**2. Classification of proteinases**

Proteinases are named for their action, i.e. to cleave the internal peptide bonds of polypeptides. In human biology they are classified into 4 groups based on the chemical

Corresponding Author

Diverse Activities for Proteinases in the

Pathogenesis of Chronic Obstructive Pulmonary Disease 49

Fig. 2. (a) Mechanism by which NE cleaves a target protein. The NE molecule has two complex carbohydrate side chains attached to Asn95 and Asn144. The catalytic site of the NE

nature of their active site: serine, metallo-, cysteine, and aspartic proteinases. Serine proteinases and MMPs are the major players in extracellular proteolysis and are optimally active at neutral pH. Cysteine and aspartic proteinases work mainly in the cell in the breakdown of proteins in lysosomes. These are optimally active at acidic pH. These acid proteinases can potentially breakdown extracellular proteins if they can keep catalytic activity at neutral pH or are released into an environment having an acidic pH, such as the pericellular environment of activated macrophages (5,6). Proteinase inhibitors are generally specific to individual classes of proteinases. Proteinases of the serine, metalloand cysteine proteinase classes have been shown to have activities that contribute to COPD pathogenesis.

### **2.1 Serine proteinases**

Members of this group that are implicated in COPD include PMN-derived serine proteinases, urokinase-type plasminogen activator, granzymes, and plasmin.

#### *PMN-derived serine proteinases*

Neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) make up this group. The proteinases are stored in an inactive form within granules in PMN (Figure 1) and proinflammatory monocytes {8}. When the cells are stimulated by pro-inflammatory mediators they degranulate releasing the enzymes (7,8). These serine proteinases have a broad action against extracellular matrix (ECM) proteins (especially elastin) and non-ECM proteins (7). Figure 2 illustrates how the catalytic triad at the active site of NE (His41-Asp99-Ser173) cleaves the internal peptide bonds of proteins.

#### *Urokinase type plasminogen activator (uPA)*

This enzyme is expressed by PMN, monocytes, and macrophages. This enzyme is also stored in and released from the specific granules of PMN. The expression of uPA is regulated at the transcriptional level in mononuclear phagocytes by pro-inflammatory

nature of their active site: serine, metallo-, cysteine, and aspartic proteinases. Serine proteinases and MMPs are the major players in extracellular proteolysis and are optimally active at neutral pH. Cysteine and aspartic proteinases work mainly in the cell in the breakdown of proteins in lysosomes. These are optimally active at acidic pH. These acid proteinases can potentially breakdown extracellular proteins if they can keep catalytic activity at neutral pH or are released into an environment having an acidic pH, such as the pericellular environment of activated macrophages (5,6). Proteinase inhibitors are generally specific to individual classes of proteinases. Proteinases of the serine, metalloand cysteine proteinase classes have been shown to have activities that contribute to

Members of this group that are implicated in COPD include PMN-derived serine

Neutrophil elastase (NE), proteinase 3 (PR3), and cathepsin G (CG) make up this group. The proteinases are stored in an inactive form within granules in PMN (Figure 1) and proinflammatory monocytes {8}. When the cells are stimulated by pro-inflammatory mediators they degranulate releasing the enzymes (7,8). These serine proteinases have a broad action against extracellular matrix (ECM) proteins (especially elastin) and non-ECM proteins (7). Figure 2 illustrates how the catalytic triad at the active site of NE (His41-Asp99-Ser173) cleaves

Fig. 1. Structure of neutrophil: Proteinases are stored in an activated form in the azurophilic

This enzyme is expressed by PMN, monocytes, and macrophages. This enzyme is also stored in and released from the specific granules of PMN. The expression of uPA is regulated at the transcriptional level in mononuclear phagocytes by pro-inflammatory

proteinases, urokinase-type plasminogen activator, granzymes, and plasmin.

COPD pathogenesis.

