RESUMO
Proteolysis within the membrane is a recent concept in biology. Rhomboid intramembrane serine proteases are conserved in evolution and serve as key switches in diverse cellular pathways ranging from signaling to protein degradation. Since deregulation of intramembrane proteolysis can lead to severe diseases including neurodegenerative disorders, dissecting their enzymatic function and specificity becomes crucial. As membrane proteins, their solubilization, and purification are technically challenging. As a start point for a comprehensive in vitro characterization of eukaryotic rhomboid proteases, we depict in this chapter a robust workflow to find the best conditions to obtain pure and active enzymes from a bacterial expression system. To monitor the integrity of their active site and visualize substrate cleavage, various established activity assays including activity-based labeling and gel-based cleavage assays are described. These methods are illustrated by use of the Escherichia coli rhomboid protease GlpG and human RHBDL2 as an example.
Assuntos
Proteínas de Ligação a DNA/isolamento & purificação , Biologia Molecular/métodos , Proteólise , Serina Proteases/isolamento & purificação , Domínio Catalítico , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Eucariotos , Humanos , Serina Proteases/química , Serina Proteases/metabolismo , Especificidade por SubstratoRESUMO
Intramembrane proteolysis - cleavage of proteins within the plane of a membrane - is a widespread phenomenon that can contribute to the functional activation of substrates and is involved in several diseases. Although different families of intramembrane proteases have been discovered and characterized, we currently do not know how these enzymes discriminate between substrates and non-substrates, how site-specific cleavage is achieved, or which factors determine the rate of proteolysis. Focusing on γ-secretase and rhomboid proteases, we argue that answers to these questions may emerge from connecting experimental readouts, such as reaction kinetics and the determination of cleavage sites, to the structures and the conformational dynamics of substrates and enzymes.
Assuntos
Proteínas de Membrana/química , Peptídeo Hidrolases/metabolismo , Proteólise , Secretases da Proteína Precursora do Amiloide/química , Membrana Celular/metabolismo , Humanos , Cinética , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/químicaRESUMO
Signal sequences of human MHC class I molecules are a unique source of epitopes for newly synthesized nonclassical HLA-E molecules. Binding of such conserved peptides to HLA-E induces its cell surface expression and protects cells from NK cell attack. After cleavage from the pre-protein, we show that the liberated MHC class I signal peptide is further processed by signal peptide peptidase in the hydrophobic, membrane-spanning region. This cut is essential for the release of the HLA-E epitope-containing fragment from the lipid bilayer and its subsequent transport into the lumen of the endoplasmic reticulum via the TAP.
Assuntos
Epitopos/biossíntese , Antígenos HLA/biossíntese , Antígenos de Histocompatibilidade Classe I/biossíntese , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Sinais Direcionadores de Proteínas , Serina Endopeptidases/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Epitopos/metabolismo , Antígenos HLA/metabolismo , Antígenos HLA-A/metabolismo , Antígeno HLA-A3 , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Hidrólise , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , Ligação Proteica/imunologia , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional/imunologia , Especificidade por Substrato/imunologia , Antígenos HLA-ERESUMO
Signal peptides of secretory and membrane proteins are generated by proteolytic processing of precursor proteins after insertion into the endoplasmic reticulum membrane. Liberated signal peptides can be further processed, and the resulting N-terminal fragments are released toward the cytosol, where they may interact with target proteins like calmodulin. We show here that the processing of signal peptides requires a protease activity distinct from signal peptidase. This activity is inhibited specifically with a newly developed cysteine protease inhibitor, 1, 3-di-(N-carboxybenzoyl-l-leucyl-l-leucyl)amino acetone ((Z-LL)(2) ketone). Inhibitor studies revealed that the final, (Z-LL)(2) ketone-sensitive cleavage event occurs within the hydrophobic transmembrane region of the signal peptide, thus promoting the release of an N-terminal fragment into the cytosol.