Showing papers in "Essays in Biochemistry in 2002"

Matrix metalloproteinases in cancer.

Abstract: The extracellular matrix (ECM) holds cells together and maintains the three-dimensional structure of the body. It also plays critical roles in cell growth, differentiation, survival and motility. For a tumour cell to metastasize from the primary tumour to other organs, it must locally degrade ECM components that are the physical barriers for cell migration. The key enzymes responsible for ECM breakdown are matrix metalloproteinases (MMPs). To date, 23 MMP genes have been identified in humans and many are implicated in cancer. ECM degradation by MMPs not only enhances tumour invasion, but also affects tumour cell behaviour and leads to cancer progression. This review highlights recent developments with regard to the cellular and molecular mechanisms of MMPs that influence tumour cell growth, invasion and metastasis.

Precursor convertases in the secretory pathway, cytosol and extracellular milieu.

Abstract: Precursor proteins that transit through the secretory pathway often require processing at specific sites in order to release their bioactive entities. The most prevalent limited proteolysis occurs at single or paired basic residues, and is achieved by one or more of the seven subtilisin-like proprotein convertases (PCs); Furin, PC1, PC2, PACE4 (paired basic amino acid converting enzyme 4), PC4, PC5 and PC7. Other types of cleavages occur at hydophobic residues, some of which are performed by subtilisin/kexin-like isozyme-1 (SKI-1), which is also known as site-1 protease. Together, the PCs and SKI-1 regulate the activity of a large variety of cellular proteins, including growth factors, neuropeptides, receptors, enzymes and even toxins and glycoproteins from infectious retroviruses. These processing events are exquisitely regulated by multiple zymogen-activation steps, as well as by specific subcellular localization signals. The above mentioned convertases are implicated in a number of pathologies such as cancer, neurodegenerative diseases, endocrine disorders and inflammation. Recently, it was recognized that the metalloendopeptidase N-arginine dibasic convertase (NRDc; nardilysin), which cleaves at the N-terminus side of basic residues in dibasic pairs, is localized both in the cytosol and at the cell surface or in the extracellular milieu. While NRDc binds heparin-binding epidermal growth factor (HB-EGF) at the cell surface and potentiates its physiological effect, HB-EGF potently inhibits the NRDc's activity. NRDc could represent the equivalent of the PCs in the cytosol or the extracellular space.

Caspases and apoptosis

Abstract: The ability of metazoan cells to undergo programmed cell death is vital to both the precise development and long-term survival of the mature adult. Cell deaths that result from engagement of this programme end in apoptosis, the ordered dismantling of the cell that results in its 'silent' demise, in which packaged cell fragments are removed by phagocytosis. This co-ordinated demise is mediated by members of a family of cysteine proteases known as caspases, whose activation follows characteristic apoptotic stimuli, and whose substrates include many proteins, the limited cleavage of which causes the characteristic morphology of apoptosis. In vertebrates, a subset of caspases has evolved to participate in the activation of pro-inflammatory cytokines, and thus members of the caspase family participate in one of two very distinct intracellular signalling pathways.

Shedding of membrane proteins by ADAM family proteases.

Abstract: Many membrane-bound proteins undergo proteolytic release from the membrane, a process known as 'shedding'. Some of the processing events are carried out by enzymes of the ADAM (a disintegrin and metalloproteinase) family, which are also membrane bound. One of the most well known ADAM family members is TACE (tumour necrosis factor-alpha-converting enzyme. TACE was the first ADAM family member to have a known physiological substrate, namely, precursor tumour necrosis factor-alpha. Inhibitors of TACE block the release of the soluble form of this inflammatory cytokine, and are currently being studied in drug discovery projects for the treatment of arthritis. Since the discovery of TACE, physiological substrates for other ADAMs have been determined. This review focuses on the shedding events carried out by TACE and other ADAM family proteinases.

Proteases in blood clotting.

