EF-hand Calcium-binding Domain Research Articles

A Phaseolus vulgaris EF-hand calcium-binding domain is induced early in the defense response against Colletotrichum lindemuthianum and by abiotic stress: Sequences shared between interacting partners

Early in the plant defense response against pathogens, transcription of specific genes is activated. We studied the genes up-regulated in the early resistance reaction of Phaseolus vulgaris against Colletotrichum lindemuthianum. The effect of the light–dark transition, ultraviolet-(A–B) light, and temperature stress on these genes was also evaluated. A calcium-binding protein and previously identified SUMO genes were over-expressed in the plant reaction to pathogen and under environmental stress. These genes appear to act as integrative signals and convergence points in early plant responses to pathogens and several forms of environmental stress. The calcium-binding protein gene was present in both plant and fungi genomes.

Transamidation by Transglutaminase 2 Transforms S100A11 Calgranulin into a Procatabolic Cytokine for Chondrocytes

In osteoarthritis (OA), low-grade joint inflammation promotes altered chondrocyte differentiation and cartilage catabolism. S100/calgranulins share conserved calcium-binding EF-hand domains, associate noncovalently as homodimers and heterodimers, and are secreted and bind receptor for advanced glycation end products (RAGE). Chondrocyte RAGE expression and S100A11 release are stimulated by IL-1beta in vitro and increase in OA cartilage in situ. Exogenous S100A11 stimulates chondrocyte hypertrophic differentiation. Moreover, S100A11 is covalently cross-linked by transamidation catalyzed by transglutaminase 2 (TG2), itself an inflammation-regulated and redox stress-inducible mediator of chondrocyte hypertrophic differentiation. In this study, we researched mouse femoral head articular cartilage explants and knee chondrocytes, and a soluble recombinant double point mutant (K3R/Q102N) of S100A11 TG2 transamidation substrate sites. Both TG2 and RAGE knockout cartilage explants retained IL-1beta responsiveness. The K3R/Q102N mutant of S100A11 retained the capacity to bind to RAGE and chondrocytes but lost the capacity to signal via the p38 MAPK pathway or induce chondrocyte hypertrophy and glycosaminoglycans release. S100A11 failed to induce hypertrophy, glycosaminoglycan release, and appearance of the aggrecanase neoepitope NITEGE in both RAGE and TG2 knockout cartilages. We conclude that transamidation by TG2 transforms S100A11 into a covalently bonded homodimer that acquires the capacity to signal through the p38 MAPK pathway, accelerate chondrocyte hypertrophy and matrix catabolism, and thereby couple inflammation with chondrocyte activation to potentially promote OA progression.

Involvement of a 25 kDa Tetrahymena Ca2+-binding Protein in Pronuclear Exchange

The Tetrahymena Ca2+-binding protein of 25 kDa (TCBP-25) is a calmodulin family protein containing four EF-hand type calcium-binding domains. TCBP-25 is localized in the whole cell cortex and around both the migratory and stationary pronuclei at the pronuclear exchange stage during conjugation. TCBP-25 is expected to play an important role in conjugation, though its function during sexual reproduction has not been elucidated. According to the localization of this protein and its timing, three possible roles of TCBP-25 are proposed. TCBP-25 may play a role in 1) differentiating the two functional pronuclei from the degenerative post-meiotic nuclei, 2) the process of pronuclear exchange and 3) pronuclear fusion. To test these hypotheses, the localization of TCBP-25 in conjugation mutants (cnj10, cnj7 and bcd2) was examined. The results ruled out the first and the third hypotheses and suggested that TCBP-25 may play a role in pronuclear exchange. In the next step we succeeded in reducing expression of the TCBP-25 gene using the antisense ribosome system, and we analyzed the phenotype of the transformants. The knock down of TCBP-25 function also suggests that TCBP-25 plays a role in the pronuclear exchange and in the maintenance of cell shape.

Tuftelin: enamel mineralization and amelogenesis imperfecta.

