a portion of the N-terminal domain, the A1 and B1 domains, and a large portion of the A2 domain. Real-time quantitative polymerase chain reaction analysis of multiple cancers showed widespread expression of full-length CEA in these tumors, with less frequent but concordant expression of the CEA splice variant. Because the epitope was largely absent from the CEA splice variant, MEDI-565 did not bind or mediate T-cell killing of cells solely expressing this form of CEA. In addition, the splice variant did not interfere with MEDI-565 binding or activity when co-expressed with full-length CEA. Thus MEDI-565 may broadly target CEA-positive tumors without regard for expression of the short splice variant of CEA. Together our data suggest that MEDI-565 activity will neither be impacted by SNPs nor by a splice variant of CEA. 相似文献
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