Increased TIMP-3 expression alters the cellular secretome through dual inhibition of the metalloprotease ADAM10 and ligand-binding of the LRP-1 receptor

The tissue inhibitor of metalloproteinases-3 (TIMP-3) is a major regulator of extracellular matrix turnover and protein shedding by inhibiting different classes of metalloproteinases, including disintegrin metalloproteinases (ADAMs). Tissue bioavailability of TIMP-3 is regulated by the endocytic receptor low-density-lipoprotein receptor-related protein-1 (LRP-1). TIMP-3 plays protective roles in disease. Thus, different approaches have been developed aiming to increase TIMP-3 bioavailability, yet overall effects of increased TIMP-3 in vivo have not been investigated. Herein, by using unbiased mass-spectrometry we demonstrate that TIMP-3-overexpression in HEK293 cells has a dual effect on shedding of transmembrane proteins and turnover of soluble proteins. Several membrane proteins showing reduced shedding are known as ADAM10 substrates, suggesting that exogenous TIMP-3 preferentially inhibits ADAM10 in HEK293 cells. Additionally identified shed membrane proteins may be novel ADAM10 substrate candidates. TIMP-3-overexpression also increased extracellular levels of several soluble proteins, including TIMP-1, MIF and SPARC. Levels of these proteins similarly increased upon LRP-1 inactivation, suggesting that TIMP-3 increases soluble protein levels by competing for their binding to LRP-1 and their subsequent internalization. In conclusion, our study reveals that increased levels of TIMP-3 induce substantial modifications in the cellular secretome and that TIMP-3-based therapies may potentially provoke undesired, dysregulated functions of ADAM10 and LRP-1

Interleukin 13 (IL-13)-regulated expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging cartilage

Objective: The adamalysin metalloproteinase 15 (ADAM15) has been shown to protect against development of osteoarthritis in mice. Here, we have investigated factors that control ADAM15 levels in cartilage. Design: Secretomes from wild-type and Adam15-/- chondrocytes were compared by label-free quantitative mass spectrometry. mRNA was isolated from murine knee joints, either with or without surgical induction of osteoarthritis on male C57BL/6 mice, and the expression of Adam15 and other related genes quantified by RT-qPCR. ADAM15 in human normal and osteoarthritic cartilage was investigated similarly and by fluorescent immunohistochemistry. Cultured HTB94 chondrosarcoma cells were treated with various anabolic and catabolic stimuli, and ADAM15 mRNA and protein levels evaluated. Results: There were no significant differences in the secretomes of chondrocytes from WT and Adam15-/- cartilage. Expression of ADAM15 was not altered in either human or murine osteoarthritic cartilage relative to disease-free controls. However, expression of ADAM15 was markedly reduced upon aging in both species, to the extent that expression in joints of 18-month-old mice was 45-fold lower than in that 4.5-month-old animals. IL-13 increased expression of ADAM15 in HTB94 cells by 2.5-fold, while modulators of senescence and autophagy pathways had no effect. Expression of Il13 in the joint was reduced with aging, suggesting this cytokine may control ADAM15 levels in the joint. Conclusion: Expression of the chondroprotective metalloproteinase ADAM15 is reduced in aging human and murine joints, possibly due to a concomitant reduction in IL-13 expression. We thus propose IL-13 as a novel factor contributing to increased osteoarthritis risk upon aging

The Chalcogen Bond in Crystalline Solids: A World Parallel to Halogen Bond

ConspectusThe distribution of the electron density around covalently bonded atoms is anisotropic, and this determines the presence, on atoms surface, of areas of higher and lower electron density where the electrostatic potential is frequently negative and positive, respectively. The ability of positive areas on atoms to form attractive interactions with electron rich sites became recently the subject of a flurry of papers. The halogen bond (HaB), the attractive interaction formed by halogens with nucleophiles, emerged as a quite common and dependable tool for controlling phenomena as diverse as the binding of small molecules to proteinaceous targets or the organization of molecular functional materials. The mindset developed in relation to the halogen bond prompted the interest in the tendency of elements of groups 13-16 of the periodic table to form analogous attractive interactions with nucleophiles.This Account addresses the chalcogen bond (ChB), the attractive interaction formed by group 16 elements with nucleophiles, by adopting a crystallographic point of view. Structures of organic derivatives are considered where chalcogen atoms form close contacts with nucleophiles in the geometry typical for chalcogen bonds. It is shown how sulfur, selenium, and tellurium can all form chalcogen bonds, the tendency to give rise to close contacts with nucleophiles increasing with the polarizability of the element. Also oxygen, when conveniently substituted, can form ChBs in crystalline solids. Chalcogen bonds can be strong enough to allow for the interaction to function as an effective and robust tool in crystal engineering. It is presented how chalcogen containing heteroaromatics, sulfides, disulfides, and selenium and tellurium analogues as well as some other molecular moieties can afford dependable chalcogen bond based supramolecular synthons. Particular attention is given to chalcogen containing azoles and their derivatives due to the relevance of these moieties in biosystems and molecular materials. It is shown how the interaction pattern around electrophilic chalcogen atoms frequently recalls the pattern around analogous halogen, pnictogen, and tetrel derivatives. For instance, directionalities of chalcogen bonds around sulfur and selenium in some thiazolium and selenazolium derivatives are similar to directionalities of halogen bonds around bromine and iodine in bromonium and iodonium compounds. This gives experimental evidence that similarities in the anisotropic distribution of the electron density in covalently bonded atoms translates in similarities in their recognition and self-assembly behavior. For instance, the analogies in interaction patterns of carbonitrile substituted elements of groups 17, 16, 15, and 14 will be presented. While the extensive experimental and theoretical data available in the literature prove that HaB and ChB form twin supramolecular synthons in the solid, more experimental information has to become available before such a statement can be safely extended to interactions wherein elements of groups 14 and 15 are the electrophiles. It will nevertheless be possible to develop some general heuristic principles for crystal engineering. Being based on the groups of the periodic table, these principles offer the advantage of being systematic

