Elucidating the function of non catalytic domains of collagenases and aggrecanases

Metalloproteinases that degrade extracellular matrix molecules play important roles in development and progression of various diseases. Among them, collagenases are unique as they have an ability to degrade triple helical interstitial collagens into 3/4 and 1/4 fragments, a crucial step for collagenolysis in the tissue. Collagenases, consisting of a catalytic domain and a hemopexin domain, requires both domains for collagenolysis. The enzymes unwind triple helical collagen before they hydrolyze the peptide bonds. Aggrecanases are also multidomain metalloproteinases belonging to the ADAMTS family, and the noncatalytic ancillary domains also play an important role in recognition of aggrecan and their activities. Attenuation of collagenase and aggrecanase activities will be achieved by inhibitors or antibodies that interact directly with those noncatalytic ancillary domains (exosite inhibitors). Such molecules will be attractive for therapy as they will be highly selective because they are based on the unique mechanism of each proteinase.     

Membrane receptors for soluble defense collagens

Membrane receptors for the soluble 'defense collagens' - naturally occurring chimeric molecules that contain a recognition domain contiguous with a collagen-like triple helical domain and play a role in protecting the host from pathogens entering the blood, lung and other tissues - are being isolated. These receptors are key to understanding the mechanisms by which defense collagens influence cellular responses in order to either provide rapid 'stealth clearance' of cellular debris or to initiate the responses that lead to the destruction of harmful microbes.     

Contributions of Gla and EGF-like domains to the function of vitamin K-dependent coagulation factors

Blood coagulation is a response to vascular injury leading to the activation of platelets and coagulation factors with the ultimate formation of a fibrin plug. Several coagulation factors are zymogens of serine proteases that require vitamin K for normal biosynthesis. The active forms of these proteases and their cofactors form membrane-bound macromolecular complexes. In the final step prothrombin is activated to thrombin by active factor X in complex with its cofactor, factor V. Thrombin then cleaves designated peptide bonds in fibrinogen, resulting in the formation of fibrin monomers that polymerize to insoluble fibrin strands. This process is regulated by an anticoagulant counterpart, the so-called protein C anticoagulant system. Balance between the two systems is crucial to avoid bleeding on the one hand and thrombosis on the other. The coagulation and anticoagulation proteases, factors VII, IX, and X, and protein C, have a common domain structure with an N-terminal gamma-carboxyglutamic acid (Gla)-containing domain that is followed by two domains that are homologous to the epidermal growth factor (EGF), whereas the C-terminal half of each protein is occupied by a trypsin-like serine protease domain. Prothrombin also has an N-terminal Gla domain and a C-terminal serine protease domain, but they are separated by two so-called kringle domains rather than EGF-like domains. Finally, the vitamin K-dependent cofactor protein S has one domain with thrombin-sensitive bonds and four EGF-like domains in tandem between the Gla domain and a C-terminal domain that is homologous to plasma steroid hormone-binding proteins. The N-terminal noncatalytic Gla and EGF-like domains that provide the coagulation serine proteases with unique properties, such as affinity for certain biological membranes, and also mediate protein-protein interactions, are the subject of this review.     

In silico study of whey-acidic-protein domain containing oral protease inhibitors

Since whey-acidic-protein domain (WAP) containing protease inhibitors such as SLPI (secretory leukocyte protease inhibitor) and elafin (elastase-specific inhibitor) have antimicrobial activities and are thought to play critical roles in mucosal defenses, we are interested in these protease inhibitors. By accessing the Novartis mouse expression database, we found that the four WAP family members, SLPI, WFDC2, WFDC5, and WFDC12, are highly expressed in the oral organs, such as the trachea, tongue, and salivary glands. Since their WAP domains play pivotal roles in the antimicrobial and/or antiprotease activities and their application in therapeutics are expected to have practical value, we collected 98 WAP homologues and tried to predict their physiological functions by analyzing their amino acid sequence structures. From the multiple alignments of amino acid sequences, we predicted that most of the mammalian N-terminal WAP domains derived from SLPIs and the WAP domains derived from WFDC12s have antimicrobial activities, whereas most of the mammalian C-terminal WAP domains derived from SLPIs and the WAP domains derived from elafins have antiprotease activities. From the phylogenetic tree, it was revealed that an ancestral WAP protein initially diverged into the WFDC5-C WAP domain and the ancestral protein for the other WAP domains. Subsequently, the ancestral protein for the other WAP domains diverged into two ancestral proteins, one for elafin and SLPI-C WAP domains and the other, for SLPI-N, WFDC15b, WFDC12, and WFDC5-N WAP domains, respectively. Moreover, the tree indicated that the WFDC5-N and WFDC12 WAP domains share a common ancestral protein.     