Tuftelin is a novel acidic enamel protein thought to play a major role in enamel mineralization. Its identity and localization has been confirmed by amino acid composition, enzyme-linked immunosorbant assay, Western blots, indirect immunohistochemistry and high resolution protein-A gold immunocytochemistry. The deduced tuftelin protein (pI 5.2) contains 389 amino acids and has a calculated peptide molecular mass of 43,814 Da. Immunological studies suggest conservation of tuftelin structure between species throughout vertebrate evolution. The cDNA sequence encodes for several putative post-translation sites including one N-glycosylation consensus site, seven O-glycosylation sites and seven phosphorylation sites, as well as an EF-hand calcium-binding domain (with mismatch), localized towards the N-terminal region. At the C-terminal region (residues 252-345) tuftelin contains structurally relevant determinants for self assembly. We recently cloned and partially sequenced the human tuftelin gene (four exons have now been sequenced). These sequences include exon 1 and over 1000 bases of the putative promoter region. Employing fluorescent in situ hybridization, we mapped the human tuftelin gene to chromosome 1q 21-31. Localization of the human tuftelin gene to a well-defined cytogenetic region may be important in understanding the aetiology of autosomally inherited amelogenesis imperfecta, the most common enamel hereditary disease.

Transient Receptor Potential Channel A1 Is Directly Gated by Calcium Ions

Members of the superfamily of transient receptor potential (TRP) channels are proposed to play important roles in sensory physiology. As an excitatory ion channel TRPA1 is robustly activated by pungent irritants in mustard and garlic and is suggested to mediate the inflammatory actions of environmental irritants and proalgesic agents. Here, we demonstrate that, in addition to pungent natural compounds, Ca(2+) directly gates heterologously expressed TRPA1 in whole-cell and excised-patch recordings with an apparent EC(50) of 905 nm. Pharmacological experiments and site-directed mutagenesis indicate that the N-terminal EF-hand calcium-binding domain of the channel is involved in Ca(2+)-dependent activation. Furthermore, we determine Ca(2+) as prerequisite for icilin activity on TRPA1.

Identification and Characterization of a Biomineralization Related Gene PFMG1 Highly Expressed in the Mantle of Pinctada fucata

To elucidate the mechanism of nacre biomineralization, the mantle of Pinctada fucata (P. fucata) from the South China Sea was used. Using the mantle cDNA library and the ESTs we have cloned through suppression subtractive hybridization (SSH), ten novel genes including PFMG1 were obtained through nested PCR. Bioinformative results showed that PFMG1 had a high homology (40%) with Onchocerca volvulus calcium-binding protein CBP-1 and had two EF-hand calcium-binding domains from the 81st to the 93rd amino acid and from the 98th to the 133rd amino acid in the deduced amino acid sequence. The results of multitissue RT-PCR and in situ hybridization demonstrated the high expression of PFMG1 in the mantle of P. fucata and confirmed the SSH method. The results of GST-PFMG1 on CaCO3 crystallization showed significant effects on nucleation and precipitation of CaCO3. PFMG1 was cloned into the pcDNA.3.1/myc-HisA vector and was subsequently transfected into MC3T3-E1 cells. RT-PCR revealed upregulation of the marker genes related to cell growth, differentiation, and mineralization, and BMP-2, osterix, and osteopontin were upregulated as a result. This research work suggests that PFMG1 plays an important role in the nacre biomineralization, and the SSH method can pave the way for the bulk cloning and characterization of new genes involved in biomineralization in P. fucata and may accelerate research on the mechanism of pearl formation.

Characterization of a novel EF-hand homologue, CnidEF, in the sea anemone Anthopleura elegantissima

