C. Graham Knight

Structural Basis of Collagen Recognition by Integrin α2β1

We have determined the crystal structure of a complex between the I domain of integrin alpha2beta1 and a triple helical collagen peptide containing a critical GFOGER motif. Three loops on the upper surface of the I domain that coordinate a metal ion also engage the collagen, with a collagen glutamate completing the coordination sphere of the metal. Comparison with the unliganded I domain reveals a change in metal coordination linked to a reorganization of the upper surface that together create a complementary surface for binding collagen. Conformational changes propagate from the upper surface to the opposite pole of the domain, suggesting both a basis for affinity regulation and a pathway for signal transduction. The structural features observed here may represent a general mechanism for integrin-ligand recognition.

A novel coumarin‐labelled peptide for sensitive continuous assays of the matrix metalloproteinases

(7‐methoxycoumarin‐4‐yl)Acetyl‐Pro‐Leu‐Gly‐Leu‐(3‐[2,4‐dinitrophenyl]‐l‐2,3‐diaminopropionyl)‐Ala‐Arg‐NH2 (Mca‐Pro‐Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly flourescent 7‐methoxycoumarin group is efficiently quenched by energy transfer to the 2,4‐dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3,4,24,23) cleaves the substrate at the Gly‐Leu bond with a 190‐fold increase in fluorescence (λcm 328 nm, λcm 393 nm). In assays of the human matrix metalloproteinases, Mca‐Pro‐Leu‐Gly‐Leu‐Dpa‐Ala‐Arg‐NH2 is about 50 to 100 times more sensitive than dinitrophenyl‐Pro‐Leu‐Gly‐Leu‐Trp‐Ala‐d‐Arg‐NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (k cal/K m) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.

TNF‐α converting enzyme (TACE) is inhibited by TIMP‐3

TNF‐α converting enzyme (TACE; ADAM‐17) is a membrane‐bound disintegrin metalloproteinase that processes the membrane‐associated cytokine proTNF‐α to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP‐3 but not by TIMP‐1, ‐2 and ‐4. These results suggest that TIMP‐3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF‐α secretion is involved.

The in vitro activity of ADAM-10 is inhibited by TIMP-1 and TIMP-3

A recombinant soluble form of the catalytic domain of human ADAM‐10 was expressed as an Fc fusion protein from myeloma cells. The ADAM‐10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described ‘metallosheddase’ cleavage sites of tumour necrosis factor α, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM‐10. The natural MMP inhibitors, TIMP‐2 and TIMP‐4 were unable to inhibit ADAM‐10, but TIMP‐1 and TIMP‐3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM‐10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP‐1) and 0.9 nM (TIMP‐3). The TIMP‐1 inhibition of ADAM‐10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP‐3, in cell based assays.

Collagen-platelet interaction : Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen

OBJECTIVE Peptides consisting of a repeat Gly-Pro-Hyp sequence are potent platelet agonists. The aim of this study was: (1) to examine the specificity of this sequence for platelet activation; (2) to confirm its recognition by platelet glycoprotein VI; and (3) to assess with suitable peptides the relative importance of glycoprotein VI and integrin alpha 2 beta 1 in platelet activation by collagen. METHODS Peptides were synthesized by standard Fmoc chemistry and tested for their ability to support adhesion of human platelets and HT 1080 cells, induce platelet aggregation, bind integrin alpha 2 subunit A-domain and to cause tyrosine phosphorylation of platelet proteins. RESULTS (1) Peptides consisting of a repeat Gly-Pro-Pro, Gly-Pro-Ala or Gly-Pro-Arg sequence exhibited little if any platelet-reactivity. (2) The platelet-reactive peptide consisting of a repeating Gly-Pro-Hyp sequence failed to induce tyrosine phosphorylation in glycoprotein VI-deficient platelets. Platelet adhesion to this peptide was inhibited by intact anti-glycoprotein VI antibody and its Fab fragment. The latter inhibited aggregation by the peptide and fibres of both collagens I and III. (3) A peptide containing a 15-mer alpha 2 beta 1-binding sequence in a repeat Gly-Pro-Pro structure supported alpha 2 beta 1-mediated platelet and HT 1080 cell adhesion and bound alpha 2 A-domain, but failed to activate platelets or to induce tyrosine phosphorylation. Conversely, a peptide containing this sequence but with an essential Glu replaced by Ala and inserted in a repeat Gly-Pro-Hyp structure did not recognize alpha 2 beta 1, but was highly platelet activatory. CONCLUSIONS Platelet activation by collagen involves the highly-specific recognition of the Gly-Pro-Hyp sequence by platelet glycoprotein VI. Recognition of alpha 2 beta 1 is insufficient to cause activation. Interaction between collagen and glycoprotein VI is unique since Gly-Pro-Hyp is common in collagens but occurs rarely in other proteins, and glycoprotein VI may be expressed solely by platelets. This sequence could provide a basis for a highly-specific anti-thrombotic reagent to control thrombosis associated with plaque rupture.

