Nigel M. Hooper

A Human Homolog of Angiotensin-converting Enzyme CLONING AND FUNCTIONAL EXPRESSION AS A CAPTOPRIL-INSENSITIVE CARBOXYPEPTIDASE

A novel human zinc metalloprotease that has considerable homology to human angiotensin-converting enzyme (ACE) (40% identity and 61% similarity) has been identified. This metalloprotease (angiotensin-converting enzyme homolog (ACEH)) contains a single HEXXH zinc-binding domain and conserves other critical residues typical of the ACE family. The predicted protein sequence consists of 805 amino acids, including a potential 17-amino acid N-terminal signal peptide sequence and a putative C-terminal membrane anchor. Expression in Chinese hamster ovary cells of a soluble, truncated form of ACEH, lacking the transmembrane and cytosolic domains, produces a glycoprotein of 120 kDa, which is able to cleave angiotensin I and angiotensin II but not bradykinin or Hip-His-Leu. In the hydrolysis of the angiotensins, ACEH functions exclusively as a carboxypeptidase. ACEH activity is inhibited by EDTA but not by classical ACE inhibitors such as captopril, lisinopril, or enalaprilat. Identification of the genomic sequence of ACEH has shown that the ACEH gene contains 18 exons, of which several have considerable size similarity with the first 17 exons of human ACE. The gene maps to chromosomal location Xp22. Northern blotting analysis has shown that the ACEH mRNA transcript is ∼3.4 kilobase pairs and is most highly expressed in testis, kidney, and heart. This is the first report of a mammalian homolog of ACE and has implications for our understanding of cardiovascular and renal function.

Families of zinc metalloproteases

A scheme based on the zinc binding site [1992, FEBS Lett. 312, 110–114] has been extended to classify zinc metalloproteases into distinct families. The gluzincins, defined by the HEXXH motif and a glutamic acid as the third zinc ligand, include the thermolysin, endopeptidase‐24.11, aminopeptidase, angiotensin converting enzyme, endopeptidase‐24.15, and tetanus and botulinum neurotoxin families. The metzincins, defined by the HEXXH motif, a histidine as the third zinc ligand and a Met‐turn, include the astacin, serralysin, reprolysin and matrixin families. The inverted zincin motif, HXXEH, defines the inverzincin family of insulin‐degrading enzymes, the HXXE motif defines the carboxypeptidase family, and the HXH Motif dd‐carboxypeptidase.

ADAMs family members as amyloid precursor protein α‐secretases

In the non‐amyloidogenic pathway, the Alzheimers amyloid precursor protein (APP) is cleaved within the amyloid‐β domain by α‐secretase precluding deposition of intact amyloid‐β peptide. The large ectodomain released from the cell surface by the action of α‐secretase has several neuroprotective properties. Studies with protease inhibitors have shown that α‐secretase is a zinc metalloproteinase, and several members of the adamalysin family of proteins, tumour necrosis factor‐α convertase (TACE, ADAM17), ADAM10, and ADAM9, all fulfil some of the criteria required of α‐secretase. We review the evidence for each of these ADAMs acting as the α‐secretase. What seems to be emerging from numerous studies, including those with mice in which each of the ADAMs has been knocked out, is that there is a team of zinc metalloproteinases able to cleave APP at the α‐secretase site. We also discuss how upregulation of α‐secretase activity by muscarinic agonists, cholesterol‐lowering drugs, steroid hormones, non‐steroidal anti‐inflammatory drugs, and metal ions may explain some of the therapeutic actions of these agents in Alzheimers disease.

DETERGENT-INSOLUBLE GLYCOSPHINGOLIPID/CHOLESTEROL-RICH MEMBRANE DOMAINS, LIPID RAFTS AND CAVEOLAE (REVIEW)

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.

Exclusively targeting beta-secretase to lipid rafts by GPI-anchor addition up-regulates beta-site processing of the amyloid precursor protein.

