Search

Collagenolysis is essential in extracellular matrix homeostasis, but its structural basis has long been shrouded in mystery. We have developed a novel docking strategy guided by paramagnetic NMR that positions a triple-helical collagen V mimic (synthesized with nitroxide spin labels) in the active site of the catalytic domain of matrix metalloproteinase-12 (MMP-12 or macrophage metalloelastase) primed for catalysis. The collagenolytically productive complex forms by utilizing seven distinct subsites that traverse the entire length of the active site. These subsites bury ∼1,080 Å(2)of surface area, over half of which is contributed by the trailing strand of the synthetic collagen V mimic, which also appears to ligate the catalytic zinc through the glycine carbonyl oxygen of its scissile G∼VV triplet. Notably, the middle strand also occupies the full length of the active site where it contributes extensive interfacial contacts with five subsites. This work identifies, for the first time, the productive and specific interactions of a collagen triple helix with an MMP catalytic site. The results uniquely demonstrate that the active site of the MMPs is wide enough to accommodate two strands from collagen triple helices. Paramagnetic relaxation enhancements also reveal an extensive array of encounter complexes that form over a large part of the catalytic domain. These transient complexes could possibly facilitate the formation of collagenolytically active complexes via directional Brownian tumbling., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The physiological roles of macrophages and dendritic cells (DCs) in lean white adipose tissue homeostasis have received little attention. Because DCs are generated from bone marrow progenitors in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF), we used GM-CSF-deficient (Csf2(-/-)) mice fed a low fat diet to test the hypothesis that adipose tissue DCs regulate the development of adipose tissue. At 4 weeks of age, Csf2(-/-) mice had 75% fewer CD45(+)Cd11b(+)Cd11c(+)MHCII(+) F4/80(-) DCs in white adipose tissue than did wild-type controls. Furthermore, the Csf2(-/-) mice showed a 30% increase in whole body adiposity, which persisted to adulthood. Adipocytes from Csf2(-/-) mice were 50% larger by volume and contained higher levels of adipogenesis gene transcripts, indicating enhanced adipocyte differentiation. In contrast, adipogenesis/adipocyte lipid accumulation was inhibited when preadipocytes were co-cultured with CD45(+)Cd11b(+)Cd11c(+)MHCII(+)F4/80(-) DCs. Medium conditioned by DCs, but not by macrophages, also inhibited adipocyte lipid accumulation. Proteomic analysis revealed that matrix metalloproteinase 12 and fibronectin 1 were greatly enriched in the medium conditioned by DCs compared with that conditioned by macrophages. Silencing fibronectin or genetic deletion of matrix metalloproteinase 12 in DCs partially reversed the inhibition of adipocyte lipid accumulation. Our observations indicate that DCs residing in adipose tissue play a critical role in suppressing normal adipose tissue expansion., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Matrix metalloproteases (MMPs) have attracted considerable attention as critical mediators of pathological tissue remodeling processes. However it remains an unresolved challenge to detect their active forms in biological samples. To prove the efficacy of a recently developed MMP activity-based probe, we examined the content in MMP active forms of bronchoalveolar lavage fluids (BALf) from male C57BL/6 mice exposed to ultrafine carbon black nanoparticles, a model of chronic obstructive pulmonary disease. This probe was shown to label proteins, mostly expressed in BALf of mice exposed to nanoparticles. Using competition assays with a selective MMP-12 inhibitor as well as MMP-12 knock-out mice, one of these proteins was identified as the active form of the catalytic domain of MMP-12. This new probe can detect the active form of MMP-12 down to a threshold of 1 fmol. Radioactive counting showed the concentration of the active form of MMP-12 to be around 1 fmol/μl in BALf from nanoparticle-treated mice. A less sensitive probe would therefore not have detected MMP-12. As the probe can detect other MMPs in the femtomolar range, it is a potentially powerful tool for monitoring the levels of MMP active forms in various diseases.

After the disappointment of clinical trials with early broad spectrum synthetic inhibitors of matrix metalloproteinases (MMPs), the field is now resurging with a new focus on the development of selective inhibitors that fully discriminate between different members of the MMP family with several therapeutic applications in perspective. Here, we report a novel class of highly selective MMP-12 inhibitors, without a phosphinic zinc-binding group, designed to plunge deeper into the S(1)' cavity of the enzyme. The best inhibitor from this series, identified through a systematic chemical exploration, displays nanomolar potency toward MMP-12 and selectivity factors that range between 2 and 4 orders of magnitude toward a large set of MMPs. Comparison of the high resolution x-ray structures of MMP-12 in free state or bound to this new MMP-12 selective inhibitor reveals that this compound fits deeply within the S(1)' specificity cavity, maximizing surface/volume ratios, without perturbing the S(1)' loop conformation. This is in contrast with highly selective MMP-13 inhibitors that were shown to select a particular S(1)' loop conformation. The search for such compounds that fit precisely to preponderant S(1)' loop conformation of a particular MMP may prove to be an alternative effective strategy for developing selective inhibitors of MMPs.

