My first project after joining Genentech in 1997 was on blood coagulation proteases, specifically on antibody-based and small molecule inhibitors of tissue factor and factor VIIa. I have worked on proteases and pseudo-proteases (hepatocyte growth factor) ever since.
PostDocs have always been a cornerstone of the research in my lab. I truly enjoy mentoring them over the course of about 4 years and seeing them mature into independent scientists. There is also a wonderful camaraderie among the many postdocs in our research organization, which often results in productive collaborations.
The focus of the current PD project is the newly discovered trypsin fold protease, called neutrophil serine protease 4 (NSP4). Little is known about it and there are only a handful of papers on NSP4. The former Postdoc Jack Lin was able to gain a clear understanding of the unusual catalytic mechanism of this protease by solving the first crystal structures of NSP4. Based on the strong evolutionary conservation of NSP4 and its unique substrate specificity we anticipate that NSP4 may be involved in highly interesting biological processes, which we aim to unravel.
Currently we are studying the serine proteases HtrA1 in ocular disease, PCSK9 in lipid metabolism and the neutrophil serine protease 4 (see under PostDoc commentary). In studying these proteases, we interact with many collaborators in other departments, allowing us to widen the range of technologies necessary to reach our scientific goals. This includes antibody engineering, biochemistry/biophysics, peptide phage display, crystallography and electron microscopy, as well as mouse genetic disease models.
HtrA1: Human genetics studies have implicated HtrA1 in age-related macular degeneration. This disease affects the photoreceptors in the retina of the eye and leads to vision impairment and even blindness. Therefore, neutralizing the enzyme activity of HtrA1 with antibodies may slow down disease progression. However the trimeric composition of HtrA1, having three active sites in close proximity, added to the challenge of generating neutralizing antibodies. Building on our structural knowledge gained with other anti-protease antibodies (Wu et al. PNAS 2007; Ganesan et al. Structure 2009), we were able to identify the first function-blocking antibodies to HtrA1, which formed a unique "cage-like" inhibition complex (Ciferri et al. Biochem. J. 2015).
PCSK9: PCSK9 is a serine protease with a subtilisin fold. As a negative regulator of liver LDL receptors, it controls plasma levels of LDL ("bad cholesterol"). Antibodies that inhibit PCSK9 binding to liver LDL receptors dramatically reduce LDL levels. In collaboration with Yingnan Zhang's lab we have used peptide phage display methods to obtain peptidic inhibitors of PCSK9, such as Pep2-8, which mimics the PCSK9-interacting domain of the LDL receptor. Recently, we discovered a cryptic site on PCSK9 located next to the main LDL receptor binding site, where Pep2-8 binds. In phage-display experiments, Pep2-8 was used as an "anchor" peptide to attach extension peptide libraries directed toward this cryptic site, which is an open groove that normally harbors the P' helix of the PCSK9 catalytic domain. Guided by structural information, we further engineered the identified groove-binding peptides into antagonists, which were able to restore LDL receptors on the surface of immortalized liver cells. This study exemplifies the collaborative and highly creative spirit of scientists here at Genentech and the results pave the way for new approaches to develop PCSK9 small-molecule inhibitors as new cardiovascular therapeutics.