**2.1 Serine proteinases** 

*PMN-derived serine proteinases* 

the internal peptide bonds of proteins.

granules within the neutrophil

*Urokinase type plasminogen activator (uPA)* 

Fig. 2. (a) Mechanism by which NE cleaves a target protein. The NE molecule has two complex carbohydrate side chains attached to Asn95 and Asn144. The catalytic site of the NE

Diverse Activities for Proteinases in the

fibroblasts produce MMPs-2, -9 and -14.

accomplishes by binding to integrins (25).

**2.3 Cysteine proteinases** 

17 is inhibited by TIMP-3 but not TIMP-1 or -2 (30,31).

also the cystatin superfamily and the kininogens (32).

**3. Evidence for activities for proteinases in COPD** 

degrade elastin (7).

*ADAM* 

Pathogenesis of Chronic Obstructive Pulmonary Disease 51

MMPs can be synthesized de novo by cells activated by pro-inflammatory mediators or growth factors. PMN, however, store preformed MMP-8, MMP-9, and MT6-MMP (MMP-25) in their cytoplasmic granules, and release the enzymes when they degranulate (7). Macrophages express MMPs-1, -3, -7, -9, -12, and -14 (21,22), and lung epithelial cells and

MMPs are classified into 6 groups based upon a similar domain organization and substrate specificity: 1) the interstitial collagenases (MMPs-1, -8, and -13); 2) the gelatinases (MMPs-2 and -9); 3) the stromelysins (MMPs-3, -10, and -11); 4) matrilysin (MMP-7); 5) metalloelastase (MMP-12); and 6) membrane-type MMPs (MT-MMPs), integral membrane proteinases with either a transmembrane domain or a glycosylphosphatidyl-inositol anchor to the cell membrane (23,24). The interstitial collagenases degrade interstitial collagens. The other subgroups have a broader range of substrates including denatured collagens (gelatins), basement membrane proteins, and pro-inflammatory mediators. MMPs-7, -9 and -12 also

This is a family of type I transmembrane proteinases, named ADAMs because they contain **a d**isintegrin and **a m**etalloproteinase domain (25). The metalloproteinase domain of ADAMs plays a role in regulation of inflammation, apoptosis and possibly fibrotic processes by shedding membrane-anchored cytokines such as protumor necrosis factor (TNF-), other cytokines, growth factors, apoptosis ligands and receptors for these molecules from cell surfaces (25-27). The disintegrin domain is involved in cell adhesion and migration which it

The inhibitors of the MMPs include the universal inhibitor, α2-M, and the four members of the tissue inhibitors of metalloproteinases family (TIMPs1-4), which are synthesized by connective tissue cells and leukocytes and form non-covalent complexes with MMPs (28,29). Although the inhibitors of ADAMs have not been fully elucidated, it is known that ADAM-

This group includes the cathepsins B, H, L, and S, which have been implicated in COPD. Cathepsin S and L are potent elastases *in vitro* (5,6) and contribute to macrophage-mediated ECM degradation. Inhibitors for this group, again include the universal inhibitor, α2-M, but

The proteinase/anti-proteinase hypothesis for the pathogenesis of COPD is not a new concept. It dates back to experimental work done over 50 years ago. The basis of the concept was 2 key observations. The first came from the keen observations by Laurell and Eriksson who noted that deficiency of AAT was associated with early onset, severe panlobular emphysema (33). AAT has since been shown to be the major inhibitor of NE in the lower respiratory tract. The second observation was made when instillation of papain (an enzyme with elastase activity) into rat lungs was shown to cause progressive airspace enlargement (34). Over the years, other elastolytic proteinases have been shown to cause airspace

molecule is an indentation of the molecule and is composed of the molecule and the triad His41-Asp88-Ser173, in which the -oxygen of serine becomes a powerful nucleophile able to attack a suitably located carbonyl group on the target substrate. The bond to be cleaved must fit into the active site pocket of the NE held there by charge interactions mediated by the residues forming the pocket. The peptide bond under attack is between two amino acid residues recognized by their side chains R1 and R2. (b) An acyl-enzyme intermediate molecule is formed between serine and the carbonyl group on the target protein. (c) The acyl-enzyme complex is hydrolysed with subsequent regeneration of active NE and cleavage of the protein.

