Meng-Huee Lee

Matrix metalloproteinases at a glance

The matrix metalloproteinases (MMPs) are one of the major families of proteinases that play key roles in the responses of cells to their microenvironment. Most notably the MMPs have the combined capacity to degrade all the components of the extracellular matrix. Besides modulating tissue structure

What are the roles of metalloproteinases in cartilage and bone damage

A role for metalloproteinases in the pathological destruction in diseases such as rheumatoid arthritis and osteoarthritis, and the irreversible nature of the ensuing cartilage and bone damage, have been the focus of much investigation for several decades. This has led to the development of broad spectrum metalloproteinase inhibitors as potential therapeutics. More recently it has been appreciated that several families of zinc dependent proteinases play significant and varied roles in the biology of the resident cells in these tissues, orchestrating development, remodelling, and subsequent pathological processes. They also play key roles in the activity of inflammatory cells. The task of elucidating the precise role of individual metalloproteinases is therefore a burgeoning necessity for the final design of metalloproteinase inhibitors if they are to be employed as therapeutic agents.

Unveiling the surface epitopes that render tissue inhibitor of metalloproteinase-1 inactive against membrane type 1-matrix metalloproteinase.

Membrane type 1-matrix metalloproteinase (MT1-MMP) is a zinc-dependent, membrane-associated endoproteinase of the metzincin family. The enzyme regulates extracellular matrix remodeling and is capable of cleaving a wide variety of transmembrane proteins. The enzymatic activity of MT1-MMP is regulated by endogenous inhibitors, the tissue inhibitor of metalloproteinases (TIMP). To date, four variants of mammalian TIMP have been identified. Whereas TIMP-2-4 are potent inhibitors against MT1-MMP, TIMP-1 displays negligible inhibitory activity against the enzyme. The rationale for such selectivity is hitherto unknown. Here we identify the surface epitopes that render TIMP-1 inactive against MT1-MMP. We show that TIMP-1 can be transformed into an active inhibitor against MT1-MMP by the mutation of a single residue, namely threonine 98 to leucine (T98L). The resultant mutant displayed inhibitory characteristics of a typical slow, tight binding inhibitor. The potency of the mutant could be further enhanced by the introduction of valine 4 to alanine (V4A) and proline 6 to valine (P6V) mutations. Indeed, the inhibitory profile of the triple mutant (V4A/P6V/T98L) is indistinguishable from those of other TIMPs. Our findings suggest that threonine 98 is critical in initiating MMP binding and complex stabilization. Our findings also provide a potential mechanistic explanation for MMP-TIMP selectivity.

Identification of the Extracellular Matrix (ECM) Binding Motifs of Tissue Inhibitor of Metalloproteinases (TIMP)-3 and Effective Transfer to TIMP-1

Tissue inhibitor of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent endopeptidases of the matrix metalloproteinase families. There are four mammalian TIMPs (TIMP-1 to -4) but only TIMP-3 is sequestered to the extracellular matrix (ECM). The molecular basis for the TIMP-3:ECM association has never been fully investigated until now. In this report, we identify the unique amino acid configuration that constitutes the basis of the ECM binding motif in TIMP-3. By systematically exchanging the subdomains of the TIMPs and exhaustive mutation of TIMP-3, we have identified the surface residues directly responsible for ECM association. Contrary to the accepted view, we have found that TIMP-3 interacts with the ECM via both its N- and C-terminal domains. The amino acids involved in ECM binding are all basic in nature: Lys-26, Lys-27, Lys-30, Lys-76 of the N-terminal domain and Arg-163, Lys-165 of the C-terminal domain. Replacement of these residues with glutamate (E) and glutamine (Q) (K26/27/30/76E + R163/K165Q) resulted in a soluble TIMP-3 devoid of ECM-adhering ability. Using the ECM binding motif derived from TIMP-3, we have also created a TIMP-1 mutant (K26/27/30 + K76 transplant) capable of ECM association. This is the first instance of TIMPs being intentionally rendered soluble or ECM-bound. The ability to prepare TIMPs in soluble or ECM-bound forms also opens new avenues for future TIMP research.

Total Conversion of Tissue Inhibitor of Metalloproteinase (TIMP) for Specific Metalloproteinase Targeting FINE-TUNING TIMP-4 FOR OPTIMAL INHIBITION OF TUMOR NECROSIS FACTOR-α-CONVERTING ENZYME

Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the matrix metalloproteinases, the ADAMs (adisintegrin and metalloproteinase) and the ADAM-TS (ADAM with thrombospondin repeats) proteinases. There are four mammalian TIMPs (TIMP-1 to -4), and each TIMP has its own profile of metalloproteinase inhibition. TIMP-4 is the latest member of the TIMPs to be cloned, and it has never been reported to be active against the tumor necrosis factor-α-converting enzyme (TACE, ADAM-17). Here we examined the inhibitory properties of the full-length and the N-terminal domain form of TIMP-4 (N-TIMP-4) with TACE and showed that N-TIMP-4 is a far superior inhibitor than its full-length counterpart. Although full-length TIMP-4 displayed negligible activity against TACE, N-TIMP-4 is a slow tight-binding inhibitor with low nanomolar binding affinity. Our findings suggested that the C-terminal subdomains of the TIMPs have a significant impact over their activities with the ADAMs. To elucidate further the molecular basis that underpins TIMP/TACE interactions, we sculpted N-TIMP-4 with the surface residues of TIMP-3, the only native TIMP inhibitor of the enzyme. Transplantation of only three residues, Pro-Phe-Gly, onto the AB-loop of N-TIMP-4 resulted in a 10-fold enhancement in binding affinity; the Ki values of the resultant mutant were almost comparable with that of TIMP-3. Further mutation at the EF-loop supported our earlier findings on the preference of TACE for leucine at this locus. Drawing together our previous experience in TACE-targeted mutagenesis by using TIMP-1 and -2 scaffolds, we have finally resolved the mystery of the selective sensitivity of TACE to TIMP-3.