The superfamily of EF-hand proteins is comprised of a large and diverse group of proteins that contain one or more characteristic EF-hand calcium-binding domains. This study describes and characterizes a novel EF-hand cDNA, CnidEF, from the sea anemone Anthopleura elegantissima (Phylum Cnidaria, Class Anthozoa). CnidEF was found to contain two EF-hand motifs near the C-terminus of the deduced amino acid sequence and two regions near the N-terminus that could represent degenerate EF-hand motifs. CnidEF homologues were also identified from two other sea anemone species. A combination of bioinformatic and molecular phylogenetic analyses was used to compare CnidEF to EF-hand proteins in other organisms. The closest homologues identified from these analyses were a luciferin binding protein (LBP) involved in the bioluminescence of the anthozoan Renilla reniformis, and a sarcoplasmic calcium-binding protein (SARC) involved in fluorescence of the annelid worm Nereis diversicolor. Predicted structure and folding analysis revealed a close association with bioluminescent aequorin (AEQ) proteins from the hydrozoan cnidarian Aequorea aequorea. Neighbor-joining analyses grouped CnidEF within the SARC lineage along with AEQ and other cnidarian bioluminescent proteins rather than in the lineage containing calmodulin (CAM) and troponin-C (TNC).

Bla g 6: A troponin C allergen from Blattella germanica with IgE binding calcium dependence

The known cockroach allergens do not appear to account for the full repertoire of IgE responses. To identify and investigate the importance of other Blattella germanica allergens contributing to cockroach allergy. A B germanica cDNA library was screened with pooled sera from patients with cockroach allergy. Three isoallergens of troponin C (Bla g 6) were cloned and expressed in Pichia pastoris. Homology modeling was performed by using Swiss-Model. IgE responses to purified allergens were simultaneously measured in 104 sera by using a fluorescent multiplex array system. The effect of calcium on IgE binding was investigated by ELISA. Three isoallergens, Bla g 6.0101, Bla g 6.0201, and Bla g 6.0301, were identified which share homology with insect troponin Cs and vertebrate calmodulins (61% to 78% and 42% to 44% amino acid identity, respectively) and have 2 EF-hand calcium binding domains. Molecular models of Bla g 6 showed 2 structurally homologous lobes connected by a linker that confers flexibility to the allergen. The prevalence of IgE binding to recombinant Bla g 6 was 14%. Calcium depletion by 10 mmol/L ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraacetic acid did not significantly affect IgE binding in most cases. Interestingly, addition of 10 mmol/L CaCl2 after calcium depletion increased IgE binding by approximately 2-fold, a finding not previously reported for calcium binding allergens. Bla g 6 is a troponin allergen with a calcium dependent IgE reactivity that may be involved in muscle contraction. Bla g 6 homologous allergens may occur among other insects and cause cosensitization or allergenic cross-reactivity.

The calcium‐binding domain of the stress protein SEP53 is required for survival in response to deoxycholic acid‐mediated injury

Stress protein responses have evolved in part as a mechanism to protect cells from the toxic effects of environmental damaging agents. Oesophageal squamous epithelial cells have evolved an atypical stress response that results in the synthesis of a 53 kDa protein of undefined function named squamous epithelial-induced stress protein of 53 kDa (SEP53). Given the role of deoxycholic acid (DCA) as a potential damaging agent in squamous epithelium, we developed assays measuring the effects of DCA on SEP53-mediated responses to damage. To achieve this, we cloned the human SEP53 gene, developed a panel of monoclonal antibodies to the protein, and showed that SEP53 expression is predominantly confined to squamous epithelium. Clonogenic assays were used to show that SEP53 can function as a survival factor in mammalian cell lines, can attenuate DCA-induced apoptotic cell death, and can attenuate DCA-mediated increases in intracellular free calcium. Deletion of the highly conserved EF-hand calcium-binding domain in SEP53 neutralizes the colony survival activity of the protein, neutralizes the protective effects of SEP53 after DCA exposure, and permits calcium elevation in response to DCA challenge. These data indicate that the squamous cell-stress protein SEP53 can function as a modifier of the DCA-mediated calcium influx and identify a novel survival pathway whose study may shed light on mechanisms relating to squamous cell injury and associated cancer development.

