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(2020-21 BY2PA2) Molecular Pathology > Matrix Metalloproteinases > Flashcards

Matrix Metalloproteinases Flashcards

1
Q

What are matrix metalloproteinases?

A
  • Enzymes that catalyse the degradation of matrix components of connective tissue.
  • They have a role in normal processes (e.g. tissue remodelling and wound healing)
  • Loss of activity control→ role in pathological conditions (e.g., arthritis, CVD, cancer, Multiple Sclerosis)
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2
Q

Describe the ECM

A
  • Extracellular space consists of network of macromolecules which makes up the extracellular matrix (ECM).
  • ECM is an organised meshwork of proteins and polysaccharides secreted locally by cells in the matrix (mainly fibroblasts). Its function is to provide a structural scaffold.
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3
Q

Two main classes of extracellular macromolecules make up the ECM:

A
  • Glycosaminoglycans (GAGs) + proteins = proteoglycan (e.g. aggrecan and decorin) i.e. carbohydrate-containing molecules (also called proteoglycans)
  • Fibrous proteins (e.g., collagen, elastin, fibronectin and laminin). These have structural and adhesive functions.
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4
Q

A schematic view of Proteoglycans

A
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5
Q

A more 3-dimensional impression of the nature of the ECM

A
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6
Q

Describe the properties of collagen

A
  • A family of fibrous proteins
  • Act to hold cells together, provide tensile strength and elasticity
  • Collagen is the most abundant protein in animals
  • Left-handed helix with a repeating sequence
  • Triple stranded helical structure
  • Depending on tissue, the fibres of collagen are arranged differently to give 16 different forms in humans.
  • Type I is most common
  • Type IV is found in basement membranes
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7
Q
A
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8
Q

Describe the structure of collagen

A
  • Long, rigid, triple helix structure
  • 3 polypeptide chains form a rope like superhelix
  • Rich in proline and glycine
  • Proline – stabilizes and confers rigidity
  • Glycine – allows tight conformation
  • It has a repeating sequence: Gly-X-Y, where X = proline and Y = hydroxyproline or hydroxylysine (pro, pro-OH, lys-OH)
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9
Q

Describe collagen in the ECM

A
  • Collagen synthesis and degradation is very important in tissue remodelling
  • Tissue remodelling is a physiological process (needed in development and wound healing) but can contribute to disease also.
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10
Q
A
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11
Q

There are 25 recognized members of the MMP family in vertebrates that can be divided into 6 groups:

A

i. Collagenases (MMP1, 8, 13 and 18)
ii. Gelatinases (MMP2, 9)
iii. Stromelysins (MMP3, 10)
iv. Matrilysins (MMP7, 26)
v. Membrane-Type MMPs (14, 15, 16, 17, 24 and 25)
vi. Others (MMP 12, 19, 20)

Note that the numbers are not sequential, as some were “discovered” and then found to be the same as others, or not MMPs.

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12
Q

What were the first members of MMPs identified?

A

Collagenases

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13
Q

What are the function and structure of MMPs?

A
  • They are proteases (also called proteinases)
  • Specifically, zinc-dependent endopeptidases that hydrolyse peptide bonds
  • The Zn is bound to 3 His residues and is involved in the catalytic mechanism which is thought to be a base hydrolysis of a peptide bond in the substrate. The Zn interacts with a catalytic glutamate in the active site.
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14
Q

Describe the structural domains in MMP

A
  • All MMPs contain a propeptide and a catalytic (C) domain
  • The propeptide maintains the enzyme in an inactive state
  • The catalytic domain contains a Zn binding domain and confers substrate specificity
  • The Fibronectin domains anchor the enzyme to the ECM
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15
Q