mediators (9,10). On release from cells, uPA binds to a specific receptor (uPA receptor) on phagocyte surfaces, where it functions as a cell-associated proteinase. The main action of uPA is to activate the serine proteinase, plasmin from its inactive form, plasminogen. Plasmin lyses blood clots by breaking down fibrin, but also cleaves and activates latent growth factors, latent pro-metalloproteinases (MMP), and protease-activated receptor-1 (PAR-1) on macrophages, which drives macrophage MMP-12 production (11-14). Through this action, it plays an important role in ECM degradation and fibrotic processes in the lung.

#### *Granzymes (GRZ)*

These **gran**ule-associated en**zymes** are predominantly expressed by CD8+ T lymphocytes and are stored in the lytic granules (15). The main GRZ family members in human CD8+ T cells are GRZ A and B. Once activated by antigen, the CD8+ T cells commence rapid exocytosis of GRZ and perforin-containing granules. Release of perforin alters the properties of the cell membrane of the target cells, heralding the entry of GRZ into the target cell, and GRZ A and GRZ B then initiate caspase-independent and caspase-dependent apoptosis, respectively.

For this group of proteinases there exists naturally occurring inhibitors. Serine proteinase inhibitors (Serpins) in plasma and interstitial fluids include α1-anti-trypsin (AAT), α1 antichymotrypsin, plasminogen activator inhibitors, α2-plasmin inhibitor, and the universal inhibitor, α2-macroglobulin (α2-M), which inhibits all four classes of enzymes (16). Secretory leukocyte proteinase inhibitor (SLPI) and elafin are inhibitors synthesized locally in the respiratory tract by epithelial cells.

### **2.2 Metalloproteinases**

Included in this group of proteinases are the MMPs and the members of the ADAMs family.

#### *MMPs*

These proteinases have an NH2 terminal pro domain, an active site zinc atom, and a COOH terminal hemopexin domain that regulates the binding of the enzymes to their substrates. They are stored in a latent form as the inactive proenzymes or proMMPS, a state maintained by an interaction between the active site zinc atom and the cystein residue in the pro domain. Disruption of this interaction is required for activation of the proMMPs. This is facilitated by the actions of other proteinases and oxidants in the extracellular space (17,18)]. The intracellular serine proteinase, furin, is responsible for activation of some MMPs (19,20).

MMPs can be synthesized de novo by cells activated by pro-inflammatory mediators or growth factors. PMN, however, store preformed MMP-8, MMP-9, and MT6-MMP (MMP-25) in their cytoplasmic granules, and release the enzymes when they degranulate (7). Macrophages express MMPs-1, -3, -7, -9, -12, and -14 (21,22), and lung epithelial cells and fibroblasts produce MMPs-2, -9 and -14.

MMPs are classified into 6 groups based upon a similar domain organization and substrate specificity: 1) the interstitial collagenases (MMPs-1, -8, and -13); 2) the gelatinases (MMPs-2 and -9); 3) the stromelysins (MMPs-3, -10, and -11); 4) matrilysin (MMP-7); 5) metalloelastase (MMP-12); and 6) membrane-type MMPs (MT-MMPs), integral membrane proteinases with either a transmembrane domain or a glycosylphosphatidyl-inositol anchor to the cell membrane (23,24). The interstitial collagenases degrade interstitial collagens. The other subgroups have a broader range of substrates including denatured collagens (gelatins), basement membrane proteins, and pro-inflammatory mediators. MMPs-7, -9 and -12 also degrade elastin (7).

