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Constitutive shedding of the amyloid precursor protein ectodomain is up-regulated by tumour necrosis factor-alpha converting enzyme.

ABSTRACT: The amyloid precursor protein (APP) of Alzheimer's disease is a transmembrane protein that is cleaved within its extracellular domain, liberating a soluble N-terminal fragment (sAPP alpha). Putative mediators of this process include three members of the ADAM (a disintegrin and metalloprotease) family, ADAM9, ADAM10 and ADAM17/TACE (tumour necrosis factor-alpha converting enzyme). Tumour necrosis factor-alpha protease inhibitor (TAPI-1), an inhibitor of ADAMs, reduced constitutive and muscarinic receptor-stimulated sAPP alpha release in HEK-293 cells stably expressing M3 muscarinic receptors. However, the former was less sensitive to TAPI-1 (IC(50)=8.09 microM) than the latter (IC(50)=3.61 microM), suggesting that these processes may be mediated by different metalloproteases. Constitutive sAPP alpha release was increased several-fold in cells transiently transfected with TACE, and this increase was proportional to TACE expression. In contrast, muscarinic-receptor-activated sAPP alpha release was not altered in TACE transfectants. TACE-dependent constitutive release of co-transfected APP(695) was inhibited by TAPI-1 with an IC(50) of 0.92 microm, a value significantly lower than the IC(50)s for inhibition of either constitutive or receptor-regulated sAPP alpha shedding mediated by endogenous secretases. The results indicate that TACE is capable of catalysing constitutive alpha-secretory cleavage of APP, but it is likely that additional members of the ADAM family mediate endogenous constitutive and receptor-coupled release of sAPP alpha in HEK-293 cells.

SUBMITTER: Slack BE

PROVIDER: S-EPMC1222008 | biostudies-other | 2001 Aug

REPOSITORIES: biostudies-other

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Tumour necrosis factor alpha-converting enzyme mediates ectodomain shedding of Vps10p-domain receptor family members.

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Systematic evaluation of candidate ligands regulating ectodomain shedding of amyloid precursor protein.

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Selective ectodomain phosphorylation and regulated cleavage of beta-amyloid precursor protein.

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Structural insights into calmodulin-regulated L-selectin ectodomain shedding.

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Up regulated expression of tumour necrosis factor {alpha} converting enzyme in peripheral monocytes of patients with early systemic sclerosis.

Project description:Systemic sclerosis (SSc) is accompanied by abnormalities in humoral and cellular immune systems.To determine the genes specifically expressed in the immune system in SSc by analysis of the gene expression profile of peripheral blood mononuclear cells (PBMC) from patients with SSc, including those treated with haematopoietic stem cell transplantation (HSCT). Additionally, to investigate the clinical significance of the up regulation of tumour necrosis factor alpha (TNFalpha) converting enzyme (TACE).PBMC from patients with SSc (n = 23) and other autoimmune diseases (systemic lupus erythematosus (SLE, n = 16), rheumatoid arthritis (RA, n = 29)), and from disease-free controls (n = 36) were examined. Complementary DNA arrays were used to evaluate gene expression of PBMC, in combination with real time quantitative polymerase chain reactions. TACE protein expression in PBMC was examined by fluorescence activated cell sorter (FACS).In patients with SSc 118 genes were down regulated after HSCT. Subsequent comparative analysis of SSc without HSCT and healthy controls indicated SSc-specific up regulation for three genes: monocyte chemoattractant protein-3 (p = 0.0015), macrophage inflammatory protein 3alpha (p = 0.0339), and TACE (p = 0.0251). In the FACS analysis, TACE protein was mainly expressed on CD14(+) monocytes both in patients with SSc and controls. TACE expression on CD14(+) cells was significantly increased in patients with early SSc (p = 0.0096), but not in those with chronic SSc, SLE, or RA. TACE protein levels in SSc monocytes correlated with the intracellular CD68 levels (p = 0.0016).Up regulation of TACE expression was a unique profile in early SSc, and may affect the function of TNFalpha and other immunoregulatory molecules.

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Constitutive and regulated alpha-secretase cleavage of Alzheimer's amyloid precursor protein by a disintegrin metalloprotease.