Explain the structural domain in MMP

A
  • S = signal peptide, which directs the propeptide to the secretory pathway or the cell membrane for insertion (if it’s a membrane-bound form) – lost on maturation.
  • Pro = propeptide (cleaved during activation of the zymogen); contains a cysteine residue that binds to the Zn in the catalytic domain and prevents it from being active.
  • V = vitronectin insert
  • Cat = catalytic domain, which includes the Zn binding site, and sometimes also Fn domains
  • Fn = fibronectin domain
  • Hpx = hemopexin domain, responsible for substrate binding and specificity
  • I = type I transmembrane domain
  • II = type II transmembrane domain
  • Ca = cysteine array region (NOT Ca2+ binding)
  • G = GPI anchor (for membrane-linked MMPs)
  • Ig = IgG-like domain

There is commonly a hinge region between the catalytic domain and the substrate-binding domain, which may be up to 75 residues in length.

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16
Q

There are 4 basic mechanisms by which MMPs can be regulated:

A
  • Transcription (i.e. gene expression regulated by transcription factors)
  • Compartmentalisation
  • Activation of the propeptide (zymogen) by proteolytic cleavage
  • Endogenous inhibitors (e.g. TIMP)
17
Q

Describe the regulation of MMPs by transcription

A
  • Growth factors and cytokines activate AP-1
  • AP-1 is a transcription factor and a leucine zipper protein
  • AP-1 consists of heterodimers encoded by c-fos and c-jun gene families (or other components – see gene expression lecture)
  • AP-1 regulated genes include cell cycle entry proteins and MMP-1 (collagenase) so AP-1 caused increased synthesis of MMP-1 mRNA

AP-1= Activator protein-1, mRNA = messenger RNA

18
Q

Describe the regulation of AP-1 level and activity to up-regulate MMP expression

A
  • Resting cells express low levels of c-jun and c-fos.
  • Addition of growth factors increases fos and jun mRNA within 15 minutes – PKC dependent.
  • The signalling is also redox sensitive transcription due to presence of a regulatory cysteine residue on c-jun
  • c-jun can also be activated by dephosphorylation (by nuclear phosphatase) on serine and threonine residues, which increases +ve charge and enhances DNA binding.

PKC = Protein kinase C

19
Q

Describe how compartmentalisation regulates MMPs

A

Neutrophil collagenase is stored in vesicles and its release is controlled to prevent cellular damage.

20
Q

Describe zygmogen as a regulating factor of MMPs

A
  • Pro-enzyme activation involves loss of a 10 kDa N-terminal pro-peptide
  • This is initiated by trypsin and may proceed through autocatalysis.
  • The pro-domain contains a cysteine residue that binds to the active site zinc and prevents its catalytic action.
21
Q

Describe how MMPs are also regulated by inhibitors

A
  • TIMPs are Tissue Inhibitors of MMPs
  • They are found in connective tissue, usually at much higher concentrations than the MMPs themselves
  • There is a family of four specific proteins (TIMP-1, 2, 3 and 4)
  • They bind active MMPs in 1:1 ratio with high affinity (Ki = 10-10)
  • TIMPs inactivate MMPs by binding to their catalytic domain.
  • There is concerted regulation of MMP activity through TGF-β (↓ MMP mRNA and ↑ TIMP mRNA).
22
Q

Describe rhe Generic Structure of TIMPs

A
  • The N-terminus of TIMPs 1-4 binds to the catalytic domain of most activated MMPs and inhibits function.
  • The C-terminus of TIMP1 and TIMP2 binds to the hemopexin domain of proMMP2 and proMMP9, respectively; this binding regulates MMP function.
23
Q

Describe MMPs in normal physiology

A
  • Any kind of tissue remodelling requires the action of MMPs to degrade the ECM and allow cell migration followed by new ECM deposition by fibroblasts.
  • In embryogenesis, many tissues are remodelled throughout the process (e.g. formation of fingers and toes, organ development).
  • In wound healing, MMPs facilitate influx of immune cells to protect the wound. MMP-3 is necessary for wound contraction and MMP-1 is important in keratinocyte migration later in healing. MMPs also assist in re-vascularization of the tissue.
24
Q

Explain MMPs and joint disease

A
  • Destruction of cartilage (composed of ECM) and bones is a feature of Rheumatoid Arthritis
  • Proteolytic degradation of the ECM of cartilage is a key step in joint destruction evident in RA
  • Increased collagenase activity (MMP-1) in RA synovial membrane and fluid
  • Collagenase is localised to bone and cartilage
  • Enhanced in TNFa transgenic mice
25
Q

TRUE or FALSE: Cartilage is an extracellular matrix, consisting mainly of collagens and proteoglycans (largely aggrecan)

A

TRUE

26
Q

How do MMPs play a role in joint disease?