β-Secretase (BACE, Asp-2) is a transmembrane aspartic proteinase responsible for cleaving the amyloid precursor protein (APP) to generate the soluble ectodomain sAPPβ and its C-terminal fragment CTFβ. CTFβ is subsequently cleaved by γ-secretase to produce the neurotoxic/synaptotoxic amyloid-β peptide (Aβ) that accumulates in Alzheimers disease. Indirect evidence has suggested that amyloidogenic APP processing may preferentially occur in lipid rafts. Here, we show that relatively little wild-type BACE is found in rafts prepared from a human neuroblastoma cell line (SH-SY5Y) by using Triton X-100 as detergent. To investigate further the significance of lipid rafts in APP processing, a glycosylphosphatidylinositol (GPI) anchor has been added to BACE, replacing the transmembrane and C-terminal domains. The GPI anchor targets the enzyme exclusively to lipid raft domains. Expression of GPIBACE substantially up-regulates the secretion of both sAPPβ and amyloid-β peptide over levels observed from cells overexpressing wild-type BACE. This effect was reversed when the lipid rafts were disrupted by depleting cellular cholesterol levels. These results suggest that processing of APP to the amyloid-β peptide occurs predominantly in lipid rafts and that BACE is the rate-limiting enzyme in this process. The processing of the APP695 isoform by GPI-BACE was up-regulated 20-fold compared with wild-type BACE, whereas only a 2-fold increase in the processing of APP751/770 was seen, implying a differential compartmentation of the APP isoforms. Changes in the local membrane environment during aging may facilitate the cosegregation of APP and BACE leading to increased β-amyloid production.

Evaluation of angiotensin-converting enzyme (ACE), its homologue ACE2 and neprilysin in angiotensin peptide metabolism

In the RAS (renin-angiotensin system), Ang I (angiotensin I) is cleaved by ACE (angiotensin-converting enzyme) to form Ang II (angiotensin II), which has effects on blood pressure, fluid and electrolyte homoeostasis. We have examined the kinetics of angiotensin peptide cleavage by full-length human ACE, the separate N- and C-domains of ACE, the homologue of ACE, ACE2, and NEP (neprilysin). The activity of the enzyme preparations was determined by active-site titrations using competitive tight-binding inhibitors and fluorogenic substrates. Ang I was effectively cleaved by NEP to Ang (1-7) (kcat/K(m) of 6.2x10(5) M(-1) x s(-1)), but was a poor substrate for ACE2 (kcat/K(m) of 3.3x10(4) M(-1) x s(-1)). Ang (1-9) was a better substrate for NEP than ACE (kcat/K(m) of 3.7x10(5) M(-1) x s(-1) compared with kcat/K(m) of 6.8x10(4) M(-1) x s(-1)). Ang II was cleaved efficiently by ACE2 to Ang (1-7) (kcat/K(m) of 2.2x10(6) M(-1) x s(-1)) and was cleaved by NEP (kcat/K(m) of 2.2x10(5) M(-1) x s(-1)) to several degradation products. In contrast with a previous report, Ang (1-7), like Ang I and Ang (1-9), was cleaved with a similar efficiency by both the N- and C-domains of ACE (kcat/K(m) of 3.6x10(5) M(-1) x s(-1) compared with kcat/K(m) of 3.3x10(5) M(-1) x s(-1)). The two active sites of ACE exhibited negative co-operativity when either Ang I or Ang (1-7) was the substrate. In addition, a range of ACE inhibitors failed to inhibit ACE2. These kinetic data highlight that the flux of peptides through the RAS is complex, with the levels of ACE, ACE2 and NEP dictating whether vasoconstriction or vasodilation will predominate.

The angiotensin–converting enzyme gene family: genomics and pharmacology

Modulation of the renin-angiotensin system (RAS), and particularly inhibition of angiotensin-converting enzyme (ACE), a zinc metallopeptidase, has long been a prime strategy in the treatment of hypertension. However, other angiotensin metabolites are gaining in importance as our understanding of the RAS increases. Recently, genomic approaches have identified the first human homologue of ACE, termed ACEH (or ACE2). ACEH differs in specificity and physiological roles from ACE, which opens a potential new area for discovery biology. The gene that encodes collectrin, a homologue of ACEH, is upregulated in response to renal injury. Collectrin lacks a catalytic domain, which indicates that there is more to ACE-like function than simple peptide hydrolysis.

