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By,Prarthana.Pai
1
Matrix Metalloproteinases
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CONTENTS
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Introduction
Structure
Classification
Individual MMPs Regulation of activation of MMPs
Activation of Pro-MMPs
Inhibitors of MMPs
MMPs and periodontal disease Conclusion
References
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Introduction
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Extracellular matrix (ECM) macromolecules are important for
creating the cellular environments required during
development and morphogenesis.
Matrix Metalloproteinases (MMPs), collectively called
Matrixins, is a large family of calcium-dependent zinc-
containing endopeptidases, that participate in ECM
degradation.
Under normal physiological conditions, the activities of MMPs
are precisely regulated at the level of:
Transcription
Activation of the precursor zymogens
Interaction with specific ECM components
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A loss of activity control may result in diseases such
as arthritis, cancer, atherosclerosis, aneurysms,nephritis, tissue ulcers, periodontal destruction and
fibrosis.
MMPs are excreted by a variety of connective tissue
and pro-inflammatory cells including fibroblasts,
osteoblasts, endothelial cells, macrophages,
neutrophils, and lymphocytes.
These enzymes are expressed as zymogens, which
are subsequently processed by other proteolytic
enzymes to generate the active forms.
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The various physiological and pathological processes for whichMMPs have been implicated are:
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History
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The earliest descriptions of MMPs were in 1949 asdepolymerizing enzymes which, it was proposed,
could facilitate tumor growth by making connective
tissue stroma, including that of small blood vessels,
more fluid.
About after 13 years, in 1962 Jerome Gross andCharles Lapire found an active enzyme in the
culture media of tissue fragments of tail fin skin that
degraded the triple helix of native type I collagen.
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During the next 20 years, several mammalian
enzymes were partially purified, but it was not until
1985 that the field really developed when structural
homologies became apparent, allowing many newmembers to be identified through the techniques of
molecular biology.
Since then, at least 26 human MMPs have been
characterized.
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Structure of MMPs
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The MMPs starting from the Nterminus, show the
following features: Massova et al., 1998
Signal peptide
Propeptide Furin-cleavage site insert
Catalytic domain
Fibronectin-like repeats
Hinge region Hemopexin domain
Membrane insertion extension
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Signal peptide:
It is typically a stretch of 17 to 20 residues, rich in
hydrophobic amino acids, that serves as a signal forsecretion into the endoplasmic reticulum for eventual
export from the cell.
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Propeptide domain:
It consists of approximately 80 to 90 amino acids
containing a cysteine residue, which interacts with thecatalytic zinc atom via its side chain thiol group.
A highly conserved sequence is present in the
propeptide.
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Removal of the propeptide by proteolysis results in
proenzyme activation, as all members of the MMPs familyare produced in a latent form.
The Cys within this sequence (the cysteine switch)
ligates the catalytic zinc to maintain the latency of pro-MMPs
Borden and Heller, 1997
This cysteine is found in all MMPs, including those that
have the furin-cleavage site.
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Furin-cleavage site insert:
This stretch of about nine residues includes the consensussequence of RXKR/RRKR that leads to intracellular cleavageby furin.
MMP-11, -14,-15, -16, and-17 possess this sequence
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In the remaining enzymes, a cleavage by externalproteases occurs in the middle of the propeptide,
partially exposing the zinc and leading to autolytic
cleavage of the remainder of the propeptide.
The exact site of final cleavage may vary within a given
enzyme leading to different degrees of activation,particularly in the case of MMP-1.
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The catalytic domain:
It is made of about 170 amino acids.
It contains two zinc ions and at least one calcium ion
coordinated to various residues.
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One of the two zinc ions is present in the active site andis involved in the catalytic processes of the MMPs.
The catalytic zinc ion is essential for the proteolyticactivity of MMPs; the three histidine residues thatcoordinate with the catalytic zinc are conserved among
all the MMPs.
The 50 to 54 residues at the C-terminal end of thecatalytic domain include the site of binding of the
catalytic zinc.
Little is known about the roles of the second zinc ion andthe calcium ion within the catalytic domain, but the
MMPs are shown to possess high affinities for structuralzinc and calcium ions (Bode et al., 1994).