Systematic Identification of Novel Protein Domain Families Associated with Nuclear Functions

A systematic computational analysis of protein sequences containing known nuclear domains led to the identification of 28 novel domain families. This represents a 26% increase in the starting set of 107 known nuclear domain families used for the analysis. Most of the novel domains are present in all major eukaryotic lineages, but 3 are species specific. For about 500 of the 1200 proteins that contain these new domains, nuclear localization could be inferred, and for 700, additional features could be predicted. For example, we identified a new domain, likely to have a role downstream of the unfolded protein response; a nematode-specific signalling domain; and a widespread domain, likely to be a noncatalytic homolog of ubiquitin-conjugating enzymes.     

The Effect of Cis-4-Hydroxy-L-Proline on Intracellular Degradation of Newly Synthesized Collagen by Freshly Isolated Chick Tendon Fibroblasts

Freshly isolated embryonic chick tendon cells were incubated for 6 hrs in suspension culture in the presence of the proline analogue, cis-4-hydroxyproline to cause the cells to synthesize collagen which was incapable of becoming triple helical. The cells were evaluated for the percentage of total protein synthesis devoted to collagen and for the percentage of newly synthesized collagen which was rapidly degraded. Collagen production in the presence of cis-4-hydroxyproline was reduced from 25% to 7% of total protein synthesis. Under normal conditions the cells degraded 8% of their newly synthesized collagen but when the cells were incubated in the presence of cis-4-hydroxyproline, 25% of their total collagen synthesized was degraded to dialyzable peptides. The enhanced degradation of nonhelical, analogue containing collagen was inhibited by inhibitors of lysosome function. These observations provide support for the concept that fibroblasts are able to recognize and degrade a portion of their newly synthesized collagen, and that defective collagen may be selectively degraded by an intracellular lysosomal process. Enhanced intracellular degradation can in part explain the decrease in collagen production by freshly isolated tendon fibroblasts incubated with cis-4-hydroxyproline.     

Proteases and glioma angiogenesis

Angiogenesis, the process by which new branches sprout from existing vessels, requires the degradation of the vascular basement membrane and remodeling of the ECM in order to allow endothelial cells to migrate and invade into the surrounding tissues. Serine, metallo, and cysteine proteinases are 3 types of a family of enzymes that proteolytically degrade various components of extracellular matrix. These proteases release various growth factors and also increase adhesive molecules and signaling pathway molecules upon their activation, which plays a significant role in angiogenesis. Downregulation of these molecules by antisense/siRNA or synthetic inhibitors decreases the levels of these molecules, inhibits the release of growth factors, and decreases the levels of various signaling pathway molecules, thereby leading to the inhibition of angiogenesis. Furthermore, MMPs degrade specific substrates and release angiogenic inhibitors which inhibit angiogenesis. Downregulation of 2 molecules, such as uPA and uPAR, uPAR and MMP-9, or Cathepsin B and MMP-9, are more effective to inhibit angiogenesis rather than downregulation of single molecules. However, careful testing of these combinations are most important because multiple effects of these combinations play a significant role in angiogenesis.     