Project description:Amyloid beta peptide (Abeta), the principal proteinaceous component of amyloid plaques in brains of Alzheimer's disease patients, is derived by proteolytic cleavage of the amyloid precursor protein (APP). Proteolytic cleavage of APP by a putative alpha-secretase within the Abeta sequence precludes the formation of the amyloidogenic peptides and leads to the release of soluble APPsalpha into the medium. By overexpression of a disintegrin and metalloprotease (ADAM), classified as ADAM 10, in HEK 293 cells, basal and protein kinase C-stimulated alpha-secretase activity was increased severalfold. The proteolytically activated form of ADAM 10 was localized by cell surface biotinylation in the plasma membrane, but the majority of the proenzyme was found in the Golgi. These results support the view that APP is cleaved both at the cell surface and along the secretory pathway. Endogenous alpha-secretase activity was inhibited by a dominant negative form of ADAM 10 with a point mutation in the zinc binding site. Studies with purified ADAM 10 and Abeta fragments confirm the correct alpha-secretase cleavage site and demonstrate a dependence on the substrate's conformation. Our results provide evidence that ADAM 10 has alpha-secretase activity and many properties expected for the proteolytic processing of APP. Increases of its expression and activity might be beneficial for the treatment of Alzheimer's disease.

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Characterization of angiotensin-converting enzyme 2 ectodomain shedding from mouse proximal tubular cells.

Project description:Angiotensin-converting enzyme 2 (ACE2) is highly expressed in the kidney proximal tubule, where it cleaves angiotensin (Ang) II to Ang-(1-7). Urinary ACE2 levels increase in diabetes, suggesting that ACE2 may be shed from tubular cells. The aim of this study was to determine if ACE2 is shed from proximal tubular cells, to characterize ACE2 fragments, and to study pathways for shedding. Studies involved primary cultures of mouse proximal tubular cells, with ACE2 activity measured using a synthetic substrate, and analysis of ACE2 fragments by immunoblots and mass spectrometry. The culture media from mouse proximal tubular cells demonstrated a time-dependent increase in ACE2 activity, suggesting constitutive ACE2 shedding. ACE2 was detected in media as two bands at ∼ 90 kDa and ∼ 70 kDa on immunoblots. By contrast, full-length ACE2 appeared at ∼ 100 kDa in cell lysates or mouse kidney cortex. Mass spectrometry of the two deglycosylated fragments identified peptides matching mouse ACE2 at positions 18-706 and 18-577, respectively. The C-terminus of the 18-706 peptide fragment contained a non-tryptic site, suggesting that Met(706) is a candidate ACE2 cleavage site. Incubation of cells in high D-glucose (25 mM) (and to a lesser extent Ang II) for 48-72 h increased ACE2 activity in the media (p<0.001), an effect blocked by inhibition of a disintegrin and metalloproteinase (ADAM)17. High D-glucose increased ADAM17 activity in cell lysates (p<0.05). These data indicate that two glycosylated ACE2 fragments are constitutively shed from mouse proximal tubular cells. ACE2 shedding is stimulated by high D-glucose, at least partly via an ADAM17-mediated pathway. The results suggest that proximal tubular shedding of ACE2 may increase in diabetes, which could enhance degradation of Ang II in the tubular lumen, and increase levels of Ang-(1-7).

| S-EPMC3893316 | biostudies-literature

The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity.

Project description:sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the k(cat) for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates.

| S-EPMC1180724 | biostudies-literature

Roles of the juxtamembrane and extracellular domains of angiotensin-converting enzyme in ectodomain shedding.

Project description:Angiotensin-converting enzyme (ACE) is one of a growing number of integral membrane proteins that is shed from the cell surface through proteolytic cleavage by a secretase. To investigate the requirements for ectodomain shedding, we replaced the glycosylphosphatidylinositol addition sequence in membrane dipeptidase (MDP) - a membrane protein that is not shed - with the juxtamembrane stalk, transmembrane (TM) and cytosolic domains of ACE. The resulting construct, MDP-STM(ACE), was targeted to the cell surface in a glycosylated and enzymically active form, and was shed into the medium. The site of cleavage in MDP-STM(ACE) was identified by MS as the Arg(374)-Ser(375) bond, corresponding to the Arg(1203)-Ser(1204) secretase cleavage site in somatic ACE. The release of MDP-STM(ACE) and ACE from the cells was inhibited in an identical manner by batimastat and two other hydroxamic acid-based zinc metallosecretase inhibitors. In contrast, a construct lacking the juxtamembrane stalk, MDP-TM(ACE), although expressed at the cell surface in an enzymically active form, was not shed, implying that the juxtamembrane stalk is the critical determinant of shedding. However, an additional construct, ACEDeltaC, in which the N-terminal domain of somatic ACE was fused to the stalk, TM and cytosolic domains, was also not shed, despite the presence of a cleavable stalk, implying that in contrast with the C-terminal domain, the N-terminal domain lacks a signal required for shedding. These data are discussed in the context of two classes of secretases that differ in their requirements for recognition of substrate proteins.

| S-EPMC1222046 | biostudies-other

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