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Fibronectin-like repeats:
These specialized structures aid the binding of enzyme
to gelatin and collagen substrates
Steffensen et al., 1995
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Hinge region:
The catalytic domain is connected to the following
hemopexin domain by a linker region usually referred to
as the hinge region.
It ranges in length from 0 to 75 residues.
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The longest hinge is found in MMP-9 and shows
considerable homology to type V collagen in that it is rich
in proline.
A typical hinge contains about 16 residues including a
number of proline residues
Pendas et al., 1997
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The hemopexin-like domain:
It is made up of about 210 amino acids.
It is highly conserved and shows sequence similarity to
the plasma protein, hemopexin.
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The hemopexin-like domain has been shown to play a
functional role in substrate binding and/or in interactionswith the TIMPs, a family of specific MMP protein
inhibitors
Gomis-Rth et al., 1997
The hemopexin domain is an absolute requirement for
collagenases to cleave triple helical interstitial collagens(Bode, 1995), although the catalytic domains alone
retain proteolytic activity toward other substrates.
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Membrane insertion extension:
Although most MMPs have their C terminus at the end of
the hemopexin domain, the four MT-MMPs have a further
extension that governs insertion of these proteases into
the cell membrane.
Its length ranges from about 80 to 110 residues.
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Classification
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According to their substrate specificity, sequence similarity,
and domain organization (Massova et al.,1998)
Collagenases (MMP-1, 8, 13 and 18)Gelatinases (MMP-2 and 9)
Stromelysins (MMP-3, 10, 11)
Elastases (MMP-7 and 12), and
Membrane type MMP (MT-mmps, MMP-14, 15,
16 and 17)
Other MMPs
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Individual MMPs
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Collagenases
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MMP-1, MMP-8, MMP-13, and MMP-18 (Xenopus) are in
this group.
The key feature of these enzymes is their ability to
cleave interstitial collagens I, II, and III at a specific sitethree-fourths from the N-terminus.
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Collagenases can also digest a number of other ECM
and non-ECM molecules
Collagenases are actively taking part in the tissue
destruction and granulation tissue formation in chronicperiodontitis
Wahlgren, 2003
The collagenases differ in their substrate specificities
and functional roles.
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MMP 1
Interstitial collagenase/ collagenase 1
Most often associated collagenase with normal tissueremodeling
Expressed by fibroblasts, endothelial cells, macrophages,
hepatocytes,chondrocytes, osteoblasts, odontoclasts,
tumors cells and migrating epidermal keratinocytes.
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Expression can be induced in certain inflammatory
diseases and cancers
Giambernardi et al., 1998
MMP-1 gene is localized on chromosome 11q22
Brinckerhoff et al., 2000
It is constitutively expressed at low levels under normal
physiological conditions & its expression may increase
markedly in pathological conditions.
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MMP-8
It was first cloned from messenger RNA (mRNA)
extracted from the peripheral leucocytes of a patient with
chronic granulocytic leukemia
Hasty et al., 1990
Synthesized in polymorphonuclear leukocytes during their
maturation in bone marrow and stored in specific
intracellular granules, out of which it is secreted to the cellenvironment in response to external triggering stimuli and
the enzyme thus plays a key role in tissue destruction
during inflammatory diseases
Ding et al., 1997
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In periodontitis, the MMP-8 is expressed in variousinflammatory cells, such as PMNs, macrophages, plasma
cells and in endothelial cells.
In pulpitis, MMP-8 it is considered to originate from both
PMN and non-PMN lineage cells.
Wahlgren, 2003
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The major collagenase species detected in inflamedhuman periodontium is MMP-8
Romanelli et al., 1999
MMP-8 is expressed in vivo by:-o
Bronchial epithelial cells and macrophages involved inbronchiectasis,
o Chondrocytes in rheumatoid arthritis and osteoarthriticlesions
o Fibroblasts in human gingival sulcular epithelial cells
o Cells of human atheromao Plasma cells associated with oral keratocysts
o Cells in proliferating and migratory wound epithelia, indermal fibroblasts and inflammatory cells
Tervahartiala et al., 2000
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MMP-13
Originally cloned from human breast tumor cDNA library
by Hasty et al., 1990
It has the widest substrate selection among the interstitialcollagenases and, in addition to collagens, it is able to
cleave various Basement Membrane components.