Rare coding variants in the phospholipase D3 gene confer risk for Alzheimer's disease

Genome-wide association studies (GWAS) have identified several risk variants for late-onset Alzheimers disease (LOAD). These common variants have replicable but small effects on LOAD risk and generally do not have obvious functional effects. Low-frequency coding variants, not detected by GWAS, are predicted to include functional variants with larger effects on risk. To identify low-frequency coding variants with large effects on LOAD risk, we carried out whole-exome sequencing (WES) in 14 large LOAD families and follow-up analyses of the candidate variants in several large LOAD case–control data sets. A rare variant in PLD3 (phospholipase D3; Val232Met) segregated with disease status in two independent families and doubled risk for Alzheimer’s disease in seven independent case–control series with a total of more than 11,000 cases and controls of European descent. Gene-based burden analyses in 4,387 cases and controls of European descent and 302 African American cases and controls, with complete sequence data for PLD3, reveal that several variants in this gene increase risk for Alzheimer’s disease in both populations. PLD3 is highly expressed in brain regions that are vulnerable to Alzheimer’s disease pathology, including hippocampus and cortex, and is expressed at significantly lower levels in neurons from Alzheimer’s disease brains compared to control brains. Overexpression of PLD3 leads to a significant decrease in intracellular amyloid-β precursor protein (APP) and extracellular Aβ42 and Aβ40 (the 42- and 40-residue isoforms of the amyloid-β peptide), and knockdown of PLD3 leads to a significant increase in extracellular Aβ42 and Aβ40. Together, our genetic and functional data indicate that carriers of PLD3 coding variants have a twofold increased risk for LOAD and that PLD3 influences APP processing. This study provides an example of how densely affected families may help to identify rare variants with large effects on risk for disease or other complex traits.

The prion protein and lipid rafts (Review)

Prions are the causative agent of the transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrPC) undergoes a post-translational conformational conversion to the infectious form (PrPSc). PrPC associates with cholesterol- and glycosphingolipid-rich lipid rafts through association of its glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and through interaction of its N-terminal region with an as yet unidentified raft associated molecule. PrPC resides in detergent-resistant domains that have different lipid and protein compositions to the domains occupied by another GPI-anchored protein, Thy-1. In some cells PrPC may endocytose through caveolae, but in neuronal cells, upon copper binding to the N-terminal octapeptide repeats, the protein translocates out of rafts into detergent-soluble regions of the plasma membrane prior to endocytosis through clathrin-coated pits. The current data suggest that the polybasic region at its N-terminus is required to engage PrPC with a transmembrane adaptor protein which in turn links with the clathrin endocytic machinery. PrPC associates in rafts with a variety of signalling molecules, including caveolin-1 and Fyn and Src tyrosine kinases. The clustering of PrPC triggers a range of signal transduction processes, including the recruitment of the neural cell adhesion molecule to rafts which in turn promotes neurite outgrowth. Lipid rafts appear to be involved in the conformational conversion of PrPC to PrPSc, possibly by providing a favourable environment for this process to occur and enabling disease progression.

Cellular prion protein regulates beta-secretase cleavage of the Alzheimer's amyloid precursor protein

Proteolytic processing of the amyloid precursor protein (APP) by β-secretase, β-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid β (Aβ) peptide, which is involved in the pathogenesis of Alzheimers disease. The normal cellular function of the prion protein (PrPC), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt–Jakob disease in humans, remains enigmatic. Because both APP and PrPC are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrPC in the proteolytic processing of APP. Cellular overexpression of PrPC inhibited the β-secretase cleavage of APP and reduced Aβ formation. Conversely, depletion of PrPC in mouse N2a cells by siRNA led to an increase in Aβ peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Aβ levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the β-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrPC on the β-secretase cleavage of APP required the localization of PrPC to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrPC via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Aβ is regulated by PrPC and may have implications for both Alzheimers and prion diseases.