Download Characterisation of the TNFalpha Converting Enzyme (TACE

Biochemical Society Transactions (1999) 27
H2 Characterisation of the TNFa Converting
Enzyme (TACEIADAM17) Deficient Mouse and
Its Role in the Shedding of Cell Surface Proteins.
D.P. Cerratt, C. Rauch, K. Kozlosky, N. Nelson, P. Reddy, J.
Peschon, J. Slack, R. Paxton, and R. Black
lmmunex Corporation, 51 University Street, Seattle, WA
98101 USA
Tumor necrosis factor (TNF)a is released from cells by a
proteolytic cleavage of the membrane-bound (26 kDa)
precursor. Hydroxamate-based inhibitors of metalloproteases block this process. We have now purified and
cloned a novel metalloprotease, called the TNFa converting
enzyme (TACE), that specifically cleaves 26 kDa TNFa at
the physiological processing site. Sequence of the cDNA
indicates that TACE is a member of the adamalysin (snake
venom)
subfamily
of
zinc
binding metallopro t eases/d isi nt eg ri ns (A DAM s) whose ma m ma1ia n
members are type 1 transmembrane proteins. TACE
mRNA was found to be expressed in many tissues. A
soluble form (extracellular domain) of the enzyme was
expressed and found to cleave pro-TNFa at it's processing
site. Moreover, T-cells, myeloid cells, and fibroblasts whose
TACE gene was inactivated have a reduced ability to shed
TNFa (80 to go%), confirming that this protein is the TNFa
converting enzyme. In addition, through additional
biochemical analysis and a study of transgenic animals, we
have discovered that TACE/ADAM17 is required for
multiple shedding events.
H4 Angiotensin converting enzyme and the amyloid
precursor protein secretases
Nigel M. Hower, S.Parvathy, Eric H. K a m * & Anthony J. Turner
School of Biochemistry and Molecular Biology, University of Leeds,
Leeds LS2 9JT, U.K. and *Neurosciences Research, SmithKline
Beecham Pharmaceuticals, Harlow CM19 5AW, U.K.
Angiotensin converting enzyme (ACE) and the Alzheimer's amyloid
precursor protein (APP) are both Type I integral membrane glycoproteins
that are subject to prdeolytic cleavage and release from the membrane by
secretases. In the case of APP it is cleavage wahin the amyloid peptide
domain by a-secretase that releases the large ectodomain of APP
(sAPPa) which has neuroprotective properties. ACE plays a key role in the
control of blood pressure homeostasis and inhibitors of the enzyme are used
clinically in the treatment of hypertension and congestive heart failure. Bdh
ACE secretase and a-secretasecleave their respective substrates betweer
a basic and a hydrophobic residue (Arg-Leu and Lys-Leu, respectively)
and are stimulated by phorbol esters. Furthermore, both secretases appear
to be integral membrane zinc metalloprateinases, inhibited by 1,lOphenanthroline, resistant to removal from the membrane by high salt, and
solubilized by the detergents Triton X-1 00 and CHAPS but not by octyl
glucoside. Studies with synthetic lipid vesicles indicate that ACE
secretase has an absolute requirement for its substrate to be membrane
inserted. Recently we have shown that a-secretase and ACE secretase
&splay a similar mhibition profile with a range of hydroxamic acid-based
zinc metalloproteinase inhibitors. The release of APP from neurcmal cells
by a-secretase and the release of ACE from kidney microvillar
membranes or ACE transfectedneuronal cells was blocked by batimastat,
marimastat, BB2116 and other related synthetic hydroxamic acid-based
zinc metallopratease inhibitors with 150 values in the low micromolar
range. Removal of the thienothiomethyl substituent adjacent to the
hydroxamic acid moiety or the substitution of the P i substituent
decreased the lnhibitory potency of batimastat towards both a-secretase
and ACE secretase. Thus a-secretase and ACE secretase appear to be
identical, or closely related, integral membrane zinc metalloproteinases.
H3 Interleukin-6 Receptor (IL-6R) Shedding: A Role for Members
of the ADAM Family. Peter I. Cro&
Fengfei Wang and
Philip G. Hargreaves. Division of Biochemical a n d
Musculoskeletal Medicine, University of Sheffield Medical School,
Beech Hill Road, Sheffield, UK, S10 2RX.