MMP-13 cleaves type II collagen more efficiently thantypes I and III, and among interstitial collagenases, it is
most effective in cleaving gelatin (Ding et al., 1997).
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Gelatinases
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Gelatinase A (MMP-2) and gelatinase B (MMP-9) belongto this group
They readily digest the denatured collagens, gelatins.
These enzymes have three repeats of a type II
fibronectin domain inserted in the catalytic domain, which
bind to gelatin, collagens, and laminin.
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MMP-2, but not MMP-9, digests type I, II, and III
collagens.
Although MMP-2 null mice develop without any
apparent abnormality, mutations in human MMP-2
resulting in the absence of active enzyme are linkedwith an autosomal recessive form of multicentric
osteolysis, a rare genetic disorder that causes
destruction and resorption of the affected bones.
Martignetti JA, 2000
This suggests that MMP-2 in humans is important for
osteogenesis.
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Stromelysins
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Stromelysin 1 (MMP-3) and stromelysin 2 (MMP-10) bothhave similar substrate specificities, but MMP-3 has a
proteolytic efficiency higher than that of MMP-10 in
general.
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Besides digesting ECM components, MMP-3 activates anumber of proMMPs, and its action on a partially
processed proMMP-1 is critical for the generation of fully
active MMP-1.
MMP-11 is called stromelysin 3, but it is usually groupedwith otherMMPsbecause the sequence and substrate
specificity diverge from those of MMP-3
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ECM components, MMP-7 processes cell surface
molecules such as pro-defensin, Fas-ligand, protumor
necrosis factor (TNF)-, and E-cadherin.
Matrilysin 2 (MMP-26) also digests a number of ECM
components.
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Membrane-Type MMPs
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There are six membrane-type MMPs (MT-MMPs)
Four are type I transmembrane proteins (MMP-14,
MMP-15, MMP- 16, and MMP-24)
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Two are glycosylphosphatidylinositol (GPI) anchored
proteins (MMP-17 and MMP-25).
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With the exception of MT4-MMP, they are all capable of
activating proMMP-2.
These enzymes can also digest a number of ECM
molecules, and MT1-MMP has collagenolytic activity ontype I, II, and III collagens.
MT1-MMP null mice exhibit skeletal abnormalities during
postnatal development that are most likely due to lack ofcollagenolytic activity.
It also plays an important role in angiogenesis.
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MT5- MMP is brain specific and is mainly expressed in
the cerebellum.
MT6-MMP (MMP-25) is expressed almost exclusively in
peripheral blood leukocytes and in anaplastic
astrocytomas and glioblastomas but not in meningiomas
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Enamelysin (MMP-20), which digests amelogenin, is
primarily located within newly formed tooth enamel.
Amelogenin imperfecta, a genetic disorder caused by
defective enamel formation, is due to mutations at MMP-
20 cleavage sites.
MMP-22 was first cloned from chicken fibroblasts, and a
human homologue has been identified on the basis of
EST sequences. The function of this enzyme is notknown.
MMP-23, also called cysteine array MMP, is mainlyex ressed in re roductive tissues.
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The enzyme lacks the cysteine switch motif in the
prodomain.
It also lacks the hemopexin domain; instead, it has acysteine-rich domain followed by an immunoglobulin-like
domain.
It is proposed to be a type II membrane protein harboring
the transmembrane domain in the N-terminal part of the
propeptide.
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Regulation of activity of MMPs
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The activity of MMPs is regulated at several different
stages:
By transcriptional regulation of MMP genes -
endogenous growth factors and cytokines
By precursor activation Disruption of cysteine-
Zn++bond.
By differences in substrate specificity
By MMP inhibitors -macroglobulin and TIMPs-
1&2.
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Activation of precursors Transcriptional Regulationof MMP genes
Differences in substrate
specificity MMP inhibitors
MMPActivity
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Activation of ProMMPs
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MMPs can be activated by proteinases or in vitro by
chemical agents, such as thiol-modifying agents (4-aminophenylmercuric acetate, HgCl2, and N-
ethylmaleimide), oxidized glutathione, SDS, chaotropic
agents, and reactive oxygens.