The catalytic domain of metalloelastase (matrix metalloproteinase-12 or MMP-12) is unique among MMPs in exerting high proteolytic activity upon fibrils that resist hydrolysis, especially elastin from lungs afflicted with chronic obstructive pulmonary disease or arteries with aneurysms. How does the MMP-12 catalytic domain achieve this specificity? NMR interface mapping suggests that α-elastin species cover the primed subsites, a strip across the β-sheet from β-strand IV to the II-III loop, and a broad bowl from helix A to helix C. The many contacts may account for the comparatively high affinity, as well as embedding of MMP-12 in damaged elastin fibrils in vivo. We developed a strategy called BINDSIght, for bioinformatics and NMR discovery of specificity of interactions, to evaluate MMP-12 specificity without a structure of a complex. BINDSIght integration of the interface mapping with other ambiguous information from sequences guided choice mutations in binding regions nearer the active site. Single substitutions at each of ten locations impair specific activity toward solubilized elastin. Five of them impair release of peptides from intact elastin fibrils. Eight lesions also impair specific activity toward triple helices from collagen IV or V. Eight sites map to the "primed" side in the III-IV, V-B, and S1' specificity loops. Two map to the "unprimed" side in the IV-V and B-C loops. The ten key residues circumscribe the catalytic cleft, form an exosite, and are distinctive features available for targeting by new diagnostics or therapeutics.

Matrix metalloproteinase (MMP)-12 (or metalloelastase) efficiently hydrolyzed the gelatinase-selective alpha1(V)436-447 fluorescent triple helical peptide (THP) when the substrate was submicromolar. The sequence of this THP was derived from collagen V, a component of collagen I fibrils. The hemopexin domains of MMP-12 and -9 each increased k(cat)/K(m) toward this substrate by decreasing K(m), just as the hemopexin domain of MMP-1 enhances its triple helical peptidase activity. Non-fluorescent alpha1(V) THP subtly perturbed amide NMR chemical shifts of MMP-12 not only in the active site cleft but also at remote sites of the beta-sheet and adjoining loops. The alpha1(V) THP protected MMP-12 from the NMR line broadening effects of Gd .EDTA in the active site cleft and more dramatically in the V-B loop next to the primed subsites. Mutagenesis of the exosite in the V-B loop at Thr-205 and His-206 that vary among MMP sequences established that this site supports the high specific activity toward alpha1(V) fluorescent THP without affecting general MMP activity. Surprisingly the alpha1(V) THP also protected novel surfaces in the S-shaped metal-binding loop and beta-strands III and V that together form a pocket on the remote side of the zinc binding site. The patterns of protection suggest bending of the triple helical peptide partly around the catalytic domain to reach novel exosites. Partial unwinding or underwinding of the triple helix could accompany this to facilitate its hydrolysis.

We have demonstrated that bronchoalveolar lavage fluid from chronic obstructive pulmonary disease patients contains higher levels of interferon-gamma compared with controls. Interferon-gamma is a potent inducer of various cathepsins and matrix metalloproteases. Therefore, we postulated that interferon-gamma could induce protease expression by macrophages in acute and chronic lung disease. Chronic obstructive pulmonary disease patients had greater levels of cathepsin S and matrix metalloprotease-12 in their bronchoalveolar lavage fluid. Macrophages incubated with chronic obstructive pulmonary disease bronchoalveolar lavage fluid exhibited increased expression of cathepsin S and matrix metalloprotease-12, which was inhibited by the addition of interferon-gamma-neutralizing immunoglobulin. Human secretory leukocyte protease inhibitor is an 11.7-kDa cationic non-glycosylated antiprotease synthesized and secreted by cells at the site of inflammation. We have demonstrated that secretory leukocyte protease inhibitor can inhibit interferon-gamma-induced cathepsin S production by macrophages. Pretreatment of macrophages with secretory leukocyte protease inhibitor inhibited interferon-gamma-induced inhibitor kappaB beta degradation and activation of nuclear factor kappaB. Secretory leukocyte protease inhibitor may prove to be therapeutically important as a potential inhibitor of protease expression in chronic obstructive pulmonary disease.