Catálogo de publicaciones - libros

The site-2 protease (S2P) is a highly hydrophobic integral membrane protean required for cleavage of various mambrane-bound transcription factors within a membrane-spanning helix. S2P was the first intramembrane-cleaving protease to be recognized but more has been learned about other such proteins. Fundamental questions about the role and function of S2P remain unanswered. S2P plays a crucial role in mammalian lipid metabolism and the unfolded protein response. Thus, finding the answers has implications for our understanding of human health and disease. Recent advances with rhomboid proteins and gamma secretase indicate that the technical challenges to getting the answers can be overcome

Signal peptide peptidases (SPPs) are the most recently identified members of a protease family of integral membrane proteins that includes the intensively studied presenilin 1 (PS1) and presenilin (PS2) proteins. There are 5 human genes encoding SPPs which can be divided into two branches based on homology and initial functional studies. One branch, which is the focus of this chapter, consists of the SPP and SPPL3 proteins. The second branch will be the focus of a subsequent chapter, and consists of the three SPPL2 proteins (SPPL2a, b, and c). The SPP proteins are conserved through evolution with family members found in fungi, archaea and plants. Presenilins (PSs) and SPPs cleave substrate polypeptides within a transmembrane region, but differ in that PSs cleave type 1 membrane proteins whereas SPPs cleave type 2 membrane proteins. SPPs and PSs have low overall sequence homology, yet exhibit considerable structural similarity as well as strict conservation of several small motifs. They are both multipass membrane proteins that contain two conserved active site motifs YD and GxGD in adjacent membrane-spanning domains and a conserved PAL motif of unknown function near their C-termini. They differ in that the active site topology of SPPs is inverted relative to PSs. Moreover, SPP and SPPL3 appear to function as proteases without the need for additional cofactors. In contrast, PSs function as the UPgamma-secretase protease only when complexed with three accessory proteins. Although the biological roles of PSs are reasonably well understood, the biological roles of SPP are largely unknown, and only a few endogenous substrates for SPP have been identified. SPP and possibly SPPL3 appear to cleave a number of endogenous type 2 signal peptides and these genes are essential genes in the development of several model organisms. In addition, in many human parasites, there is only a single SPP gene that is most closely related to the human SPP. Thus, SPPs may be novel antiviral drug targets in humans and represent a novel drug target for major human pathogens such as malaria

Among the known intramembrane-cleaving proteases (I-CLiPs), the aspartate proteases are unique. Unlike I-CLiPs of the serine- and metalloprotease-type, which share their respective active site motifs with their classical counterparts, the aspartate protease I-CLiPs acquired a novel characteristic GxGD active site motif during evolution. These so-called GxGD-type proteases include the presenilin (PS), signal peptide peptidase (SPP), SPP-like protease (SPPL) families and the related type IV prepilin peptidase family, bacterial leader peptidases, which share the same active site motif, but which cleave their substrates directly at, rather than within, the membrane. PS, SPP and SPPLs adopt a similar, but inverted membrane topology with respect to their active site orientation. PS is the founding member of the GxGD-type I-CLiPs and has been identified as the catalytic subunit of Γ-secretase. The major function of this protease complex appears to be the clearance of the remnants of a large number of type I membrane proteins that have undergone shedding of their ectodomains. For some substrates of Γ-secretase, most prominently for the cell surface receptor Notch, Γ-secretase cleavage is coupled with signaling by the release of a nuclear-targeted intracellular domain (ICD). In the case of Notch, the ICD functions in the nucleus as a key transcriptional regulator for cell differentiation in development and adulthood. In addition, Γ-secretase is a pivotal enzyme in Alzheimer’s disease (AD), responsible for the liberation of the AD-causing amyloid β-peptide from its precursor protein. SPP and SPPLs exert similar functions, which, however, use type II membrane proteins as substrates consistent with their opposite topologies compared to PS. Thus, the major function of SPP is likely to be to clear the ER membrane of signal peptides of secretory proteins, whereas SPPL2a and b have recently been shown to cleave tumor necrosis factor UPalpha to release an ICD that triggers interleukin-12 signaling. Despite the similarities in their overall biological functions, the major difference is that PS requires partner proteins for its proteolytic function, whereas SPP and probably also the SPPLs do not

