RESUMEN
Rhomboid proteases are intramembrane enzymes that hydrolyze peptide bonds of transmembrane proteins in the lipid bilayer. They play a variety of roles in key biological events and are linked to several disease states. Over the last decade a great deal of structural and functional knowledge has been generated on this fascinating class of proteases. Both structural and kinetic analyses require milligram amounts of protein, which may be challenging for membrane proteins such as rhomboids. Here, we present a detailed protocol for optimization of expression and purification of three rhomboid proteases from Escherichia coli (ecGlpG), Haemophilus influenzae (hiGlpG), and Providencia stuartii (AarA). We discuss the optimization of expression conditions, such as concentration of inducing agent, induction time, and temperature, as well as purification protocol with precise details for each step. The provided protocol yields 1-2.5mg of rhomboid enzyme per liter of bacterial culture and can assist in structural and functional studies of intramembrane proteases.
Asunto(s)
Proteínas de Unión al ADN/aislamiento & purificación , Endopeptidasas/aislamiento & purificación , Proteínas de Escherichia coli/aislamiento & purificación , Escherichia coli/enzimología , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/aislamiento & purificación , Biología Molecular/métodos , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Endopeptidasas/biosíntesis , Endopeptidasas/química , Endopeptidasas/genética , Proteínas de Escherichia coli/biosíntesis , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Haemophilus influenzae/enzimología , Cinética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Providencia/enzimología , Relación Estructura-ActividadRESUMEN
Intramembrane serine proteases of the rhomboid family are widespread, and their gradually uncovered functions in different organisms already suggest medical relevance for infectious diseases and cancer. However, selective inhibitors that could serve as research tools for rhomboids, for validation of their disease relevance, or as templates for drug development are lacking. Here I summarize the current knowledge about rhomboid protease mechanism and specificity, overview the currently used inhibitors, and conclude by proposing avenues for future development of rhomboid protease inhibitors.
Asunto(s)
Endopeptidasas/química , Proteínas de la Membrana/química , Endopeptidasas/metabolismo , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Inhibidores de Proteasas/química , Especificidad por SustratoRESUMEN
Rhomboids are ubiquitous intramembrane serine proteases that are involved in various signaling pathways. This fascinating class of proteases harbors an active site buried within the lipid milieu. High-resolution structures of the Escherichia coli rhomboid GlpG with various inhibitors revealed the catalytic mechanism for rhomboid-mediated proteolysis; however, a quantitative characterization was lacking. Assessing an enzyme's catalytic parameters is important for understanding the details of its proteolytic reaction and regulatory mechanisms. To assay rhomboid protease activity, many challenges exist such as the lipid environment and lack of known substrates. Here, we summarize various enzymatic assays developed over the last decade to study rhomboid protease activity. We present detailed protocols for gel-shift and FRET-based assays, and calculation of KM and Vmax to measure catalytic parameters, using detergent solubilized rhomboids with TatA, the only known substrate for bacterial rhomboids, and the model substrate fluorescently labeled casein.
Asunto(s)
Proteínas de Unión al ADN/química , Endopeptidasas/química , Pruebas de Enzimas/métodos , Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , Proteolisis , Dominio Catalítico , Membrana Celular/enzimología , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/aislamiento & purificación , Endopeptidasas/metabolismo , Escherichia coli/enzimología , Proteínas de Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Unión Proteica , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Aspartic proteinases share a conserved network of hydrogen bonds (termed "fireman's grip"), which involves the hydroxyl groups of two threonine residues in the active site Asp-Thr-Gly triplets (Thr26 in the case of human immunodeficiency virus type 1 (HIV-1) PR). In the case of retroviral proteinases (PRs), which are active as symmetrical homodimers, these interactions occur at the dimer interface. For a systematic analysis of the "fireman's grip," Thr26 of HIV-1 PR was changed to either Ser, Cys, or Ala. The variant enzymes were tested for cleavage of HIV-1 derived peptide and polyprotein substrates. PR(T26S) and PR(T26C) showed similar or slightly reduced activity compared to wild-type HIV-1 PR, indicating that the sulfhydryl group of cysteine can substitute for the hydroxyl of the conserved threonine in this position. PR(T26A), which lacks the "fireman's grip" interaction, was virtually inactive and was monomeric in solution at conditions where wild-type PR exhibited a monomer-dimer equilibrium. All three mutations had little effect when introduced into only one chain of a linked dimer of HIV-1 PR. In this case, even changing both Thr residues to Ala yielded residual activity suggesting that the "fireman's grip" is not essential for activity but contributes significantly to dimer formation. Taken together, these results indicate that the "fireman's grip" is crucial for stabilization of the retroviral PR dimer and for overall stability of the enzyme.