A Flagellum-specific Calcium Sensor

The flagellar calcium-binding protein (FCaBP) of the flagellated protozoan Trypanosoma cruzi associates with the flagellar membrane via its N-terminal myristate and palmitate moieties in a calcium-modulated, conformation-dependent manner. This mechanism of localization is similar to that described for neuronal calcium sensors, which undergo calcium-dependent changes in conformation, which modulate the availability of the acyl groups for membrane interaction and partner association. To test whether FCaBP undergoes a calcium-dependent conformational change and to explore the role of such a change in flagellar targeting, we first introduced point mutations into each of the two EF-hand calcium-binding sites of FCaBP to define their affinities. Analysis of recombinant EF-3 mutant (E151Q), EF-4 mutant (E188Q), and double mutant proteins showed EF-3 to be the high affinity site (Kd approximately 9 microM) and EF-4 the low affinity site (Kd approximately 120 microM). These assignments also correlated with partial (E188Q), nearly complete (E151Q), and complete (E151Q,E188Q) disruption of calcium-induced conformational changes determined by NMR spectrometry. We next expressed the FCaBP E151Q mutant and the double mutant in T. cruzi epimastigotes. These transproteins localized to the flagellum, suggesting the existence of a calcium-dependent interaction of FCaBP that is independent of its intrinsic calcium binding capacity. Several proteins were identified by FCaBP affinity chromatography that interact with FCaBP in a calcium-dependent manner, but with differential dependence on calcium-binding by FCaBP. These findings may have broader implications for the calcium acyl switch mechanism of protein regulation.

Conservation of novel Mahya genes shows the existence of neural functions common between Hymenoptera and Deuterostome

Honeybees have been shown to exhibit cognitive performances that were thought to be specific to some vertebrates. However, the molecular and cellular mechanisms of such cognitive abilities of the bees have not been understood. We have identified a novel gene, Mahya, expressed in the brain of the honeybee, Apis mellifera, and other Hymenoptera. Mahya orthologues are present in Deuterostomes but are absent or highly diverged in nematodes and, intriguingly, in two dipteran insects (fruit fly and mosquito) and Lepidoptera (silk moth). Mahya genes encode novel secretory proteins with a follistatin-like domain (Kazal-type serine/threonine protease inhibitor domain and EF-hand calcium-binding domain), two immunoglobulin domains, and a C-terminal novel domain. Honeybee Mahya is expressed in the mushroom bodies and antennal lobes of the brain. Zebra fish Mahya orthologues are expressed in the olfactory bulb, telencephalon, habenula, optic tectum, and cerebellum of the brain. Mouse Mahya orthologues are expressed in the olfactory bulb, hippocampus, and cerebellum of the brain. These results suggest that Mahya may be involved in learning and memory and in processing of sensory information in Hymenoptera and vertebrates. Furthermore, the limited existence of Mahya in the genomes of Hymenoptera and Deuterostomes supports the hypothesis that the genes typically represented by Mahya were lost or highly diverged during the evolution of the central nervous system of specific Bilaterian branches under the specific selection and subsequent adaptation associated with different ecologies and life histories.

The Three-dimensional Solution Structure of Ca 2+-bound S100A1 as Determined by NMR Spectroscopy

S100A1 is an EF-hand-containing Ca(2+)-binding protein that undergoes a conformational change upon binding calcium as is necessary to interact with protein targets and initiate a biological response. To better understand how calcium influences the structure and function of S100A1, the three-dimensional structure of calcium-bound S100A1 was determined by multidimensional NMR spectroscopy and compared to the previously determined structure of apo. In total, 3354 nuclear Overhauser effect-derived distance constraints, 240 dihedral constraints, 160 hydrogen bond constraints, and 362 residual dipolar coupling restraints derived from a series of two-dimensional, three-dimensional, and four-dimensional NMR experiments were used in its structure determination (>21 constraints per residue). As with other dimeric S100 proteins, S100A1 is a symmetric homodimer with helices 1, 1', 4, and 4' associating into an X-type four-helix bundle at the dimer interface. Within each subunit there are four alpha-helices and a short antiparallel beta-sheet typical of two helix-loop-helix EF-hand calcium-binding domains. The addition of calcium did not change the interhelical angle of helices 1 and 2 in the pseudo EF-hand significantly; however, there was a large reorientation of helix 3 in the typical EF-hand. The large conformational change exposes a hydrophobic cleft, defined by residues in the hinge region, the C terminus, and regions of helix 3, which are important for the interaction between S100A1 and a peptide (TRTK-12) derived from the actin-capping protein CapZ.