Interleukin-6 exerts its biological effect by binding to the IL-6R. This
complex associates with a transmembrane molecule, known as gp130,
which is responsible for transducing a signal to the cell. A soluble form
of the IL-6R (sIL-6R) has been identified which is unusual amongst
soluble cytokine receptors in that it retains biological activity and acts
as an agonist by binding IL-6, associating with gp130 and allowing
signal transduction. The sIL-6R provides a novel means of regulating
cellular responses to IL-6, since cells that do not express the IL-6R but
express gp130 can, in the presence of sIL-~R,respond to IL-6.
A number of cell types constitutively produce the sIL-6R, although
our understanding of the mechanism by which the sIL-6R is generated
is incomplete. An alternate spliced form of the IL-R, lacking the
sequence predicted to encode the transmembrane domain, has been
identified. However, studies have also shown that sIL-6R can be
produced by proteolytic cleavage of the membrane-bound IL-6R.
Treatment of cells with phorbol 12-myristate 13-acetate (PMA)
promotes a significant decrease in membrane-bound IL-6R and the
appearance of IL-6R in the cell supernatant. Inhibitors of serine and
cysteine proteinases or the naturally occurring tissue inhibitors of
matrix metalloproteinases (TIMP-1 and TIMP-2) have no effect on IL6R shedding. In contrast, TIMP-3 and hydroxamate-based
metalloproteinase inhibitors are able to inhibit both constitutive and
PMA-induced IL-6R shedding, suggesting that a non-matrix type
metalloproteinase is responsible for this activity. Although the identity
of this proteinase is unknown the enzyme responsible for processing
tumour necrosis factor-a (TACE) has recently been identified and
shown to be a member of the ADAM family (a disintegrin and
metalloproteinase). This activity has a very similar inhibition profile to
the ILdR secretase. Furthermore, cells that process IL-6R also express
several members of the ADAM family.
Taken together, these data suggest that the sIL-6R can be generated
by proteolytic cleavage by a non-matrix type metalloproteinase.
Members of the ADAM family of metalloproteinases currently
represent the strongest candidates for this activity.
A23
H5
Proteolytic processing of 6-amyloid precursor protein and
presenilins
Harald Steiner and Christian Haass
central Institute of Mental Health; Department of Molecular
Biology; J5; 68159 Mannheim; Germany
Alzheimer's disease is characterized by the invariable
accumulation of senile plaques that are predominantly
composed of amyloid D-peptide (AD). AD is derived by
proteolytic processing from the D-amyloid precursor protein
(DAPP). AD is liberated from DAPP by a physiological normal
process involving the combined action of two secretases, Dsecretase (cleaving at the N-terminus of the AD domain) and ysecretase (cleaving at the C-terminus of the AD-domain).
Cleavage within the AD domain by a-secretase prevents AD
generation. In some very rare cases of familial AD (FAD),
mutations have been identified within the BAPP gene. These
mutations are located close to or at the cleavage sites of all three
secretases. The DAPP mutations pathologically effect DAPP
processing by increasing AD production, specifically its 42
amino acid version (AD42). The much more abundant A040
requires reintemalization for the D-secretase cleavage and
recycling to the cell surface for the y-secretase cleavage. In
contrast to AD40, AD42 can be detected inside the cell within the
endoplasmic reticulum (ER) and the Golgi.
Intracellular
production of AD42 within the ER and the Golgi relates to the
subcellular localization of the FAD associated Presenilins, PSI
and PS2, which have been detected within the same
compartments.
Numerous mutations causing early onset
Alzheimer's disease have been identified in the Presenilin (PS)
genes, particularly the PS1 gene. Like the mutations identified
within the D-amyloid precursor protein gene, PS mutations cause
the increased generation of AD42.
PS proteins are
proteolytically processed to a N-terminal -30 kDa (NTF) and a
C-terminal -20 kDa fragment (CTF,,) which form a
heterodimeric complex. Expression of PSI is highly controlled
by multiple proteolytic activities and complex formation.
Recent work suggests that wt PS 1 proteins activate the y-secretase
cleavage. In contrast to the FAD associated PS mutations, wt
PS 1 promotes the production of both AD40 as well as AD42.