A

In RA, cartilage destruction is from outside in (synovial fibroblast) AND from inside out (chondrocyte). Both show enhanced MMP expression (mainly MMP-1 and MMP-13)

The irreversible destruction of the cartilage, tendon, and bone that comprise synovial joints is the hallmark of both rheumatoid arthritis (RA) and osteoarthritis (OA). While cartilage is made up of proteoglycans and type II collagen, tendon and bone are composed primarily of type I collagen.

27
Q

Explain which MMPs are involved in rheumatoid artritis and osteoarthristis

A
  • RA is an autoimmune disease afflicting numerous joints throughout the body; in contrast, OA develops in a small number of joints, usually resulting from chronic overuse or injury.
  • In both diseases, inflammatory cytokines such as interleukin-1 beta (IL-1 beta) and tumour necrosis factor-alpha (TNF-alpha) stimulate the production of matrix metalloproteinases (MMPs), enzymes that can degrade all components of the extracellular matrix.
  • The collagenases, MMP-1 and MMP-13, have predominant roles in RA and OA because they are rate limiting in the process of collagen degradation.
  • MMP-1 is produced primarily by the synovial cells that line the joints, and MMP-13 is a product of the chondrocytes that reside in the cartilage.
  • In addition to collagen, MMP-13 also degrades the proteoglycan molecule, aggrecan, giving it a dual role in matrix destruction.
  • Expression of other MMPs such as MMP-2, MMP-3 and MMP-9, is also elevated in arthritis and these enzymes degrade non-collagen matrix components of the joints.
  • Significant effort has been expended in attempts to design effective inhibitors of MMP activity and/or synthesis with the goal of curbing connective tissues destruction within the joints.
  • To date, however, no effective clinical inhibitors exist. Increasing our knowledge of the crystal structures of these enzymes and of the signal transduction pathways and molecular mechanisms that control MMP gene expression may provide new opportunities for the development of therapeutics to prevent the joint destruction seen in arthritis.
28
Q

How do MMPs contribute both to tissue injury & restructuring in CVD?

A
  • Foam cells (lipid-laden macrophages) are a major source of MMP-9
  • High systemic levels of MMP-9 correlate with poor outcome.
  • MMP-9 also contributes to left ventricular remodelling of the heart in myocardial infarction.

MMP-9 is gelatinase B

29
Q

A proposed role of MMPs in multiple sclerosis (MS).

A

Schematic sketch showing the role of matrix metalloproteinases (MMPs) in multiple sclerosis (MS). MMPs produced by leukocytes impair the blood–brain barrier (BBB) initiating a cascade of inflammatory response leading to demyelination and scarring in the central nervous system (CNS)

MMP-9,MMP-2,MMP-7 present at higher levels in disease

30
Q

For cancer metastasis to occur, what must happen to the ECM?

A
  • For cancer metastasis to occur, the ECM must be degraded so that cancer cells lose adherence to it and are able to leave the tissue.
  • Remodelling of tissue in the tumour is also needed to allow vascularization and a good blood supply.
31
Q

Which MMP is important in tumour resistance?

A

MMP-9 is important in tumour resistance and metastasis, involved in cleavage of ICAM-1 (needed for cell-cell adherence)

32
Q

TRUE or FALSE: Angiogenesis, metastasis and invasion all involve tissue remodelling and therefore action of MMPs

A

TRUE

(2020-21 BY2PA2) Molecular Pathology (13 decks)

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