Ontogenetic and Phylogenetic Analysis of Myosin Light Chain Proteins from Skeletal Muscles of Loach Misgurnus fossilis

mRNAs of all three types of myosin light chain proteins are expressed in skeletal muscles of both larval and adult stages of loach Misgurnus fossilis (Cobitidae) and these proteins are encoded by different genes (mlc1, mlc2, and mlc3). No difference was revealed between transcripts from larval stage and adult fish for all three mlc proteins. Our approach (RT-PCR with fish-specific mlc1, mlc2, and mlc3 primers) failed to reveal the larval form of myosin light chain protein found previously by protein electrophoresis of loach fry muscle extract. Comparative analysis of the protein structure shows high homology of MLC1 and MLC3 proteins sharing a large EF-hand calcium-binding domain. Phylogenetic analysis of MLC1 from skeletal muscles of fish and other vertebrate species is concordant with the traditional phylogeny of the group. Within the Teleostei, loach MLC1 had the highest homology with other Cyprinidae, and least with Salmonidae fishes.

Structural Characterization of a Novel Cbl Phosphotyrosine Recognition Motif in the APS Family of Adapter Proteins

The Cbl adapter proteins typically function to down-regulate activated protein tyrosine kinases and other signaling proteins by coupling them to the ubiquitination machinery for degradation by the proteasome. Cbl proteins bind to specific tyrosine-phosphorylated sequences in target proteins via the tyrosine kinase-binding (TKB) domain, which comprises a four-helix bundle, an EF-hand calcium-binding domain, and a non-conventional Src homology-2 domain. The previously derived consensus sequence for phosphotyrosine recognition by the Cbl TKB domain is NXpY(S/T)XXP (X denotes lesser residue preference), wherein specificity is conferred primarily by residues C-terminal to the phosphotyrosine. Cbl is recruited to and phosphorylated by the insulin receptor in adipose cells through the adapter protein APS. APS is phosphorylated by the insulin receptor on a C-terminal tyrosine residue, which then serves as a binding site for the Cbl TKB domain. Using x-ray crystallography, site-directed mutagenesis, and calorimetric studies, we have characterized the interaction between the Cbl TKB domain and the Cbl recruitment site in APS, which contains a sequence motif, RA(V/I)XNQpY(S/T), that is conserved in the related adapter proteins SH2-B and Lnk. These studies reveal a novel mode of phosphopeptide interaction with the Cbl TKB domain, in which N-terminal residues distal to the phosphotyrosine directly contact residues of the four-helix bundle of the TKB domain.

The ability of AIF-1 to activate human vascular smooth muscle cells is lost by mutations in the EF-hand calcium-binding region

Allograft Inflammatory Factor-1 (AIF-1) is a cytoplasmic calcium-binding protein expressed in vascular smooth muscle cells (VSMC) in response to injury or cytokine stimulation. AIF-1 contains a partially conserved EF-hand calcium-binding domain, and participates in VSMC activation by activation of Rac1 and induction of Granulocyte-Colony Stimulating Factor (G-CSF) expression; however, the mechanism whereby AIF-1 mediates these effects is presently uncharacterized. To determine if calcium binding plays a functional role in AIF-1 activity, a single site-specific mutation was made in the EF-hand calcium-binding domain to abrogate binding of calcium (AIF-1ΔA), which was confirmed by calcium overlay. Functionally, similar to wild-type AIF-1, AIF-1ΔA was able to polymerize F-actin in vitro. However, in contrast to wild-type AIF-1, over-expression of AIF-1ΔA was unable to increase migration or proliferation of primary human VSMC. Further, it was unable to activate Rac1, or induce G-CSF expression to the degree as wild-type AIF-1. Taken together, modification of the wild-type EF-hand domain and native calcium-binding activity results in a loss of AIF-1 function. We conclude that appropriate calcium-binding potential is critical in AIF-1-mediated effects on VSMC pathophysiology, and that AIF-1 activity is mediated by Rac1 activation and G-CSF expression.