Low pH and heat treatment can also lead to activation.
These agents most likely work through the disturbance of
the cysteine-zinc interaction of the cysteine switch.
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Latency of the virgin enzyme is maintained, at least in
part, by virtue of a putative Cys-Zn2+ bond that links the
unpaired propeptide Cys residue to the active site Zn2+and displaces the H2O molecule, which is necesssary for
catalysis.
Springman et al., 1990; Van Wart and Birkedal- Hansen,
1990
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Organomercurials, metal ions, thiol reagents, and
oxidants presumably interact directly with the propeptide
cys-residue to shift the equilibrium between the "closed"
and the "open" form toward the open form
Chaotropic agents (3M KI, 3M nascn) and detergents (1to 2% SDS) induce polypeptide chain conformational
changes that also shift the equilibrium entirely toward the
open form.
Proteolytic enzymes excise a portion of the propepeptide
so that the switch opens, most likely because of an
entro effect.
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Proteolytic activation of MMPs is stepwise.
The initial proteolytic attack occurs at an exposed loopregion between the first and the second helices of the
propeptide.
Th l ifi it f th b it i i di t t d b
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The cleavage specificity of the bait region is dictated by
the sequence found in each MMP.
Once a part of the propeptide is removed, this probably
destabilizes the rest of the propeptide, including the
cysteine switchzinc interaction, which allows the
intermolecular processing by partially activated MMP
intermediates or other active MMPs.
Activation of proMMPs by plasmin is a relevant pathway in
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Activation of proMMPs by plasminis a relevant pathway in
vivo.
Plasmin is generated from plasminogen by tissueplasminogen activator bound to fibrin and urokinase
plasminogen activator bound to a specific cell surface
receptor.
Both plasminogen and urokinase plasminogen activator
are membrane-associated, thereby creating localized
proMMP activation and subsequent ECM turnover.
Plasmin has been reported to activate proMMP-1,
proMMP-3, proMMP-7, proMMP-9, proMMP-10, and
proMMP-13.
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Intracellular Activation
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Most proMMPs are secreted from cells and activatedextracellularly.
Pei and Weiss first demonstrated that proMMP-11
(stromelysin 3) is activated intracellularly by furin.
ProMMP-11 possesses a furin recognition sequence,
KX(R/K)R, at the C-terminal end of the propeptide.
u vP MMP 2
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ProMMP-2
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ProMMP-2 is not readily activated by generalproteinases.
The main activation of proMMP-2 takes place on the cellsurface and is mediated by MT-MMPs.
MT1-MMPmediated activation of proMMP-2 has been
studied extensively. The unique aspect is that it requires
the assistance of TIMP-2.
ProMMP-2 forms a tight complex with TIMP-2 through their C-
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g p gterminal domains, therefore permitting the N-terminal inhibitorydomain of TIMP-2 in the complex to bind to MT1-MMP on thecell surface.
This MT1-MMPTIMP-2proMMP-2 complex is thenpresented to an adjacent free MT1-MMP for activation.
The cell surfacebound proMMP-2 is then activated by an
MT1- MMP that is free of TIMP-2.
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Major cell types of human periodontal tissues
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Major cell types of human periodontal tissues
capable of expressing unique complement of MMP
when stimulated
PMN Leukocytes
Interstitial collagenase (MMP8)
Gelatinase B (MMP9)
Keratinocytes
Fibroblast collagenase (MMP1)
Gelatinase A (MMP2)
Gelatinase B (MMP9)
Stromelysin 2 (MMP 10)
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Fibroblasts
Interstitial collagenase (MMP8)
Fibroblast collagenase (MMP1)
Stromelysin 1 (MMP3)
Stromelysin 3(MMP11)
Gelatinase A (MMP2)
Matrilysin (MMP7)
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Macrophage
Fibroblast collagenase (MMP1)
Stromelysin 1 (MMP3)
Gelatinase A (MMP2)
Gelatinase B (MMP9)
Endothelial Cell
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Osteoid is composed by type I collagen, proteoglycans,glycoproteins, and native types IV and IX collagens.
