Gary Christie

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.

Inhibitor of neurite outgrowth in humans.

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.

Neurobiology: Inhibitor of neurite outgrowth in humans

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.

Characterization of peptidyl boronic acid inhibitors of mammalian 20 S and 26 S proteasomes and their inhibition of proteasomes in cultured cells

Proteasomes are large multisubunit proteinases which have several distinct catalytic sites. In this study a series of di- and tri-peptidyl boronic acids have been tested on the chymotrypsin-like activity of purified mammalian 20 S and 26 S proteasomes assayed with succinyl-Leu-Leu-Val-Tyr-amidomethylcoumarin (suc-Leu-Leu-Val-Tyr-AMC) as substrate. The inhibition of 20 S proteasomes is competitive but only slowly reversible. The K(i) values for the best inhibitors were in the range 10-100 nM with suc-Leu-Leu-Val-Tyr-AMC as substrate, but the compounds tested were much less effective on other proteasome activities measured with other substrates. Free boronic acid inhibitors exhibited equivalent potency to their pinacol esters. Both benzoyl (Bz)-Phe-boroLeu and benzyloxycarbonyl (Cbz)-Leu-Leu-boroLeu pinacol ester inhibited 20 S and 26 S proteasomes with non-ideal behaviour, differences in inhibition of the two forms of proteasomes becoming apparent at high inhibitor concentrations (above 3xK(i)). Both of these compounds were also potent inhibitors of 20 S and 26 S proteasomes in cultured cells. However, gel filtration of cell extracts prepared from cells treated with radiolabelled phenacetyl-Leu-Leu-boroLeu showed that only 20 S proteasomes were strongly labelled, demonstrating differences in the characteristics of inhibition of 20 S and 26 S proteasomes. The usefulness of peptidyl boronic acid inhibitors for investigations of proteasome-mediated protein degradation was confirmed by the observation that Bz-Phe-boroLeu and Cbz-Leu-Leu-boroLeu pinacol ester inhibited NFkappaB activation with IC(50) values comparable to their K(i) values for purified proteasomes. The latter result supports the view that the chymotrypsin-like activity of proteasomes assayed with suc-Leu-Leu-Val-Tyr-AMC is a critical one for protein degradation in cells.

In search of an enzyme: the β-secretase of Alzheimer's disease is an aspartic proteinase

The deposition of beta-amyloid (Abeta) in the brain is a neuropathological feature of Alzheimers disease. Abeta is cleaved from its precursor protein (APP) by processing at its N and C termini by enzymes known as beta- and gamma-secretases,respectively. The identity of these enzymes has been elusive but the search for the N-terminal secretase might have ended recently with the almost simultaneous publication by five major laboratories claiming a transmembrane aspartic proteinase to be the long sought after beta-secretase. Even at this early stage of its characterization, this aspartic proteinase fulfils many of the key criteria necessary for beta-secretase. The race is now on to develop inhibitors that could prove effective in halting the progression of Alzheimers disease.

Endocytosis and recycling of the complex between CD23 and HLA‐DR in human B cells

The presentation of extremely low doses of antigen to T cells is enhanced by immunoglobulin E (IgE)‐dependent antigen focusing to CD23, the low‐affinity receptor for IgE, expressed on activated B cells. CD23 contains a C‐type lectin domain in its extracellular sequence and a targeting signal for coated pits, required for endocytosis, in its cytoplasmic sequence. CD23 is non‐covalently associated with the major histocompatibility complex class II antigen, human leucocyte antigen HLA‐DR, on the surface of human B cells, but the fate of this complex following endocytosis is unknown. To answer this question we have labelled these proteins on the surface of RPMI 8866 B cells and traced their route through the cytoplasm. Endocytosis mediated by anti‐CD23 antibodies (BU38 and MHM6) led to the loss of CD23 from the cells. Endocytosis mediated by an antibody to HLA‐DR (CR3/43) or an antigen–IgE complex (NP‐BSA–anti‐NP IgE), however, led to recycling of the HLA‐DR–CD23 complex to the cell surface on a time scale (3–6 hr) consistent with the recycling of HLA‐DR in antigen presentation. Along the latter pathway CD23 label was observed in cytoplasmic organelles that resembled the ‘compartments for peptide loading’ or ‘class II vesicles’ described by previous authors. Two features of the recycling process may contribute to the efficiency of antigen presentation. Peptide exchange may be facilitated by the proximity of HLA‐DR and antigen in peptide loading compartments of the endosomal network. The return of CD23 with HLA‐DR to the cell surface may then help to stabilize specific B‐cell–T‐cell interactions, contributing to T‐cell activation.

Alzheimer's disease : Correlation of the suppression of β-amyloid Peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome

Abstract: Peptide aldehyde inhibitors of the chymotrypsin‐like activity of the proteasome (CLIP) such as N‐acetyl‐Leu‐Leu‐Nle‐H (or ALLN) have been shown previously to inhibit the secretion of β‐amyloid peptide (Aβ) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on Aβ formation of a much wider range of peptide‐based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide‐based inhibitors of β‐amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress Aβ formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in Aβ formation.

In vitro cytocidal effects on Trypanosoma brucei and inhibition of Leishmania major GP63 by peptidomimetic metalloprotease inhibitors.

Peptidomimetic inhibitors of mammalian zinc metalloproteases have been tested as potential agents for intervention in disease caused by kinetoplastid protozoa. Certain metalloprotease inhibitors were able to inhibit the release of variant surface glycoprotein from cultured transgenic procyclic Trypanosoma brucei, confirming our previous identification of a cell surface zinc metalloprotease activity in this stage of the trypanosome lifecycle [Bangs, JD et al. Expression of bloodstream variant surface glycoproteins in procyclic stage Trypanosoma brucei: role of GPI anchors in secretion, EMBO J. 1997;16:4285]. Selected peptidomimetics were also found to be toxic for cultured bloodstream trypanosomes with IC50 values in the low micromolar range. The paradigm for zinc metalloproteases in kinetoplastids are the GP63 surface enzymes of Leishmania. Peptidomimetics at low micromolar concentrations were able to inhibit in vitro cleavage of a synthetic peptide substrate by purified GP63 from L. major. Our results suggest that zinc metalloproteases perform essential functions in different stages of the trypanosome lifecycle and we hypothesize that these activities may be affected by the recently discovered trypanosomal homologues of GP63 [El-Sayed, NMA and Donelson, JE. African trypanosomes have differentially expressed genes encoding homologues of Leishmania GP63 surface protease, J. Biol. Chem. 1997;272:26742]. Development of higher affinity metalloprotease inhibitors may provide a novel avenue for treatment of parasitic diseases.

Inhibitor of neurite outgrowth in humans: Neurobiology

Axons are generally believed to be incapable of regeneration in the adult central nervous system. Inhibition results from physical barriers imposed by glial scars, a lack of neurotrophic factors, and growth-inhibitory molecules associated with myelin, the insulating axon sheath. These molecules include proteoglycans, myelin-associated glycoprotein and, in bovine brain, two proteins called Nogo. We have used this bovine sequence to identify the human Nogo gene and have isolated complementary DNA clones encoding three different Nogo isoforms that are potent inhibitors of neurite outgrowth and which may help block the regeneration of the central nervous system in adults.