Solution Structure of Zinc- and Calcium-Bound Rat S100B as Determined by Nuclear Magnetic Resonance Spectroscopy,

The EF-hand calcium-binding protein S100B also binds one zinc ion per subunit with a relatively high affinity (K(d) approximately 90 nM) [Wilder et al., (2003) Biochemistry 42, 13410-13421]. In this study, the structural characterization of zinc binding to calcium-loaded S100B was examined using high-resolution NMR techniques, including structural characterization of this complex in solution at atomic resolution. As with other S100 protein structures, the quaternary structure of Zn(2+)-Ca(2+)-bound S100B was found to be dimeric with helices H1, H1', H4, and H4' forming an X-type four-helix bundle at the dimer interface. NMR data together with mutational analyses are consistent with Zn(2+) coordination arising from His-15 and His-25 of one S100B subunit and from His-85 and Glu-89 of the other subunit. The addition of Zn(2+) was also found to extend helices H4 and H4' three to four residues similar to what was previously observed with the binding of target proteins to S100B. Furthermore, a kink in helix 4 was observed in Zn(2+)-Ca(2+)-bound S100B that is not in Ca(2+)-bound S100B. These structural changes upon Zn(2+)-binding could explain the 5-fold increase in affinity that Zn(2+)-Ca(2+)-bound S100B has for peptide targets such as the TRTK peptide versus Ca(2+)-bound S100B. There are also changes in the relative positioning of the two EF-hand calcium-binding domains and the respective helices comprising these EF-hands. Changes in conformation such as these could contribute to the order of magnitude higher affinity that S100B has for calcium in the presence of Zn(2+).

Characterization of a human regulatory subunit of protein phosphatase 3 gene (PPP3RL) expressed specifically in testis

Protein phosphatase 3 (PPP3, formerly PP2B, Calcineurin), a serine/threonine protein phosphatase, is a heterodimer composed of one catalytic subunit (PPP3C, Calcineurin A) and one regulatory subunit (PPP3R, Calcineurin B). PPP3R, an EF-hand Ca2+ binding protein, contains four high-affinity EF-hand calcium-binding sites, indicating that PPP3 plays critical roles in many calcium-mediated signal transduction pathways. PPP3R has two isoforms, PPP3R1 (also known as PP2Bbeta1) and PPP3R2 (also known as PP2BB2). While PPP3R1 is ubiquitously expressed in different tissues, PPP3R2 is exclusively expressed in testis. PPP3R2 has only been identified in rat and mouse. Here we report a human homologue of PPP3R2, which is designated PPP3RL (PPP3R like protein). PPP3RL gene was predicated to encode 171 amino acid residues with four EF-hand calcium-binding domains and this putative protein shares 82.9% and 80.5% identity with PPP3R2 of rat and mouse, respectively. Our results show that PPP3RL gene localizes to human chromosome 9q22 and transcripts of PPP3RL gene are specifically expressed in the testis, moreover, this tissue-specific expression is due to demethylation of its promoter region in testis.

Cellular expression and alternative splicing of SLC25A23, a member of the mitochondrial Ca 2+-dependent solute carrier gene family

The transport of metabolites across the inner mitochondrial membrane is mediated by a large superfamily of mitochondrial solute carrier (MSC) proteins. A novel human member of the MSC gene family named SLC25A23, with homologs in mammalian and non-mammalian species has been recently identified together with two close paralogs, SLC25A24 and SLC25A25. These genes encode the human isoforms of the ATP-Mg/Pi carrier described in whole mitochondria. We report here the cellular expression and alternative splicing of SLC25A23. The gene encodes a 468 amino acids polypeptide, named SCaMC-3, with a bipartite structure typical of calcium-binding mitochondrial solute carrier (CaMSC) proteins. The amino-terminal portion harbors three canonical EF-hand calcium-binding domains while the carboxyl-terminal portion of SCaMC-3 has the characteristic features of the MSC superfamily. Northern blot analysis reveals the presence of the transcript in brain, heart, skeletal muscle, liver and small intestine. The SLC25A23 gene undergoes alternative splicing suggesting a modular nature of the encoded product. Three out of four putative protein isoforms lack a significant portion of the third mitochondrial carrier signature. The most common SCaMC-3 isoform shows a mitochondrial subcellular localization when transfected in HeLa cells and is able to bind calcium by Ca 2+-dependent mobility shift assays. We believe that our study will contribute to a better knowledge of this family of mitochondrial carriers.