Proteases and protease inhibitors: a balance of activities in host-pathogen interaction

The immune system is the collection of effector molecules and cells of the host that act against invading parasites and their products. Secreted proteases serve important roles in parasitic metabolism and virulence and the several families of protein protease inhibitors of the plasma and blood cells play an important role in immunity by inactivating and clearing the protease virulence factors of parasites. The protease inhibitors are of two classes, the active-site inhibitors and the alpha2-macroglobulins. Inhibitors for the first class bind and inactivate the active site of the target protease. Proteins of the second class bind proteases by a unique molecular trap mechanism and deliver the bound protease to a receptor-mediated endocytic system for degradation in secondary lysosomes. Proteins of the alpha2-macroglobulin family are present in a variety of animal phyla, including the nematodes, arthropods, mollusks, echinoderms, urochordates, and vertebrates. A shared suite of unique functional characteristics have been documented for the alpha2-macroglobulins of vertebrates, arthropods, and mollusks. The alpha2-macroglobulins of nematodes, arthropods, mollusks, and vertebrates show significant sequence identity in key functional domains. Thus, the alpha2-macroglobulins comprise an evolutionarily conserved arm of the innate immune system with similar structure and function in animal phyla separated by 0.6 billion years of evolution.     

Construction of antibody mimics from a noncatalytic enzyme-detection of polysialic acid

We have used a conceptually novel way to construct antibody mimics based on the binding of a noncatalytic enzyme to its substrate. Bacteriophage-derived endosialidase cleaves polysialic acid (polySia), an important oncofetal and bacterial antigen, which is poorly immunogenic. We fused to green fluorescent protein (GFP) a catalytically inactive endosialidase known to bind but not degrade polysialic acid. The fusion protein is a convenient single-step reagent in fluorescence microscopy, binding assays and immunoblots. It efficiently and specifically detected polysialic acid in developing brain, neuroblastoma cells and bacteria causing meningitis. Enzyme-substrate interactions represent an unexploited source of molecular recognition events. Some of these could be used in designing well-defined substitute antibodies for the study of target molecules which are difficult to purify, available in low quantities, are unstable or have poor immunogenity.     

Abnormal extracellular matrix in Ehlers-Danlos syndrome type IV due to the substitution of glycine 934 by glutamic acid in the triple helical domain of type III collagen

A unique substitution of glycine 934 by glutamic acid in the triple helical domain of type III collagen was identified in a proband with Ehlers-Danlos syndrome type IV. The substitution was due to the transition of G 3302 to A in α 1 (III) cDNA which is encoded by exon 46 of COL3A1. It resulted in a severe deficiency of type III collagen in fibroblast cultures and dermis. Dilatation of the endoplasmic reticulum of the dermal fibroblasts was probably due to the failure of these cells to secrete type III collagen molecules containing one or more mutant α 1(III) chains. The dermal collagen fibrils were narrow, but their constituent type III collagen molecules contained predominantly normal α 1(III) chains. As a result, the major effect of the substitution of glycine 934 by glutamic acid was to severely reduce the amount of normal type III collagen available for the formation of heterotypic collagen fibrils in the extracellular matrix.     

beta(3)-Integrin cytoplasmic binding proteins

Integrins are cell-surface adhesion receptors that play an important role in mediating numerous physiological processes,including inflammation, migration, adhesion, and proliferation. Integrin regulation by events within the cell has been termed "inside-out " signaling; this is a capacity that is unique to integrin receptors. As is typical of other cell-surface receptors, integrins can also transduce signals from outside the cell into the cytoplasm on binding extracellular ligands ("outside-in signaling "). Integrins are composed of an alpha and a beta subunit, which form a heterodimer. The beta(3)-integrin family consists of alpha(IIb)beta(3)-found on platelets and megakaryocytes, and the more widely distributed alpha(v)beta(3). beta Subunits consist of a large extracellular domain, a single transmembrane segment, and a relatively short cytoplasmic tail. The cytoplasmic domains do not contain intrinsic tyrosine kinase activity, and therefore signaling occurs primarily via recruitment of intracellular signaling molecules. Integrins form transmembrane connections, and the interactions between integrin cytoplasmic domains, intracellular factors (cytoplasmic proteins and intracellular signaling pathways), and membrane-anchored proteins play an important role in integrin-mediated events. There are at least 21 proteins that associate with integrin beta tails to regulate cell motility, proliferation, differentiation, and apoptosis. In this review, we will focus on 10 of these proteins and their function in integrin-mediated events.     

