The Wolfe lab studies intramembrane-cleaving proteases (I-CLiPs) that play critical roles both in normal biology and in human disease. The last place in the cell to expect hydrolysis is within the hydrophobic environment of the lipid bilayer. Nevertheless, a number of multi-pass membrane proteins appear to carry out this seemingly paradoxical process (see the figure). Such proteases cut within the transmembrane region of their respective substrates, and consistent with this observation, these proteases contain putative catalytic residues located within transmembrane domains.

The major focus of the lab has been on the chemistry and biology of γ-secretase. This protease is critical to the pathogenesis of Alzheimer's disease and to cell differentiation during embryonic development and adulthood. The enzyme is responsible for producing the amyloid β-peptide (Aβ) that deposits in the Alzheimer brain and is considered an important target for the development of potential therapeutics. The protease also cleaves Notch proteins as an essential part of the signaling mechanism of these receptors that regulate cell-fate determination. The Wolfe lab developed transition-state analogue inhibitors and used these as tools to characterize and identify γ-secretase. Findings from the lab implicated a membrane protein called presenilin as the catalytic component of a large γ-secretase complex. Missense mutations in presenilin cause hereditary Alzheimer's disease, and these mutations specifically affect γ-secretase activity. In the process of purifying γ-secretase, we found that an endogenous substrate also copurified, suggesting an initial substrate docking site on the protease complex distinct from the active site. Helical peptides designed to interact with this docking site can potently inhibit γ-secretase activity. The enzyme also has two distinct proteolytic functions: an endopeptidase activity and a carboxypeptidase activity that trims initially formed long Aβ peptides to shorter secreted forms. Alzheimer-causing mutations in presenilin dramatically decrease the carboxypeptidase activity of γ-secretase, leading to increased proportions of long Aβ peptides. Current efforts are aimed at further elucidating the mechanism of γ-secretase substrate recognition and its multiple proteolytic functions, understanding how genetic mutations that cause Alzheimer’s disease alter γ-secretase structure and function, and developing new chemical probes and therapeutic prototypes targeting this protease complex.

The Wolfe lab has also investigated the structure, mechanism, and inhibition of other intramembrane proteases, such as the serine protease Rhomboid and the presenilin homolog signal peptide peptidase, both of which are conserved across evolution and play critical roles in biology. In this way, the lab helped establish common biochemical principles and strategies for designing inhibitors for this family of membrane-embedded enzymes. The current focus is developing new chemical probes to understand the roles of these membrane-embedded proteases in biology, particularly in human parasites such as Plasmodium falciparum that causes malaria, and explore the potential of these proteases as therapeutic targets.

Representative I-CLiPs. Top left: Presenilin is the catalytic component of a large γ-secretase complex (inset), an aspartyl protease that cleaves within the transmembrane domain of the amyloid precursor protein, Notch receptors, and other type I integral membrane proteins. Top right: Signal peptide peptidase (SPP) is part of a family of presenilin homologs. These aspartyl proteases are active on their own and have inverted membrane topology relative to presenilins. SPP cleaves remnant signal peptides in the membrane produced by signal peptidase. Bottom left: the Site 2 Protease (S2P) family are metalloproteases, with the transmembrane motifs coordinating with zinc in the active site. The human S2P enzyme cleaves a transcription factor (SREBP) that regulates cholesterol biosynthesis. Bottom right: The Rhomboid family of serine proteases are found in virtually all forms of life and are involved in many biological processes. In Drosophila, Rhomboid cleaves a growth factor called Spitz. Figure taken from Wolfe and Kopan, Science, 2004.