Osteoblast-derived collagenase (MMP-13) seems to bemain responsible for degrading the nonmineralizedosteoid layer covering bone surfaces, exposing themineralized matrix to osteoclasts.
MMP-13 (collagenase-3) is expressed in osteoblasts,periosteal cells and fibroblasts during human fetal bonedevelopment, and postnatally in bone remodeling.
Cleavage of collagen I by MMP-13 seems to be the initialstep of the entire bone resorption process.
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In chronic periodontitis MMP-8 increased
TIMP are not increased.
In LAP ( Localized Aggressive Periodontitis)
MMP-1 is increased.
Elevated levels of TIMPs are present.
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MMP Inhibitors
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Under pathological conditions associated withunbalanced MMP activities, changes of TIMP levels are
considered to be important because they directly affect
the level of MMP activity.
TIMPs have effects on cell growth and survival thatcannot always be clearly reconciled with their ability to
abrogate MMPs activity.
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TIMP-2 inhibits proMMP-2 over 10-fold more effectivelythan TIMP-1.
However, TIMP-2 has a bi-functional effect on MMP-2,since MT-MMP mediated proMMP-2 activation requires a
tiny amount of TIMP-2 to make activation progress,
whereas a greater concentration of TIMP-2 inhibits MMP-
2
Brew et al., 2000
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High levels of TIMP-3 promote apoptosis in many cell
types in vitro and in vivo (Vaalamo et al., 1997), and this
effect is associated with death receptor modulation.
TIMP-3 inhibits at least MMP-2 and MMP-9, whereas
TIMP-4 is a good inhibitor for all classes of MMPs without
remarkable preference for specific MMPs.
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The TIMP structure
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The TIMPs are wedge-shaped molecules that are
subdivided into an N-terminal part (~120 aa), mainly
consisting of a closed five-stranded b-barrel, and a C-
terminal part (~60 aa) which forms -turn structure.
The N-terminal part resembles the so-called OB-fold
known for a number of oligosaccharide/oligonucleotide-binding proteins.
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While the N-terminal part is in intimate contact with the
active site of the target MMP, the C-terminal part seems tohave only minor impact on the tightness of the interaction
with the MMP (except for the MMP2-TIMP-2 complex).
However, it conveys other functions such as the binding
of TIMP-2 to proMMP, which is an essential step in the
activation procedure of proMMP-2 and inhibition ofangiogenesis.
Inhibition mechanism of TIMPs
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Inhibition mechanism of TIMPs
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There are different mechanisms to inhibit ordownregulate MMPs.
Inhibition may take place by:
Interaction with an active zn2+ -site
By cleaving the active enzyme
By binding it to a non-active complex form.
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To influence the MMP levels, it is also possible to down-
regulate MMPs at the transcriptional level
Birkedal-Hansen et al., 1993
The proteolytic activity of the MMPs involved in
extracellular matrix degradation must be preciselyregulated by their endogenous protein inhibitors, the
TIMPs.
Disruption of this balance results in serious diseases.
-Macroglobulins
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-Macroglobulins
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-Macroglobulins inactivate susceptible proteinases by
entrapment following cleavage of the bait region.
The proteinase cleaves one or more bonds in the 40
residue bait region and thereby initiates a conformational
change that leads to entrapment of the proteinase
Sottrup-Jensen et al, 1989
a-macroglobulins play an important role in the regulation
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a-macroglobulins play an important role in the regulation
of MMP activity.
The rapid capture rates, particularly with collagenase,
render the reaction all but instantaneous.
Cawston and Mercer (1986) showed that in a mixture ofTIMP and 2M, collagenase binds preferentially to 2M.