Platelet activation mediated through membrane glycoproteins: involvement of tyrosine kinases]

Platelet membrane glycoproteins such as GPIb and GPIa/IIa play important roles in platelet functional responses. They are the receptors for specific ligands (GPIb for von Willebrand factors, and GPIa/IIa for collagen), and the ligand-receptor interaction is the first step that elicits downstream intracellular activation signals which finally culminate in platelet aggregation. Although a variety of signal transduction pathways may be involved, tyrosine kinases appear to be most closely related to platelet activation mediated by membrane glycoproteins. Since its discovery in early 1980's, protein tyrosine phosphorylation catalyzed by tyrosine kinases has been recognized to play a role in regulating the cell function of various cells. Platelets have several Src family tyrosine kinases with an SH2 domain and an SH3 domain. Syk with two SH2 domains also appears to play an important role in platelet activation, especially in its early phase. The SH2 domain binds to a phosphorylated tyrosine residue of other proteins, and the SH3 domain recognizes proline-rich domains of target proteins, thus providing the anchoring sites for protein-protein interactions. In this article, some of the recent developments in the signal transduction pathways related with tyrosine kinases are introduced. Several signaling molecules involved in GPIb- or GPIa/IIa-mediated platelet activation have been identified. Interestingly, the members participating in these processes are distinct, suggesting a diversity of signal transduction mechanisms.     

Generation of monoclonal antibodies to cryptic collagen sites by using subtractive immunization

The extracellular matrix (ECM) plays a fundamental role in the regulation of normal and pathological processes. The most abundantly expressed component found in the ECM is collagen. Triple helical collagen is known to be highly resistant to proteolytic cleavage except by members of the matrix metalloproteinase (MMP) family of enzymes. To date little is known concerning the biochemical consequences of collagen metabolism on human diseases. This is due in part to the lack of specific reagents that can distinguish between proteolyzed and triple helical forms of collagen. Here we used the technique of Subtractive Immunization (SI) to generate two unique monoclonal antibodies (MAbs HUIV26 and HUI77) that react with denatured and proteolyzed forms of collagen, but show little if any reaction with triple helical collagen. Importantly, HUIV26 and HUI77 react with cryptic sites within the ECM of human melanoma tumors, demonstrating their utility for immunohistochemical analysis in vivo. Thus, the generation of these novel MAbs not only identify specific cryptic epitopes within triple helical collagen, but also provide important new reagents for studying the roles of collagen remodeling in normal as well as pathological processes.     

The 18-kDa form of cat allergen Felis domesticus 1 (Fel d 1) is associated with gelatin- and fibronectin-degrading activity

BACKGROUND: Fel d 1, an important allergen from domestic cats, is a significant cause of asthma. In addition to directly promoting IgE synthesis, other biological activities of allergens may contribute to either allergic sensitization or the magnitude of allergic effector responses. For example, allergens that degrade proteins have been suggested to facilitate allergen presentation by increasing parallelular permeability of airways epithelium. However, little information exists to indicate whether Fel d 1 has other activities relevant to allergic responses. OBJECTIVE: To study whether Fel d 1 is associated with enzyme activity. METHODS: Fel d 1 was obtained by a rigorous purification strategy and its identity confirmed by laser desorption mass spectrometry, cleavage and sequencing. The ability of Fel d 1 to degrade gelatin, fibronectin and the artificial substrate N-benzoyl-FVR-p-nitroanilide was studied. The effect of Fel d 1 on the morphology of tight junctions in epithelial cell monolayers was also investigated. RESULTS: The 18-kDa form of Fel d 1 caused degradation of denatured collagens (gelatin) and cleaved a 20-kDa fragment from the A chain of plasma fibronectin. Catalytic activity was not altered by inhibitors of cysteine peptidases, matrix metallopeptidases or by removal of divalent cations. In contrast, aprotinin and TLCK were inhibitors of Fel d 1. The absence of a serine peptidase catalytic triad in Fel d 1, together with the stoichiometry of the inhibition of TLCK and aprotinin, suggest that their inhibitory action may be due to noncatalytic site interactions. Alternatively, highly purified Fel d 1 may be associated with an active contaminant, although none were found. CONCLUSION: These results suggest that Fel d 1 is another example of a domestic allergen which is associated with enzyme activity. It remains to be established whether the activity resides in Fel d 1 itself or in an unresolved, and possibly related, protein.     