Abstract: The serine proteases, cofactors and cell-receptor molecules that comprise the haemostatic mechanism are highly conserved modular proteins that have evolved to participate in biochemical reactions in blood coagulation, anticoagulation and fibrinolysis. Blood coagulation is initiated by exposure of tissue factor, which forms a complex with factor VIIa and factor X, which results in the generation of small quantities of thrombin and is rapidly shutdown by the tissue factor pathway inhibitor. The generation of these small quantities of thrombin then activates factor XI, resulting in a sequence of events that lead to the activation of factor IX, factor X and prothrombin. Sufficient thrombin is generated to effect normal haemostasis by converting fibrinogen into fibrin. The anticoagulant pathways that regulate blood coagulation include the protein C anticoagulant mechanism, the serine protease inhibitors in plasma, and the Kunitz-like inhibitors, tissue factor pathway inhibitor and protease nexin 2. Finally, the fibrinolytic mechanism that comprises the activation of plasminogen into plasmin prevents excessive fibrin accumulation by promoting local dissolution of thrombi and promoting wound healing by reestablishment of blood flow.

The ubiquitin-proteasome pathway of intracellular proteolysis.

Abstract: Intracellular proteins are targeted for degradation by the covalent attachment of chains of the small protein ubiquitin; a process known as ubiquitylation. Many proteins are phosphorylated prior to ubiquitylation, and therefore ubiquitylation and degradation of these proteins is regulated by kinase activity and signalling cascades. Many ubiquitylated proteins are degraded by the 26 S proteasome complex, which is found in the cytosol and nucleus. The 26 S proteasome consists of a 20 S core with proteolytic activity and 18 S regulatory complexes containing ATPases and ubiquitin-chain-binding proteins. Proteins degraded by the ubiquitin-proteasome pathway include cyclins and other regulators of the cell cycle, and transcription factors. Abnormal polypeptides are also degraded by the ubiquitin pathway, including abnormal polypeptides in the endoplasmic reticulum, which are translocated back out of the endoplasmic reticulum prior to ubiquitylation and degradation by the proteasome. The ubiquitin-proteasome pathway is implicated in numerous diseases including cancer and neurodegenerative diseases.

Proteases: a primer

Abstract: A protease can be defined as an enzyme that hydrolyses peptide bonds. Proteases can be divided into endopeptidases, which cleave internal peptide bonds in substrates, and exopeptidases, which cleave the terminal peptide bonds. Exopeptidases can be further subdivided into aminopeptidases and carboxypeptidases. The Schechter and Berger nomenclature provides a model for describing the interactions between the peptide substrate and the active site of a protease. Proteases can also be classified as aspartic proteases, cysteine proteases, metalloproteases, serine proteases and threonine proteases, depending on the nature of the active site. Different inhibitors can be used experimentally to distinguish between these classes of protease. The MEROPs database groups proteases into families on the basis of similarities in sequence and structure. Protease activity can be regulated in vivo by endogenous inhibitors, by the activation of zymogens and by altering the rate of their synthesis and degradation.

Methionine aminopeptidases and angiogenesis.

Abstract: The initiator methionine residue of proteins is removed during synthesis by a specific and ubiquitous enzyme, methionine aminopeptidase (MetAP). Prokaryotes have a single gene, while eukaryotes have two isoforms. This family of metalloenzymes generally cleaves substrates in which the penultimate residue is one of the seven smaller amino acids (glycine, alanine, serine, threonine, proline, cysteine and valine). One of the eukaryotic isoforms (MetAP2) has an additional non-proteolytic function and is the principle target of a family of anti-angiogenic drugs that are related to fumagillin. The resulting covalent modification inhibits the protease activity of MetAP2 and blocks cell-cycle function in endothelial and some cancer cells. The role of MetAP2 in the mitogenic activity of these cells is unknown.

Protease-activated receptors: the role of cell-surface proteolysis in signalling.

