Biomedical subjects

Carrie Paquette-Straub

Publications and source records attributed to Carrie Paquette-Straub.

4 recordsLinked to original sources

PubMed2005

Scratching below the surface: wound healing and alanine mutagenesis provide unique insights into interactions between eristostatin, platelets and melanoma cells.

To study the molecular mechanism of the disintegrin eristostatin, cellular functional studies were performed using ten recombinant alanine mutants. ADP-induced platelet aggregation revealed critical contributions of seven residues within the 'RGD loop' (R24, R27, G28, N31) and C-terminus (W47, N48, G49) of this disintegrin. Using an in vitro scratch wound healing assay, four human melanoma cell lines yielded similar results when exposed to wildtype eristostatin. All eristostatin-treated cells healed less of the wounded area than control conditions. This phenomenon was reproduced when using fibronectin as the matrix. C8161 cells showed significant delay in wound closure with the N-terminal mutant P4A but not with R24A or G28A. Evidence from our laboratory and others suggests neither alpha IIb, alpha 4 nor alpha 5 integrins are directly involved in eristostatin's interactions. Eristostatin did not affect the number of melanoma cells in culture after 24 h or the development of apoptosis. However, phosphorylation studies performed after these melanoma cells were exposed to eristostatin revealed changes in several tyrosine phosphorylated molecules.

Alanine↗

PubMed2004

Disintegrins.

The existence of disintegrins, non-enzymatic, small molecular weight proteins from viper venom, has been known for 2 decades, and their impact on cellular research has been substantial and far-reaching. Disintegrins have been the molecular scaffold used in the design of therapeutics for the prevention of thrombosis and cancer. Their sequencing has provided insights into the evolution of proteins over millennia. Production of recombinant disintegrin mutants and fusion proteins has allowed investigations into molecular mechanisms at work in cell-extracellular matrix interactions. Structural homologies with non-snake proteins have shown disintegrin-like molecules in species ranging from slime mold to humans. Intracellular signaling events have been elucidated through the use of venom disintegrins, including events related to programmed cell death, motility, cell proliferation and viral pathogenesis. Disintegrin sequences (protein or genes) have been placed in microbubbles and liposomes and been found to target neovascular endothelium and metastatic tumors in two mouse models. The purpose of this review is to highlight the members of this disintegrin family discovered since 1998 as well as the increased understanding of their usefulness in therapeutics and technical assays.

Amino Acid Sequence↗

PubMed2003

Melanoma metastasis suppression by chromosome 6: evidence for a pathway regulated by CRSP3 and TXNIP.

Loss of genetic material on chromosome 6 has been associated with progression of human melanomas. We showed previously that introducing chromosome 6 into metastatic human melanoma cell lines suppresses metastasis without affecting the ability of the hybrids to form progressively growing tumors. By subtractive hybridization comparing nonmetastatic chromosome 6-containing (neo6/C8161) versus parental (C8161) metastatic cells, the KISS1 metastasis suppressor gene was isolated. However, KISS1 mapped to chromosome 1q32. To identify upstream regulator(s) of (and downstream effectors of) KISS1, microarray hybridization comparing C8161 and neo6/C8161 variants was performed. TXNIP/VDUP1, a thioredoxin-binding protein, was expressed more highly in neo6/C8161 and in nonmetastatic melanomas. Increased TXNIP expression inhibited metastasis and up-regulated KISS1. Surprisingly, TXNIP also mapped to chromosome 1q. PCR karyotyping that refined the region on chromosome 6 identified CRSP3/DRIP130, a transcriptional coactivator, as a metastasis suppressor. CRSP3 transfectant cells had up-regulated KISS1 and TXNIP expression and were suppressed for metastasis. Quantitative real-time reverse-transcription PCR of clinical melanoma samples showed that loss of CRSP3 expression correlated with decreased KISS1 expression and increased metastasis. Thus, we implicated a specific gene on chromosome 6 in the etiology of melanoma metastasis and identified potential up-stream regulators of KISS1 and TXNIP.

Animals↗

PubMed2003

Cloning, expression, and characterization of a bi-functional disintegrin/alkaline phosphatase hybrid protein.

Integrins are transmembrane heterodimeric glycoproteins responsible for cellular communication; therefore, they play an essential role in many physiological events. Viper snake venoms contain integrin antagonists called disintegrins which bind and inhibit integrin function. They present a loop containing an RGD motif responsible for integrin binding. The engineering of disintegrins fused to a reporter enzyme will be an interesting approach to build integrin markers. Even more, the disintegrin scaffold could be used to present other protein binding motifs. In this work, we have obtained alkaline phosphatase (APv) tagged eristostatin (Er) by cloning and expressing eristostatin DNA into the pLIP6-GN vector. Eristostatin, a 49 residue disintegrin, binds selectively to alphaIIbbeta3 integrin, inhibiting its binding to fibrinogen. The resulting fusion protein Er/APv was identified by SDS-PAGE and by Western blotting using both anti-Er and anti-AP antibodies. This fusion protein showed enzymatic AP activity similar to that of wild APv and its potential use for an alphaIIbbeta3 integrin assay was tested in a one-step dot blot using immobilized cells incubated with the marker and developed by AP substrate. Er/APv showed selectivity towards platelets and alphaIIbbeta3 integrin transfected cells and reacted with the same region as unlabeled Er, as analyzed in competition assays. Our data present a novel tool, Er/APv, with potential use as molecular marker in processes where the alphaIIbbeta3 integrin is involved.

Alkaline Phosphatase↗