Research
Overview
We are currently focused on three processes involved in protein secretion. The first two systems, Sec and Tat, are pathways for proteins to be translocated across membranes.The second is the essential eukaryotic process of protein glycosylation by which sugars are added specifically to proteins. We are also interested in the general problem of the over-expression and synthesis of membrane proteins.
Protein translocation pathways
There are two major protein translocation pathways across the bacterial cell membrane (Wickner and Schekman, 2005). The Sec pathway is the predominant one and is often referred to as the general secretion pathway as it is the major pathway in all kingdoms of life. The Sec channel consists of three universally conserved integral membrane proteins, called SecYEG, and is powered by the cytoplasmic ATPase, SecA, or the translating ribosome. Translocation requires energy input, either ATP or GTP, and transports unfolded protein. Recently, structural and mechanistic studies have completely changed the paradigm of how this process works (Osborne et al., 2005; Saparov et al., 2007; van den Berg et al., 2004; Wickner and Schekman, 2005).
The second major translocation pathway of the bacterial cell membrane is the twin-arginine translocation (Tat) pathway. The striking feature of this system is that it translocates folded protein across the membrane. A large variability in the pore diameter (up to ~70Å) is required to accommodate the variety of folded substrate translocated by Tat (Gohlke et al., 2005). Similar to Sec, the Tat pathway requires targeting by a signal peptide; however, Tat substrates are unique in that their signal sequences contain a highly conserved twin-arginine motif. The Tat pathway uses the proton-motive force for energy and is found only in prokaryotes and chloroplasts. Unlike the ubiquitous Sec system, Tat appears to be found in roughly half of the prokaryotic genomes currently sequenced (Dilks et al., 2003). The bulk of my research program in this area is focused on the Tat Pathway. I will discuss our efforts in both.
Twin-arginine translocation
Sec translocation
Protein glycosylation
Glycosylation plays a central role in biology and understanding its mechanism is essential for biomedical research. The glycome, the various sugar chains in an organism, is thought to be hundreds to thousands of times larger than the proteome (Freeze, 2006). In higher eukaryotes, over 50% of all proteins are glycosylated, and problems with glycosylation are linked to a number of both inherited and acquired diseases in humans (Durand and Seta, 2000). Glycosylation is involved in cell adhesion, molecular trafficking, receptor interaction, signal transduction and endocytosis, and enzymes involved in this process comprise up to 2% of the mammalian genome (Ohtsubo and Marth, 2006). In medicine, the correct glycosylation of a recombinant protein can mean the difference between functional and non-functional proteins. A classic example of this is the drug Epogen, a billion dollar success story for Amgen; in which correct glycosylation of the recombinant erythropoeitan was essential for efficacy. Current methods of synthesizing glycosylated proteins require expression in mammalian cells at a cost of $1 to $3 million dollars per kilogram (Dove, 2001). Despite an understanding of the essential role of these processes in biology, there is no clear understanding of the chemical mechanism that drives them. Our goal is to provide enough detailed information so that we can design novel mechanisms for synthesizing glycosylated proteins.
There are two main protein glycosylation pathways, O-linked glycosylation of serines/threonines and N-linked glycosylation of asparagines. Our current work focuses on the latter.