#### *ADAM*

50 Chronic Obstructive Pulmonary Disease – Current Concepts and Practice

mediators (9,10). On release from cells, uPA binds to a specific receptor (uPA receptor) on phagocyte surfaces, where it functions as a cell-associated proteinase. The main action of uPA is to activate the serine proteinase, plasmin from its inactive form, plasminogen. Plasmin lyses blood clots by breaking down fibrin, but also cleaves and activates latent growth factors, latent pro-metalloproteinases (MMP), and protease-activated receptor-1 (PAR-1) on macrophages, which drives macrophage MMP-12 production (11-14). Through this action, it plays an important role in ECM degradation and fibrotic processes in the

These **gran**ule-associated en**zymes** are predominantly expressed by CD8+ T lymphocytes and are stored in the lytic granules (15). The main GRZ family members in human CD8+ T cells are GRZ A and B. Once activated by antigen, the CD8+ T cells commence rapid exocytosis of GRZ and perforin-containing granules. Release of perforin alters the properties of the cell membrane of the target cells, heralding the entry of GRZ into the target cell, and GRZ A and GRZ B then initiate caspase-independent and caspase-dependent apoptosis,

For this group of proteinases there exists naturally occurring inhibitors. Serine proteinase inhibitors (Serpins) in plasma and interstitial fluids include α1-anti-trypsin (AAT), α1 antichymotrypsin, plasminogen activator inhibitors, α2-plasmin inhibitor, and the universal inhibitor, α2-macroglobulin (α2-M), which inhibits all four classes of enzymes (16). Secretory leukocyte proteinase inhibitor (SLPI) and elafin are inhibitors synthesized locally in the

Included in this group of proteinases are the MMPs and the members of the ADAMs family.

These proteinases have an NH2 terminal pro domain, an active site zinc atom, and a COOH terminal hemopexin domain that regulates the binding of the enzymes to their substrates. They are stored in a latent form as the inactive proenzymes or proMMPS, a state maintained by an interaction between the active site zinc atom and the cystein residue in the pro domain. Disruption of this interaction is required for activation of the proMMPs. This is facilitated by the actions of other proteinases and oxidants in the extracellular space (17,18)]. The intracellular serine proteinase, furin, is responsible for activation of some MMPs (19,20).

molecule is an indentation of the molecule and is composed of the molecule and the triad His41-Asp88-Ser173, in which the -oxygen of serine becomes a powerful nucleophile able to attack a suitably located carbonyl group on the target substrate. The bond to be cleaved must fit into the active site pocket of the NE held there by charge interactions mediated by the residues forming the pocket. The peptide bond under attack is between two amino acid residues recognized by their side chains R1 and R2. (b) An acyl-enzyme intermediate molecule is formed between serine and the carbonyl group on the target protein. (c) The acyl-enzyme complex is hydrolysed with subsequent regeneration of active NE and

cleavage of the protein.

lung.

*Granzymes (GRZ)*

respectively.

*MMPs* 

respiratory tract by epithelial cells.

**2.2 Metalloproteinases** 

This is a family of type I transmembrane proteinases, named ADAMs because they contain **a d**isintegrin and **a m**etalloproteinase domain (25). The metalloproteinase domain of ADAMs plays a role in regulation of inflammation, apoptosis and possibly fibrotic processes by shedding membrane-anchored cytokines such as protumor necrosis factor (TNF-), other cytokines, growth factors, apoptosis ligands and receptors for these molecules from cell surfaces (25-27). The disintegrin domain is involved in cell adhesion and migration which it accomplishes by binding to integrins (25).

The inhibitors of the MMPs include the universal inhibitor, α2-M, and the four members of the tissue inhibitors of metalloproteinases family (TIMPs1-4), which are synthesized by connective tissue cells and leukocytes and form non-covalent complexes with MMPs (28,29). Although the inhibitors of ADAMs have not been fully elucidated, it is known that ADAM-17 is inhibited by TIMP-3 but not TIMP-1 or -2 (30,31).