Intramembrane proteolysis catalyzed by rhomboid proteases plays key roles in such diverse cell communication events as receptor tyrosine kinase signalling during animal development, and quorum sensing during bacterial growth. In these contexts, rhomboid proteins act in the signal-sending cell to activate signal precursor proteins and initiate the signalling event. Recent biochemical advances have culminated in the first high-resolution crystal structures of an intramembrane protease, and a pure enzyme reconstitution system for studying rhomboid activity. Functional studies have expanded the cellular role of rhomboid proteins to broad biological processes, including host-cell invasion by malaria parasites, which is the first implication of these enzymes as possible therapeutic targets in human disease

Rhomboid proteins are a class of serine proteases conserved in all kingdoms of organisms. They contain six or seven transmembrane helices and control a wide range of cellular functions and developmental processes by intramembrane proteolysis. In yeast, two members of the rhomboid family are known, Rbd2 and Pcp1. Rbd2 is associated with the Golgi apparatus, but its function and its substrates are still unknown. The rhomboid protease Pcp1, located in the mitochondrial inner membrane, catalyses the second step in the proteolytic processing of cytochrome $c$ peroxidase, a mitochondrial enzyme that acts as a peroxide scavenger. Pcp1 also affects the morphology of mitochondria by acting on, Mgm1, a dynamin-related GTPase. Mgm1 is present in short and long forms, and both isoforms are required for fusion of mitochondria and the maintenance of mitochondrial DNA. The proteolytic conversion of the long to the short form is catalysed by Pcp1. The cleavage sites in their substrates are not typical transmembrane domains but hydrophobic regions that must be actively translocated into the inner mitochondrial membrane by an ATP-consuming process to make them accessible to cleavage by the rhomboid protease

Deposition of the amyloid β-protein is a defining pathological characteristic of Alzheimer’s disease, and this small protein is proteolytically produced from the amyloid β-protein precursor. ,-Secretase is responsible for the second cut, which forms the C-terminus of amyloid-β and determines how much of the transmembrane domain is included in this aggregation-prone protein. This intramembrane aspartyl protease is a complex of four different integral membrane proteins: presenilin, nicastrin, Aph-1 and Pen-2. During assembly and maturation of the protease complex, presenilin is endoproteolyzed into two subunits, each of which contributes one aspartate to the active site. A model of successive proteolysis may explain how Alzheimer-causing mutations in presenilin can both decrease enzyme activity and increase the proportion of longer, more aggregation-prone forms of amyloid-β. Substrate apparently interacts with an initial docking site before passing in whole or in part between the two presenilin subunits to the internal water-containing active site. The ectodomain of nicastrin also interacts with the N-terminus of the substrate as an essential step in substrate recognition and processing. Inhibitors and allosteric modulators of γ-secretase activity are under investigation as potential Alzheimer therapeutics. Elucidation of detailed structural features of γ-secretase is the next logical step toward understanding how this enzyme carries out intramembrane proteolysis and will set the stage for structure-based drug design

Notch proteins are evolutionary conserved transmembrane receptors used by metazoans to direct cell fate decisions, proliferation, differentiation and cell death at all stages of development, including self-renewing adult tissues. Notch signaling is a well-established example of a pathway that is mediated by Regulated Intramembrane Proteolysis (RIP). Upon binding of ligand, the Notch receptor undergoes successive proteolytic cleavages – an ectodomain shedding cleavage followed by intramembrane proteolysis by γ-secretase. This process releases the Notch intracellular domain, which translocates to the nucleus to activate its target genes. Deciphering the proteolytic mechanism for Notch activation relied on the convergence of previously independent fields of research, revealing that the Notch receptor resembled another Type I membrane protein, the amyloid-γ precursor protein, in that both are proteolytically cleaved within their transmembrane domains (TMDs) by the same protease, γ-secretase, whose catalytic center resided in the protein Presenilin. Intramembrane proteolysis has continued to emerge as an exciting research area in cell biology. Recent studies on γ-secretase function have begun to reveal the molecular details involved in ectodomain shedding and intramembrane cleavage events as well as the importance of endocytosis and endosomal sorting as key regulators of γ-secretase cleavage of Notch