Intramembrane-cleaving proteases are a special class of proteins which cleave peptide bonds inside or at the surface of the membrane. These proteins are important to the regulation of a wide range of biological processes such as transcriptional regulation, coordination of cell division, immune response, and cholesterol metabolism. Defects in intramembrane proteases are associated with diseases such as Alzheimer’s, Progeria, Parkinson’s, diabetes, and cancer.
Examples of intramembrane proteases include:
- Site-2 protease (S2P)
- CAAX proteases
M. Joanne Lemieux and her colleagues at the University of Alberta, Membrane Protein Disease Research Group studied the Rhomboid family of intramembrane serine proteases(1). The family is found in all levels of life and plays essential roles in diverse signaling events ranging from epidermal growth factor release in eukaryotes to facilitating invasion in parasites. In particular, the team’s work concentrated on AarA cleavage of psTatA.
This particular enzyme–substrate pair was selected for allosteric regulation examination because psTatA is the only identified substrate for any prokaryotic rhomboid. AarA in its active form is a dimer in the membrane. The process of substrate binding is achieved through a multi-step dynamic process:
1. Dimerization - allows for the formation of the AarA exosite
The dimerization only forms the exosite. The active site is already formed in the monomeric form.
2. Recognition - the transmembrane segment of psTatA binds to the exosite
3. Entry - binding the exosite allows for lateral entry into the AarA active site
4. Cleavage - allosteric changes to the AarA active site allow for optimal positioning and cleavage of psTatA
The above-mentioned allosteric model would also provide an elegant mechanism for enforcing specificity during recognition of transmembrane substrates for these intramembrane proteases.
In the paper, n-Dodecyl-β-D-Maltopyranoside (DDM) and n-Decyl-β-D-Maltopyranoside (DM) were used to determine whether oligomerization affects catalytic properties of rhomboids. Also referenced in the paper was an alkyl peg detergent (C12E7) which was used to determine the crystal structure of CaaX protease(2). Alkyl peg detergents have recently found a place in crystallizing membrane proteins. It has been determined that the best way to screen alkyl pegs, as well as 96 different detergents, is via the Anatrace Analytic Selector Kit.
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Arutyunova, Elena, Panwar, Pankai, Skiba, Pauline M., Gale, Nicole, Mak, Michelle W. and Lemieux, M. Joanne. Allosteric Regulation of Rhomboid Intramembrane Proteolysis. (2014) The EMBO Journal 33(17), 1869-1881.
Pryor, Edward E., Jr., Horanyi, Peter S., Clark, Kathleen, M., Fedoriw, Nadia, Connelly, Sara M., Koszelak-Rosenblum, Mary, Zhu, Guangyu, Malkowski, Michael G., Wiener, Michael C., and Dumont, Mark E. Structure of the Integral Membrane Protein CAAX Protease Ste24p. (2013) Science 339(6127), 1600-1604.