Date of Award

6-2019

Document Type

Restricted (Opt-Out)

Degree Name

Bachelor of Science

Department

Biochemistry

First Advisor

Kristin M Fox

Keywords

Substrate specificity, metacaspases, Schizophyllum commune, kinetic characterization, enzymes, AMC

Abstract

Metacaspases are cysteine-dependent enzymes that belong to the same peptidase family as caspases, which are essential regulatory proteins involved in metazoan apoptosis, the programmed cell death that occurs in animals. Though distantly related, metacaspases share many characteristics with caspases and thus are believed to be involved in the pathways of programmed cell death in fungi, protozoa, and plants. However, even though metacaspases play a similar role in apoptosis, they likely perform it through different means. Unlike caspases which show aspartate specificity, metacaspases exhibit a preference to cleave after arginine or lysine amino acid residues.

In our research, we study metacaspases found in the fungus Schizophyllum commune. Currently, we have identified and purified five Type 1 S. commune metacaspases (ScMC1-5) with and without the prodomain (∆pro). Preliminary studies carried out with a limited number of substrates revealed that ScMC1-5∆pro exhibit arginine/lysine specificity and do not cleave after aspartate but provided limited information about the amino acid preference at different binding pocket positions. To investigate the importance of substrate amino acid sequence for substrate specificity, we performed in-vitro fluorescent activity assays comparing how ScMC1- 5∆pro cleave various synthetic peptide substrates. Specifically, we examined the effect of amino acid size, charge and polarity on peptide cleavage.

ScMC1-5∆pro demonstrate a preference to cleave more readily after arginine than lysine. However, the detailed substrate specificity profiles vary among these metacaspases. For example, we observed ScMC1∆pro and ScMC4∆pro recognize a wide variety of amino acid residues. Peptides with small non-polar amino acids like glycine, proline, valine, and alanine showed good relative activities, but the highest activity was toward a substrate with the small polar threonine. On the other hand, ScMC2,3, and 5∆pro did not recognize most of those amino acid residues and the enzymes were inactive toward those substrates. They only showed some relative activity toward the large non-polar amino acid phenylalanine, the polar threonine, and had the highest activity toward the small non-polar glycine. Studying these small differences in substrate specificity can be relevant for determining the types of proteins that metacaspases cleave in vivo and for understanding their individual, physiological functions.

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