Abstract: Certain extracellular proteases, derived from the circulation and inflammatory cells, can specifically cleave and trigger protease-activated receptors (PARs), a small, but important, sub-group of the G-protein-coupled receptor super-family Four PARs have been cloned and they all share the same basic mechanism of activation: proteases cleave at a specific site within the extracellular N-terminus to expose a new N-terminal tethered ligand domain, which binds to and thereby activates the cleaved receptor Thrombin activates PAR1, PAR3 and PAR4, trypsin activates PAR2 and PAR4, and mast cell tryptase activates PAR2 in this manner Activated PARs couple to signalling cascades that affect cell shape, secretion, integrin activation, metabolic responses, transcriptional responses and cell motility PARs are 'single-use' receptors: proteolytic activation is irreversible and the cleaved receptors are degraded in lysosomes Thus, PARs play important roles in 'emergency situations', such as trauma and inflammation The availability of selective agonists and antagonists of protease inhibitors and of genetic models has generated evidence to suggests that proteases and their receptors play important roles in coagulation, inflammation, pain, healing and protection Therefore, selective antagonists or agonists of these receptors may be useful therapeutic agents for the treatment of human diseases

Proteolytic processing of the amyloid-beta protein precursor of Alzheimer's disease.

Abstract: The proteolytic processing of the amyloid-beta protein precursor plays a key role in the development of Alzheimer9s disease. Cleavage of the amyloid-beta protein precursor may occur via two pathways, both of which involve the action of proteases called secretases. One pathway, involving beta- and gamma-secretase, liberates amyloid-beta protein, a protein associated with the neurodegeneration seen in Alzheimer9s disease. The alternative pathway, involving alpha-secretase, precludes amyloid-beta protein formation. In this review, we describe the progress that has been made in identifying the secretases and their potential as therapeutic targets in the treatment or prevention of Alzheimer9s disease.

Regulated intramembrane proteolysis: from the endoplasmic reticulum to the nucleus.

Abstract: Regulated intramembrane proteolysis (Rip) is an ancient and widespread process by which cells transmit information from one compartment (the endoplasmic reticulum) to another (the nucleus). Two separate cleavages that are carried out by two separate proteases are required for Rip. The first protease cleaves its protein substrate within an extracytoplasmic domain; the second cleaves it within a membrane-spanning domain, releasing a functionally active fragment of the target protein. In eukaryotes, examples of Rip can be divided into two classes, according to the proteases that are involved and the orientation of the substrates with the membrane. Class 1 Rip involves type 1 transmembrane proteins and requires presenilin for cleavage within a membrane-spanning domain. In Class 2 Rip, the highly hydrophobic metalloprotease, site-2 protease, is required for cleavage within a membrane-spanning domain and substrates are type 2 transmembrane proteins. Both classes of Rip are implicated in diseases that are important in modern societies, such as hyperlipidaemias (via the sterol regulatory element binding protein pathway) and Alzheimer's disease (via processing of the amyloid precursor protein.)

Inhibition of peptidases in the control of blood pressure.

Abstract: The natriuretic peptide and renin-angiotensin systems are physiological counterparts with opposite roles in the regulation of electrolyte balance and blood pressure. In both systems, membrane-bound, zinc-dependent peptidases play an important role in the inactivation or activation of the system. Angiotensin-converting enzyme (ACE) converts angiotensin I into angiotensin II, and neutral endopeptidase (NEP) degrades the natriuretic peptides. Simultaneous inhibition NEP and ACE by a single molecule (a vasopeptidase inhibitor) is a new therapeutic approach in hypertension. Wider applications for vasopeptidase inhibitors being studied include their role as cardioprotective agents in heart failure, as renoprotective agents in chronic renal failure and diabetic nephropathy, and as vasculoprotective agents in endothelial dysfunction and athersclerosis.

Anatomy and pathology of HIV-1 peptidase

Abstract: The peptidase of the HIV type 1 (HIV PR) is required for the replication of and further infection by the virus. A concerted effort has taken place in the past 15 years to understand the properties of this enzyme, as it serves as an excellent drug target for control of the virus. Owing to drug pressure, many mutations arise during turnover of the virus and some of these lead to resistance to the effects of the inhibitors. Recent advances in the understanding of the changes these mutations cause to the enzyme and its interaction with substrates and inhibitors have been described. In addition, studies of closely related retroviral enzymes from simian immunodeficiency virus, feline immunodeficiency virus and HIV-2 have expanded the structure-function paradigm. The role of the flexibility of ligands and of the enzyme in active-site interactions is discussed.