In our lab, we use biochemical, cell biological, genetic, and molecular approaches in conjunction with the yeast system to better understand the function of proteases that act on isoprenylated proteins. Examples of isoprenylated proteins include the Ras family of oncoproteins, Ras-related proteins, kinases, and secreted fungal mating pheromones, among many others. Understanding the function of these proteases may lead to novel therapeutic strategies for cancer, Alzheimer's disease, and other diseases.
The CaaX Proteases: Rce1p is an ER membrane-localized protease of unknown mechanism. This protease is essential for the maturation of isoprenylated molecules that are involved in cellular transformation (e.g., Ras and RhoB). We aim to understand the proteolytic mechanism of Rce1p and to develop pharmacological inhibitors that have anti-tumor potential. Rce1p has partial overlapping function with the ER membrane-localized, the zinc-dependent Ste24p protease, which has been linked to premature aging (progeria) because of its role in lamin A production. Thus, we are also trying to understand the functional differences of Rce1p and Ste24p to better understand their relative physiological importance.
The M16A Proteases: Ste23p and Axl1p are zinc-dependent metalloproteases that are required for the maturation of the isoprenylated yeast a-factor mating pheromone. These proteases are part of the M16A subfamily of metalloproteases, which includes the insulin-degrading enzyme (IDE) that has a proposed protective function in Alzheimer's disease (AD). Our research on Ste23p and Axl1p is designed is to gain a better understanding of these largely uncharacterized yeast proteases and the M16 metalloprotease family as a whole, thus potentially providing novel insight into new methods for the treatment of AD.
Molecular and biochemical analyses of proteases required for the maturation of isoprenylated signaling molecules
Kelly K, Manandhar SP, Mullen, D, Schmidt, WK, and Distefano, MD Photoaffinity labeling of Ras converting enzyme 1 (Rce1p) using a benzophenone-containing peptide substrate. Bioorganic & Medicinal Chemistry (submitted)
Manandhar SP, Hildebrandt ER, Jacobsen WH, Santangelo GM, and Schmidt WK Chemical inhibition of CaaX protease activity disrupts yeast Ras localization.Yeast (in press)
Mokry DZ, Manandhar SP, Chicola KA,, Santangelo GM, and Schmidt WK Heterologous expression studies in yeast reveal two distinct trypanosomatid CaaX protease activities and identifies their potential targets. Eukaryotic Cell (2009), 8(12):1891-1900.
Alper BJ, Rowse JW, and Schmidt WK Yeast Ste23p shares functional similarities with mammalian insulin-degrading enzymes. Yeast (2009), 26(11):595-610.
Krishnankutty RK, Kukday SS, Castleberry AJ, Breevoort SR, and Schmidt WK
Proteolytic processing of certain CaaX motifs can occur in the absence of the Rce1p and Ste24p CaaX proteases Yeast (2009), 26(8):451-463.
Alper BJ and Schmidt WK A capillary electrophoresis method for evaluation of Abeta proteolysis in vitro. J Neurosci Methods (2009), 178:40-45.
Manandhar SP, Hildebrandt ER, and Schmidt WK Small molecule inhibitors of the Rce1p CaaX protease. J Biomolecular Screening (2007),12(7):883-893.
Porter SB, Hildebrandt ER, Breevoort SR, Dore TM, and Schmidt WK Inhibition of the CaaX proteases Rce1p and Ste24p by peptidyl (acyloxy)methyl ketones. Biochim Biophys Acta (2007), 1773(6):853-62.