CA074 methyl ester: A proinhibitor for intracellular cathepsin B

The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.

The enzymatic activity of ADAM8 and ADAM9 is not regulated by TIMPs

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane‐bound precursors and involved in modulating cell–cell and cell–matrix interactions. ADAM8 (MS2, CD156) has been identified in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane‐bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin‐γ, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membrane‐associated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane‐type metalloproteinases) which have been implicated in protein processing at the cell surface.

α11β1 Integrin Recognizes the GFOGER Sequence in Interstitial Collagens

The integrins α1β1, α2β1, α10β1, and α11β1 are referred to as a collagen receptor subgroup of the integrin family. Recently, both α1β1 and α2β1integrins have been shown to recognize triple-helical GFOGER (where single letter amino acid nomenclature is used, O = hydroxyproline) or GFOGER-like motifs found in collagens, despite their distinct binding specificity for various collagen subtypes. In the present study we have investigated the mechanism whereby the latest member in the integrin family, α11β1, recognizes collagens using C2C12 cells transfected with α11 cDNA and the bacterially expressed recombinant α11 I domain. The ligand binding properties of α11β1 were compared with those of α2β1. Mg2+-dependent α11β1 binding to type I collagen required micromolar Ca2+ but was inhibited by 1 mmCa2+, whereas α2β1-mediated binding was refractory to millimolar concentrations of Ca2+. The bacterially expressed recombinant α11 I domain preference for fibrillar collagens over collagens IV and VI was the same as the α2 I domain. Despite the difference in Ca2+ sensitivity, α11β1-expressing cells and the α11 I domain bound to helical GFOGER sequences in a manner similar to α2β1-expressing cells and the α2 I domain. Modeling of the α I domain-collagen peptide complexes could partially explain the observed preference of different I domains for certain GFOGER sequence variations. In summary, our data indicate that the GFOGER sequence in fibrillar collagens is a common recognition motif used by α1β1, α2β1, and also α11β1 integrins. Although α10and α11 chains show the highest sequence identity, α2 and α11 are more similar with regard to collagen specificity. Future studies will reveal whether α2β1 and α11β1integrins also show overlapping biological functions.

Disruption of the c-JUN-JNK Complex by a Cell-permeable Peptide Containing the c-JUN δ Domain Induces Apoptosis and Affects a Distinct Set of Interleukin-1-induced Inflammatory Genes

The transcription factor activator protein (AP)-1 plays crucial roles in proliferation, cell death, and the immune response. c-JUN is an important component of AP-1, but only very few c-JUN response genes have been identified to date. Activity of c-JUN is controlled by NH2-terminal phosphorylation (JNP) of its transactivation domain by a family of JUN-NH2-terminal protein kinases (JNK). JNK form a stable complex with c-JUN in vitro and in vivo. We have targeted this interaction by means of a cell-permeable peptide containing the JNK-binding (δ) domain of human c-JUN. This peptide strongly and specifically induced apoptosis in HeLa tumor cells, which was paralleled by inhibition of serum-induced c-JUN phosphorylation and up-regulation of the cell cycle inhibitor p21cip/waf. Application of the c-JUN peptide to interleukin (IL)-1-stimulated human primary fibroblasts resulted in up-regulation of four genes, namely COX-2, MnSOD, IκBα, and MAIL and down-regulation of 10 genes, namely CCL8, mPGES, SAA1, hIAP-1, hIAP-2, pent(r)axin-3, CXCL10, IL-1β, ICAM-1, and CCL2. Only a small group of genes, namely pent(r)axin-3, CXCL10, ICAM-1, and IL-1β, was inhibited by both the c-JUN peptide and the JNK inhibitor SP600125. Thereby, and by additional experiments using small interfering RNA to suppress endogenous c-JUN we identify for the first time three distinct groups of inflammatory genes whose IL-1-induced expression depends on c-JUN, on JNK, or on both. These results shed further light on the complexity of c-JUN-JNK-mediated gene regulation and also highlight the potential use of dissecting signaling downstream from JNK to specifically target proliferative diseases or the inflammatory response.

The collagen-platelet interaction.

Collagen-platelet interaction, occurring in hemostasis but also a cause of thrombosis, is a two-step process of adhesion and activation involving the sequential recognition of distinct receptors. Adhesion involves first the reversible recognition of collagen-bound von Willebrand factor by the platelet receptor complex Gp lb/IX/V, followed by direct interaction between collagen and the platelet integrin receptor α2β1, which binds to specific sequences in collagen in which the GER motif appears important. Platelet activation then follows from the recognition by the receptor Gp VI of GPP* sequences in collagen, culminating in signalling events unique to collagen as a platelet agonist: Gp VI leads via the novel platelet Fc receptor γ-chain to p72syk and phospholipase Cγ2.