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DIII Domain of Calpain 10 and Cpl Towards an Understanding of Calpain 10 Function

Huang, Xinhua
http://rave.ohiolink.edu/etdc/view?acc_num=mco1096641027

Abstract Details

2004, Doctor of Philosophy in Medical Sciences (Ph.D.), University of Toledo, Graduate School.
Calpains are intracellular, cysteine proteases found in vertebrates and some lower organisms. There is evidence that they are important mediators of cell adhesion and motility in animal cells. While there is evidence that the single nucleotide polymorphism in the non-coding region of the calpain 10 gene is associated with type 2 diabetes, little cellular and biochemical information is currently available on calpain 10. Because the cellular slime mold Dictyostelium discoideum is a genetically tractable model for cellular studies, in this organism we have investigated aa calpain-like protein which may provide useful clues towards an understanding of the function of mammalian calpains. Contig 13130 (Sanger Institute Dictyostelium sequencing project) was identified as a 3-exon gene that encodes a calpain-like protein. Using a custom peptide antibody to assay for the presence of this putative protein, we identified Dictyostelium calpain-like protein (Cpl) and purified it to near homogeneity. Cp1 is a 72,278 Da cytosolic protein. Weak caseinolytic activity inhibitable by cysteine protease inhibitors was co-purified with Cp1 immunoreactivity, and purified Cp1 appears to undergo autoproteolysis when transferred to inhibitor-free buffer. The major cleavage after Pro189 generated a 51,291 Ca Cp1 fragment. Cp1 domain structure resembles mammalian calpain 10 with a putative N-terminal catalytic domain followed by tandem calpain D-III domains. The knockout of Cp1 in Dictyostelium showed similar phenotypes as ablation of calpain 1 and calpain 2 in mammalian cells. Yeast two-hybrid screening of a human fetal liver library by using Ga14Bd-calpain DIIIX2 as bait revealed several positive clones, including flotillin-1. The proteins expressed by these clones specifically interacted with calpain 10 DIIIX2 when tested with bait constructs comprising DIII domains from different calpains. Among the interacting proteins identified, flotillin-1 may be a promising subject for the relationship between calpain-10 and type 2 diabetes. Flotillin-1, and its binding proteins, CAP and cbl, are involved in insulin-induced glucose transport. It is possible that calpain-10 mediates the insulin-induced glucose transport pathway via its interaction with flotillin-1. Further confirmation of protein-protein interaction by co-immunoprecipitation and cellular function studies of the positive clones are under way.
Ronald Mellgren, Ph.D. (Advisor)
126 p.
Cpl; Calpain 10; DIIIX2 domain; yeast two-hybrid

Recommended Citations

Citations

  • Huang, X. (2004). DIII Domain of Calpain 10 and Cpl Towards an Understanding of Calpain 10 Function [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1096641027

    APA Style (7th edition)

  • Huang, Xinhua. DIII Domain of Calpain 10 and Cpl Towards an Understanding of Calpain 10 Function. 2004. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1096641027.

    MLA Style (8th edition)

  • Huang, Xinhua. "DIII Domain of Calpain 10 and Cpl Towards an Understanding of Calpain 10 Function." Doctoral dissertation, University of Toledo, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1096641027

    Chicago Manual of Style (17th edition)

mco1096641027
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© 2004, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.