Structural and functional analyses of phosphoglucose isomerase from Vibrio vulnificus and its lysyl aminopeptidase activity

Phosphoglucose isomerase (PGI) with a novel lysyl aminopeptidase (LysAP) activity was recently isolated and partially characterized from the human pathogen, Vibrio vulnificus. This PGI is a heterodimer consisting of 60.8- and 23.4-kDa subunits, which together provide LysAP activity. The present study further characterizes the complex structure and functions of Vibrio PGI and draws parallels with rabbit and human PGI. A Proscan search of Vibrio PGI revealed 194 different structural motifs of which 124 and 127 were also found in rabbit and human PGI, respectively. Vibrio PGI contains motifs for the serine, histidine and aspartic acid active sites of the subtilase family of serine proteases which form a putative catalytic triad consisting of His534 and Ser159 on the 60.8-kDa subunit and Asp53 on the 23.4-kDa subunit. Together, they form one LysAP site for each heterodimer. Each active site motif is overlapped by motifs for EF-hand calcium binding domains. The LysAP activity was inhibited by the addition of ≥10 μM Ca 2+, suggesting that the EF-hand calcium-binding domain may be a natural regulatory region for LysAP activity. In contrast, PGI's isomerase activity was enhanced at Ca 2+ concentrations >100 μM. PGI-LysAP cleaved the amino-terminal lysyl residue from des-Arg 10-kallidin producing des-Arg 9-bradykinin; therefore, Vibrio PGI-LysAP may serve as a virulence factor to enhance Vibrio invasiveness. Together, these data provide a framework to account for PGI's LysAP activity and further demonstrate the structural complexity and functional importance of this molecule.

CDNA-based comparative genomic hybridization (cCGH) analysis of genomic aberrations of pancreatic cancer cells

4122 Introduction: Pancreatic carcinoma (PanCa) is the fourth leading cause of cancer mortality in US with a 5-year survival rate of 5%. urrent treatments have only a minimal survival benefit. In this study, we are using cDNA-based comparative genomic hybridization (cCGH) technologies to search for common genomic alternations in human PanCa cell lines. Methods: Genomic DNA from seven human PanCa cell lines was extracted and labeled with Cyanine 3-dUTP. Normal human genomic DNA isolated from leukocytes of healthy male and female volunteers was labeled with Cyanine 5-dUTP. The labeled probe mixtures were then hybridized with 10K (5K in replicates) cDNA slides prepared by the UT MD Anderson Cancer Genomics Core Laboratory. The gain or loss of individual genes after background subtraction and appropriate normalization. The identified genomic alternations were then verified by florescent in situ hybridization (FISH) analysis in pancreatic cells and biopsy tissues with human BAC clone DNA and the centromere controls. Results: We successfully performed cCGH analysis using 7 pancreatic cancer cell lines. Our data revealed that human hepatocyte nuclear factor-6 (HNF6), telomeric repeat binding factor 2 (TRF2), reticulocalbin 2 EF-hand calcium binding domain (RCN2), and bromodomain testis-specific (BRDT) were amplified by more than 6 copies and fucose-1-phosphate guanyltransferase (FPGT) lost two endogenous copies in 43 to 71% of the PanCa cell lines studied (see the attached table). The genomic amplifications of HNF6 and TRF2 have been validated by FISH assays. We are validating FISH analysis for RCN3, BRDT and FPGT and with PanCa tissues from primary and metastatic tumors. Conclusions: We identified 4 genes with 6+fold gain and one gene with complete loss in PanCa cell lines. Two of these genes have been validated by the FISH analysis. Using similar analyses with patient tissues, we are searching for predictive and prognostic genomic markers and new therapeutic targets for improving PanCa survival (Supported by a pilot study grant and a T32 training grant from UT MD Anderson Cancer Center and NCI). Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration M.D. Anderson Cancer Center; National Cancer Institute