Photochemical methods in which light is used to trigger the release of a bioactive compound into a physiological environment are ideal for studying biological function and enable the conduct of experiments that would be difficult by other means.
Light-driven tools enable a high level of control of the timing, location, and amplitude of an event in a relatively non-invasive manner, especially when 2-photon excitation (2PE) is employed (Figure 1). 2PE enables activation of a bioactive messenger in a small volume relative to mammalian cells (1 fL, i.e., the volume of a single E. coli bacterium). Other advantages to 2PE include less photodamage to biological tissues and the ability to conduct experiments deeper inside tissues than can be achieved using conventional 1-photon excitation (1PE).
My laboratory designs, synthesizes, studies, and develops applications for photochemically active molecules, particularly those with sensitivity to 2PE. We are applying our photochemical technology to accurately control neural activity on the cellular scale in an intact brain to understand how information propagates in the central nervous system and to explore the role of certain genes in vertebrate development.
The Ras subfamily of small GTP-binding proteins are CaaX proteins that play a prominent role in carcinogenesis; therefore, Ras proteins and Ras-regulatory proteins are considered targets for anticancer therapeutics.
The C-terminal end of the nascent protein contains a 4-residue motif where C is cysteine, a is typically an aliphatic amino acid, and X is one of several amino acids. Three post-translational modifications are critical for the proper localization and proper function of CaaX proteins (Figure 2): farnesylation by farnesyl transferase (FTase) or geranylgeranylation by geranylgeranyl transferase (GGTase); proteolysis by CaaX proteases Ras converting enzyme (Rce1p) or sterile mutant 24 (Ste24p); and carboxyl methyl esterification by isoprenylcysteine carboxyl methyltransferase (ICMT).
Ste24p and Rce1p are membrane-bound and ER localized, but have unrelated primary sequences. Rce1 and Ste24p have distinct, but partially overlapping substrate specificities; Rce1p proteolyzes Ras, but Ste24p does not. Ste24p is a zinc metalloprotease, but Rce1p’s mechanism of proteolysis has eluded classification. Little structural information on Rce1p is available. In collaboration with Walter Schmidt, we have discovered molecular scaffolds that provide the basis to design selective and potent inhibitors of Ste24p and Rce1p. Small-molecule inhibitors of these proteases would provide insight into each enzyme’s mechanism of action and could potentially have therapeutic value.