Cyclophilin inhibitors: a novel class of promising host-targeting anti-HCV agents

With the approval in 2011 of the protease inhibitors Victrelis and Incivek, direct-acting antivirals have begun to revolutionize HCV treatment. Although the addition of Incivek or Victrelis to PEGylated IFNα and ribavarin (pIFNα/RBV) may improve cure rates and shorten the treatment duration of the "old" standard of care (SOC), this triple therapy will not be suitable for patients intolerant to pIFNα or RBV. The efficacy of this triple therapy will also certainly be attenuated in pIFNα/RBV non-responders. As Incivek is inactive against genotype 3 (GT3) combined with the fact that all protease inhibitors and most of the non-nucleoside polymerase inhibitors in development are active primarily against GT1, pIFNα/RBV will remain the SOC for non-GT1 until new classes of inhibitors enter into clinical practice. GT1 patients who do not respond to this new triple therapy will have developed resistance to protease inhibitors that will limit future treatment options. There is thus an important need for the identification of new potent HCV agents. A novel class of HCV inhibitors that have great potential for the treatment for HCV has recently emerged: the host-targeting antivirals cyclophilin inhibitors.     

Multi-faceted strategies to combat disease by interference with the chemokine system

Inappropriate cell recruitment is a hallmark of all autoimmune, allergic and inflammatory diseases. The prevention of inflammation by interfering with cellular recruitment through the neutralization of cytokines and adhesion molecules has proven to be successful in the clinic. Chemokines are important potential targets owing to their central role in the cell recruitment process. Chemokines are unique among cytokines because they signal through seven transmembrane receptors, thus enabling the identification of small molecule inhibitors through high throughput screening. The object of this Review is to discuss the validity and feasibility of targeting several points of therapeutic intervention offered by the chemokine system and to assess the state of play within the field to date.     

Regulation of hematopoietic cell development and activation by adapter proteins

Adapter proteins, molecules with modular domains that mediate intermolecular interactions, play critical roles in the regulation of signaling events in all cell types. A major focus of our laboratory has been to examine the role of adapter molecules in hematopoietic cell development and activation. This review will describe the approaches we are taking to identify such proteins and to determine the mechanisms by which they exert their functions. This work represents the enormous efforts of the students and postdocs who have committed themselves to these projects, as well as the important collaborations we have developed with other investigators at the University of Pennsylvania and elsewhere.     

The cell biology of leukocyte-mediated proteolysis

Leukocyte-derived proteinases have the capacity to degrade every component of the extracellular matrix, and thereby play fundamental roles in physiological processes. However, if the activity of these proteinases is uncontrolled or dysregulated, they have the capacity to contribute to tissue injury that potentially affects every organ in the body. Although there is a substantial literature on structure and activity of these proteinases when they are free in solution, until recently there has been little information about the cell biology of proteinases and their inhibitors. Recent studies, however, have identified several mechanisms by which inflammatory cells can degrade extracellular proteins in a milieu that contains high-affinity proteinase inhibitors.     

Relevance of individual CD5 extracellular domains on antibody recognition, glycosylation and co-mitogenic signalling

CD5 is a type I glycoprotein which modulates T- and B-cell receptor-mediated signals and is expressed by thymocytes, mature T cells and a subset of mature B cells. The extracellular region of CD5 is composed of three scavenger receptor cysteine-rich domains (D1, D2, D3) for which only limited functional and structural data are available. Using cell transfectants expressing ectodomain-deficient CD5 molecules or CD5 immunoglobulin fusion proteins, we analysed individual CD5 domains with respect to monoclonal antibody binding specificity, glycosylation, and co-mitogenic signalling. Our results show the presence of N-linked oligosaccharides on D1 and D2, but not on D3. D1, the most amino-terminal domain, is predicted to be the most appropriately placed domain for an interaction with a ligand. This domain is recognised by a large panel of well characterised CD5 mAbs, reflecting its higher immunogenicity. In an attempt to develop mAbs with specificity for the more conserved membrane-proximal domains, we generated a unique mAb, named 83-C4, whose binding mapped to D3. Co-stimulatory studies revealed no significant differences between anti-D1 and anti-D3 mAbs. The high interspecies conservation of D3 implies a conserved role of this domain in CD5 function and the 83-C4 mAb promises to be a valuable tool in exploring this.