Originally discovered through genetic analysis of Drosophila embryogenesis, rhomboid proteases initiate Drosophila EGF signaling by releasing transmembrane EGF precursors Spitz, Keren and Gurken from the membrane. In contrast to the site-2 protease and γ-secretase/signal peptide peptidase intramembrane protease superfamilies, rhomboid proteins are serine proteases mechanistically, and differ functionally by releasing factors to the outside of the cell, rather than into the cytosol. Rhomboid proteins are the largest family of intramembrane proteases. Although initially thought to be rare, intramembrane proteases are now recognized to be among the most prevalent membrane proteins known, conserved from bacteria to man. Without exception, each of the three types of intramembrane proteases were discovered unintentionally, through analyses focused on cell regulation or human disease. Yet even this well-studied area of biochemistry is not immune to surprise: in the past decade an unforeseen class of proteases was discovered that reside immersed within the cell’s membrane. Moreover, serine proteases in particular have served as paradigms for understanding enzyme catalysis through integration of chemical, enzymological and structural investigations for over a century. It’s often stated that evidence of their importance is encrypted directly in our genomes: up to 5% of a genome is often devoted to encoding proteolytic components. Because of their speed, selectivity and versatility, proteolytic mechanisms have come to regulate a broad array of cellular processes. Proteases cleave proteins by catalyzing hydrolysis of peptide bonds. The modern challenge is to understand how the properties of these regulatory enzymes have been tailored to fit the specific needs of the cell. To this daunting task enzymes bring key advantages: they act rapidly and with a high degree of specificity. While most enzymes catalyze metabolic reactions, a subset evolved to function within information-processing circuits, where they are entrusted to regulate cellular processes.
As a result, enzymes have been the focus of biochemical investigation for over a century. The remarkable synthetic ability of cells relies on a vast orchestra of enzymes that transform simple chemicals from the environment into complex biomolecules and energy that sustain life. Although far from complete, studies with GlpG currently offer the best prospect for achieving a thorough and sophisticated understanding of a simplified intramembrane protease. Complex interplay between lateral substrate gating by rhomboid, substrate unwinding, and local membrane thinning leads to intramembrane proteolysis of selected target proteins. Four conserved architectural elements in particular act as ‘keystones’ to stabilize this structure, while the lateral, membrane-embedded L1 loop functions as a ‘flotation device’ to position the protease tilted in the membrane. The protein creates a central, hydrated microenvironment immersed below the membrane surface to support hydrolysis by its serine protease-like catalytic apparatus. But recent multidisciplinary approaches, including eight crystal structures, four computer simulations, and nearly one hundred engineered mutants interrogated in vivo and in vitro, are coalescing into an integrated model for one rhomboid ortholog in particular, bacterial GlpG.
Despite having key roles in animal cell signaling and microbial pathogenesis, the membrane-immersed nature of these enzymes had long imposed obstacles to elucidating their biochemical mechanisms. Rhomboid proteases are a fascinating class of enzymes that combine a serine protease active site within the core of an integral membrane protein.
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