It is also of note that the concentration of 2M in the
interstitial fluid is quite considerable and often
approaches plasma values (2.2 mg/ml)
Tollefsen and Saltvedt, 1980
2M concentrations in synovial fluids from rheumatoid
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2M concentrations in synovial fluids from rheumatoidarthritis patients range from 0.7 to 1.1 mg/ml whereasthose of osteoarthritis patients are slightly lower, 0.5 to
0.6 mg/mlVirca et al, 1982, Abe et ai, 1973
Similar determinations on gingival crevicular fluids(GCFs) from variously inflamed sites have yielded valuesfrom 0.2 to 2.4 mg/ml
Schenkein and Genco, 1977; Tollefsen and Saltvedt, 1980;
Condacci et al, 1982; Sengupta et al, 1988
These values suggest that 2M concentrations ininterstitial fluids are as much as 100- to 1000- fold greaterthan those of TIMPs
Cawston et al 1987 Ha akawa et al 1991
Inhibiting Antibodies
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Inhibiting Antibodies
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Production of blocking antibodies represents another
attractive approach to the design of effective and specific
inhibiting reagents.
Affinity purified polyclonal antibodies as well as
monoclonal antibodies are both highly effective and attain
IC50 in the 1 to 10 ug/ml range (20 to 200 nM).
The best of these are almost as potent as good synthetic
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The best of these are almost as potent as good synthetic
inhibitors and are frequently highly specific and readily
discriminate between closely related enzymes such as
FIB-CL and PMN-CL
Birkedal-Hansen etal, 1988
The inhibition obtained with monoclonal antibodies to
collagenase on collagenous substrates is virtually
complete (>90%) but similar antibodies against GLs on
gelatin substrates appear to be less effective (40 to 60%inhibition)
Hoyhtya et al, 1988; Birkedal-Hansen et al, 1992c
Synthethic inhibitors
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Synthethic inhibitors
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The development of synthetic inhibitors of MMPs has
been relied on the peptide sequence, recognized by the
targeted protease, to which have been grafted differentchemical functionalities able to interact potently with the
zinc ion of the active site.
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The requirements for a molecule to be an effective
inhibitor of the MMP class of enzymes are: Functional group [e.g., hydroxamate (CONHO--),
carboxylate (COO--), thiolate (S--), phosphinyl (PO2--) etc.]
capable of chelating the active-site zinc2+Ion (this will be
referred to as zinc binding group or ZBG)
At least one functional group that provides a hydrogen
bond interaction with the enzyme backbone
One or more side chains, which undergo effective van der
Waals interactions with the enzyme subsites.
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Natural product MMP inhibitors include tetracyclines,
pycnidione, neovastat, squalamine, genistein, nobiletin,
myricetin, curcumin, xanthorhizzol, theaflavin,resveratrol, actinonin, BE-166278 (Banyu), matlystatin B,
nicotinamide, betulinic acid, glycyrrhetinic acid,
rifampicin, catechin derivatives, and a series of
futoenone derivatives.
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Studies have demonstrated that low dose doxycycline usedas an adjunct to scaling and root planning (SRP) significantly
improves clinical attachment and reduces pocket depth,slowing the progression of periodontitis when compared withSRP alone
Caton JG, 2001
The benefits were achieved after three months of treatment,and appear to be maintained during a 3-month post-treatmentperiod
The attachment level gains in the deepest sites are modest inthese studies (~0.7 mm). The gains in deeper sites treatedwith systemic tetracyclines and SRP are twice bigger thangains achieved using MMP inhibitor.
Peterson JT. 2004
Chemically modified tetracycline's
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Chemically modified tetracycline s
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Golub and co-workers 1991, synthesized 10 differentanalogs of tetracyclines known as chemically-modified
tetracycline's (CMTs 1-10) to identify the site of the
anticollagenase property in tetracycline.
This modification, did not reduce the ability of drug to
block the activity of collagenases from a number of tissue
sources or its ability to inhibit bone resorption.
Thus, it was concluded that the C-4 moiety was not
required for the anticollagenase activity of the
tetracycline molecule.
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Investigations
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Analysis of - GCF, Serum & Saliva
Immunocytochemistry
DentoELISA
Dip stick method
CONCLUSION
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CONCLUSION
In conclusion, MMPs play an important role during the
process of periodontitis since they are responsible for thedegradation of extracellular matrix components.
In recent years the use of molecular and cellular biology
methodologies has greatly contributed to the understandingof the basic mechanisms that determine the onset andprogression of periodontal diseases.
It is likely that in a recent future they may also help in theprognosis and in the prediction of disease progression.
This will facilitate the identification of individuals at high risk