Cell membranes, which are sites of interface between the cell and the outside world, constitute major sites of signaling. Membranes also form the front lines where deadly pathogens first contact human cells and initiate infection. Our main focus is a family of enzymes, called rhomboid proteases, that function immersed within the membrane. These enzymes cut protein segments in the membrane through a hydrolytic reaction. This cleavage liberates protein domains from their transmembrane anchors, either to activate targets rapidly or to inactivate their function. Because of its speed and versatility, this basic biochemical reaction has evolved to control many cellular processes in all forms of life, from diverse bacteria to humans. Despite this, our understanding of how these enzymes achieve catalysis within the membrane, and their roles in all but a few organisms, remains rudimentary. These two fundamental questions are the focus of our research.
Rhomboid was first discovered more than 30 years ago through genetic dissection of Drosophila development. My interest in Drosophila embryogenesis led to my discovery that this classical developmental factor achieves its remarkable feats by acting as an intramembrane serine protease. This discovery put a biochemical face on a gene that was exquisitely well-understood at the genetic level: rhomboid initiates epidermal growth factor (EGF) signaling between cells by cleaving transmembrane EGF precursors, releasing their extracellular domains as active signals.
The unusual and unanticipated biochemistry of this intramembrane reaction was a point of skepticism and debate for years. As an intramembrane protease, rhomboid joined three other analogous enzymes that had been discovered at the heart of several human diseases. The first to be identified was site-2 protease, a membrane metalloenzyme that controls cholesterol and fatty acid biosynthesis in human cells. The search for the enzymes that generate the Aβ peptide in Alzheimer's disease soon identified γ-secretase as an aspartyl intramembrane protease with presenilin at its catalytic core. γ-Secretase has also been linked to many other roles, including roles in human cancer. The related, presenilin-like signal peptide peptidase was found to be responsible for cleaving hepatitis C virus core protein. Although other intramembrane proteases typically release domains, usually transcription factors, into the cytoplasm, rhomboid proteases differ functionally by releasing factors to the outside of the cell.
Despite their importance, the biochemical complexity of intramembrane proteases has long imposed obstacles to deciphering how they function. We study the biochemical principles governing how rhomboid enzymes catalyze reactions immersed within the membrane. We have reconstituted rhomboid activity with pure components, and we are using a combination of membrane biochemistry, cell biology, and chemical genetics to probe their mechanism. Through these advances, we have recently built a structural and functional framework for understanding their basic catalytic mechanism. In collaboration with Yigong Shi (Tsinghua University), we discovered that the enzyme architecture creates a water-filled microenvironment for catalysis within the membrane, protected from surrounding membrane lipid by protein segments. Water enters this membrane-embedded active site through a large opening that faces outward. Our subsequent structure-function approach identified a gating helix that tilts to allow substrate entry laterally from the membrane into the active site.
Rhomboid proteins are among the most widely conserved of all membrane proteins, although their roles in most organisms remain largely unknown. We have focused on rhomboid function in deadly human pathogens, and discovered that these proteases execute an array of key functions: malaria and related parasites use their rhomboid enzymes to invade human cells; a parasitic amoeba deploys its rhomboid protease in phagocytosis and immune evasion. Although without exception all intramembrane proteases were initially discovered through the study of animal biology, more than a dozen intramembrane proteases are now known to play central roles in viral, bacterial, and eukaryotic pathogens that cause a great deal of human suffering. These recent observations raise the exciting possibility that understanding and targeting intramembrane proteases may be a way of combating multiple infectious diseases.