C(sp3) atoms as tetrel bond donors: A crystallographic survey

The σ-hole and π-hole interactions allow for a systematic understanding of some features of the attractive interactions involving elements of groups 13–18 of the periodic table and of some other groups. Areas of depleted electron density, where the electrostatic potential can be positive, exist on these atoms and these areas can form attractive interactions with electron rich sites (nucleophiles). When the electrophilic atom belongs to groups 14, 15, 16, or 17, the resulting interactions are named tetrel, pnictogen, chalcogen, and halogen bond, respectively. Here we discuss the tetrel bonds (TtBs) formed in crystalline solids on interaction of sp3 hybridized carbon atoms with lone pair possessing atoms and anions. A mapping of the specific short contacts formed in the solid by C(sp3) atoms is realized by discussing selected structures from the Cambridge Structural Database. This mapping led to the identification of some functional groups particularly tailored to form TtBs which can affect or control the packing in crystalline solids. Specifically, it is shown that methyl and methylene groups bound to ammonium, pyridinium, and sulfonium residues can give rise to particularly short and directional TtBs. Topologically, the formed adducts can exist as discrete species or one, two, or three dimensional networks. Fluorine atoms and perfluorinated residues as well as nitro and cyano substituents can also lead to the formation of TtBs which can control molecular conformation and packing in the solid

Binding motif of ebselen in solution: Chalcogen and hydrogen bonds team up

Ebselen (2-phenyl-1,2-benzoselenazol-3(2H)one), a glutathione peroxidase mimic, is active against several RNA viruses, among others the retrovirus responsible for the COVID-19 pandemic. In this paper 77Se and 1H NMR studies of ebselen are reported and they identify the chalcogen bond (ChB) and hydrogen bond (HB) that are central in the landscape of interactions formed by the compound in solution. The selenium atom and the hydrogen atom at the C7 carbon act as ChB and HB donors and the O and N atoms of neutral molecules function as acceptors. The ChB and HB give rise to a bifurcated supramolecular synthon, which fastens the interaction acceptor opposite to the N-Se covalent bond of the selenazole ring. It is known that the biologically important reaction of ebselen with cysteine thiol groups is favoured when selenium acts as a chalcogen bond donor. This journal i

Chalcogen Bonds in Crystals of Bis(o-anilinium)diselenide Salts

The diselenide moiety is labeled as a novel and robust chalcogen bond (ChB) donor group. The molecular electrostatic potential of two prototype diselenide derivatives shows the presence of two σ-holes along both the covalent bonds in which each selenium atom is involved. The propensity of selenium atoms of diselenides to work as electrophilic sites is confirmed by computational studies on the bis(o-anilinium)diselenide cation, and single crystal X-ray analysis of salts of this cation reveals the presence of close selenium⋯anion contacts. Comparison with halogen bonds in crystal structures of ionic δ 3 -iodane derivatives supports the rationalization of these close contacts as charge-assisted ChBs. Discrete adducts or two-dimensional networks are formed, suggesting the profitable use of the diselenide moiety in ChB based crystal engineering

4,4′-Dipyridyl Dioxide·SbF3 Cocrystal: Pnictogen Bond Prevails over Halogen and Hydrogen Bonds in Driving Self-Assembly

The SbF3·4,4′-dipyridyl N,N′-dioxide cocrystal is prepared and characterized via infrared spectroscopy and 121Sb and 123Sb nuclear quadrupole resonance. Single crystal X-ray analysis proves that a major role in cocrystal formation is played by Sb···O pnictogen bonds, the attractive interactions wherein antimony and oxygen act as the electrophilic and nucleophilic sites, respectively. Molecular electrostatic potential and natural bond orbital analyses confirm the relevance of this interaction in the self-assembly process. Dipyridyl dioxide also forms hydrogen bonded and halogen bonded cocrystal, e.g., when water and 1,4-diiodo-tetrafluoro-benzene function as acceptors of electron density. Experiments of competitive cocrystal formation indicate that under the adopted conditions the pnictogen bond prevails over the halogen bond and the hydrogen bond in identifying the tecton involved in cocrystal formation with dipyridyl dioxide

Tetrel and Pnictogen Bonds Complement Hydrogen and Halogen Bonds in Framing the Interactional Landscape of Barbituric Acids

Experimental and theoretical studies of fluoro-, chloro-, and bromo-substituted derivatives of barbituric acid and indandione show that imide protons form short hydrogen bonds and bromine or, to a lesser extent, chlorine atoms form halogen bonds. The imide nitrogen atoms act as effective pnictogen bond donors, while C(sp2) and C(sp3) atoms act as tetrel bond donors; the resulting N···O and C···O close interactions are a distinctive feature of crystal lattices in all compounds. Importantly, halogen atoms promote the electrophilicity of C(sp3) sites and favor the formation of C(sp3)···O close contacts. Oxygen atoms of carbonyl groups of barbituric and indandione units or of water molecules function as the interaction acceptor sites: namely, they donate electron density to hydrogen, halogen, nitrogen, and carbon atoms. Modeling of various barbituric acid derivatives indicates that the positive electrostatic potentials of π-holes orthogonal to the C(sp2) carbons and σ-holes on the elongation of quasi-axial F/Cl/Br-C(sp3) bonds merge to produce a single well-defined point of the most positive electrostatic potential on one face of the barbituric acids. This single local maximum of the potential on the molecular face is close to the site occupied by the oxygen forming the C(sp3)···O, and C(sp2)···O, short contacts observed in crystals