The Intrinsic Protein Flexibility of Endogenous Protease Inhibitor TIMP-1 Controls Its Binding Interface and Affects Its Function.

Protein flexibility is thought to play key roles in numerous biological processes, including antibody affinity maturation, signal transduction, and enzyme catalysis, yet only limited information is available regarding the molecular details linking protein dynamics with function. A single point mutation at the distal site of the endogenous tissue inhibitor of metalloproteinase 1 (TIMP-1) enables this clinical target protein to tightly bind and inhibit membrane type 1 matrix metalloproteinase (MT1-MMP) by increasing only the association constant. The high-resolution X-ray structure of this complex determined at 2 A could not explain the mechanism of enhanced binding and pointed to a role for protein conformational dynamics. Molecular dynamics (MD) simulations reveal that the high-affinity TIMP-1 mutants exhibit significantly reduced binding interface flexibility and more stable hydrogen bond networks. This was accompanied by a redistribution of the ensemble of substrates to favorable binding conformations that fit the enzyme catalytic site. Apparently, the decrease in backbone flexibility led to a lower entropy cost upon formation of the complex. This work quantifies the effect of a single point mutation on the protein conformational dynamics and function of TIMP-1. Here we argue that controlling the intrinsic protein dynamics of MMP endogenous inhibitors may be utilized for rationalizing the design of selective novel protein inhibitors for this class of enzymes.

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: A new frontier in TIMP engineering

Tumor necrosis factor‐α (TNF‐α) converting enzyme (TACE/ADAM‐17) is responsible for the release of TNF‐α, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP‐1 to ‐4), TACE is specifically inhibited by TIMP‐3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N‐terminal domain form of TIMP‐3 (N‐TIMP‐3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N‐TIMP‐1. We succeeded in transforming N‐TIMP‐1 into an active inhibitor for TACE (Kiapp 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP‐3 AB‐loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of “super‐N‐TIMP‐3” epitopes, defined in our previous work, only impaired the affinity of N‐TIMP‐1 for TACE. Our mutagenesis results indicate that TIMP‐3‐TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.

The C-terminal domains of TACE weaken the inhibitory action of N-TIMP-3

Tumor necrosis factor‐α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) that comprises an active catalytic domain and several C‐terminal domains. We compare the binding affinity and association rate constants of the N‐terminal domain form of wild‐type tissue inhibitor of metalloproteinase (TIMP‐3; N‐TIMP‐3) and its mutants against full‐length recombinant TACE and the truncated form of its catalytic domain. We show that the C‐terminal domains of TACE substantially weaken the inhibitory action of N‐TIMP‐3. Further probing with hydroxamate inhibitors indicates that both forms of TACE have similar active site configurations. Our findings highlight the potential role of the C‐terminal domains of ADAM proteinases in influencing TIMP interactions.

A mechanistic rationalisation for the substrate specificity of recombinant mammalian 4-hydroxyphenylpyruvate dioxygenase (4-HPPD)

Abstract The isolation and purification of α-ketoisocaproate dioxygenase [α-KICD] from rat liver is described. Sequence determination of the purified protein revealed it to have complete homology to rat liver 4-hydroxyphenyl-pyruvate dioxygenase [4-HPPD] which was confirmed by the cloning and expression of the gene encoding 4-HPPD in E. coli. Examination of the substrate specificity of the resulting soluble recombinant protein revealed it to be capable of the oxidative decarboxylation of a range of ketoacids derived from proteinogenic amino acids. The significance of the turnover of these different ketoacids is discussed in relation to the mechanism of this fascinating enzyme.

Selective inhibition of ADAM12 catalytic activity through engineering of tissue inhibitor of metalloproteinase 2 (TIMP-2)

The disintegrin and metalloprotease ADAM12 has important functions in normal physiology as well as in diseases, such as cancer. Little is known about how ADAM12 confers its pro-tumorigenic effect; however, its proteolytic capacity is probably a key component. Thus selective inhibition of ADAM12 activity may be of great value therapeutically and as an investigative tool to elucidate its mechanisms of action. We have previously reported the inhibitory profile of TIMPs (tissue inhibitor of metalloproteinases) against ADAM12, demonstrating in addition to TIMP-3, a unique ADAM-inhibitory activity of TIMP-2. These findings strongly suggest that it is feasible to design a TIMP mutant selectively inhibiting ADAM12. With this purpose, we characterized the molecular determinants of the ADAM12-TIMP complex formation as compared with known molecular requirements for TIMP-mediated inhibition of ADAM17/TACE (tumour necrosis factor alpha-converting enzyme). Kinetic analysis using a fluorescent peptide substrate demonstrated that the molecular interactions of N-TIMPs (N-terminal domains of TIMPs) with ADAM12 and TACE are for the most part comparable, yet revealed strikingly unique features of TIMP-mediated ADAM12 inhibition. Intriguingly, we found that removal of the AB-loop in N-TIMP-2, which is known to impair its interaction with TACE, resulted in increased affinity to ADAM12. Importantly, using a cell-based epidermal growth factor-shedding assay, we demonstrated for the first time an inhibitory activity of TIMPs against the transmembrane ADAM12-L (full-length ADAM12), verifying the distinctive inhibitory abilities of N-TIMP-2 and engineered N-TIMP-2 mutants in a cellular environment. Taken together, our findings support the idea that a distinctive ADAM12 inhibitor with future therapeutic potential can be designed.