Proteomic elucidation of the targets and primary functions of the picornavirus 2A protease.

Picornaviruses are small RNA viruses that hijack host cell machinery to promote their replication. During infection, these viruses express two proteases, 2Apro and 3Cpro, which process viral proteins. They also subvert a number of host functions, including innate immune responses, host protein synthesis, and intracellular transport, by utilizing poorly understood mechanisms for rapidly and specifically targeting critical host proteins. Here, we used proteomic tools to characterize 2Apro interacting partners, functions, and targeting mechanisms. Our data indicate that, initially, 2Apro primarily targets just two cellular proteins: eukaryotic translation initiation factor eIF4G (a critical component of the protein synthesis machinery) and Nup98 (an essential component of the nuclear pore complex, responsible for nucleocytoplasmic transport). The protease appears to employ two different cleavage mechanisms; it likely interacts with eIF3L, utilizing the eIF3 complex to proteolytically access the eIF4G protein but also directly binds and degrades Nup98. This Nup98 cleavage results in only a marginal effect on nuclear import of proteins, while nuclear export of proteins and mRNAs were more strongly affected. Collectively, our data indicate that 2Apro selectively inhibits protein translation, key nuclear export pathways, and cellular mRNA localization early in infection to benefit viral replication at the expense of particular cell functions.

The extracellular matrix protein agrin promotes heart regeneration in mice.

The adult mammalian heart is non-regenerative owing to the post-mitotic nature of cardiomyocytes. The neonatal mouse heart can regenerate, but only during the first week of life. Here we show that changes in the composition of the extracellular matrix during this week can affect cardiomyocyte growth and differentiation in mice. We identify agrin, a component of neonatal extracellular matrix, as required for the full regenerative capacity of neonatal mouse hearts. In vitro, recombinant agrin promotes the division of cardiomyocytes that are derived from mouse and human induced pluripotent stem cells through a mechanism that involves the disassembly of the dystrophin-glycoprotein complex, and Yap- and ERK-mediated signalling. In vivo, a single administration of agrin promotes cardiac regeneration in adult mice after myocardial infarction, although the degree of cardiomyocyte proliferation observed in this model suggests that there are additional therapeutic mechanisms. Together, our results uncover a new inducer of mammalian heart regeneration and highlight fundamental roles of the extracellular matrix in cardiac repair.

Next generation matrix metalloproteinase inhibitors - Novel strategies bring new prospects.

Enzymatic proteolysis of cell surface proteins and extracellular matrix (ECM) is critical for tissue homeostasis and cell signaling. These proteolytic activities are mediated predominantly by a family of proteases termed matrix metalloproteinases (MMPs). The growing evidence in recent years that ECM and non-ECM bioactive molecules (e.g., growth factors, cytokines, chemokines, on top of matrikines and matricryptins) have versatile functions redefines our view on the roles matrix remodeling enzymes play in many physiological and pathological processes, and underscores the notion that ECM proteolytic reaction mechanisms represent master switches in the regulation of critical biological processes and govern cell behavior. Accordingly, MMPs are not only responsible for direct degradation of ECM molecules but are also key modulators of cardinal bioactive factors. Many attempts were made to manipulate ECM degradation by targeting MMPs using small peptidic and organic inhibitors. However, due to the high structural homology shared by these enzymes, the majority of the developed compounds are broad-spectrum inhibitors affecting the proteolytic activity of various MMPs and other zinc-related proteases. These inhibitors, in many cases, failed as therapeutic agents, mainly due to the bilateral role of MMPs in pathological conditions such as cancer, in which MMPs have both pro- and anti-tumorigenic effects. Despite the important role of MMPs in many human diseases, none of the broad-range synthetic MMP inhibitors that were designed have successfully passed clinical trials. It appears that, designing highly selective MMP inhibitors that are also effective in vivo, is not trivial. The challenges related to designing selective and effective metalloprotease inhibitors, are associated in part with the aforesaid high structural homology and the dynamic nature of their protein scaffolds. Great progress was achieved in the last decade in understanding the biochemistry and biology of MMPs activity. This knowledge, combined with lessons from the past has drawn new "boundaries" for the development of the next-generation MMP inhibitors. These novel agents are currently designed to be highly specific, capable to discriminate between the homologous MMPs and ideally administered as a short-term topical treatment. In this review we discuss the latest progress in the fields of MMP inhibitors in terms of structure, function and their specific activity. The development of novel highly specific inhibitors targeting MMPs paves the path to study complex biological processes associated with ECM proteolysis in health and disease. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman.

A general method for quantitative fractionation of mammalian cells.

Subcellular fractionation in combination with mass spectrometry-based proteomics is a powerful tool to study localization of key proteins in health and disease. Here we offered a reliable and rapid method for mammalian cell fractionation, tuned for such proteomic analyses. This method proves readily applicable to different cell lines in which all the cellular contents are accounted for, while maintaining nuclear and nuclear envelope integrity. We demonstrated the method's utility by quantifying the effects of a nuclear export inhibitor on nucleoplasmic and cytoplasmic proteomes.

From sensors to silencers: quinoline- and benzimidazole-sulfonamides as inhibitors for zinc proteases.

Derived from the extensive work in the area of small molecule zinc(II) ion sensors, chelating fragment libraries of quinoline- and benzimidazole-sulfonamides have been prepared and screened against several different zinc(II)-dependent matrix metalloproteinases (MMPs). The fragments show impressive inhibition of these metalloenzymes and preferences for different MMPs based on the nature of the chelating group. The findings show that focused chelator libraries are a powerful strategy for the discovery of lead fragments for metalloprotein inhibition.

Inhibition of pectin methyl esterase activity by green tea catechins.

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.

Extracellular Matrix Proteolysis by MT1-MMP Contributes to Influenza-Related Tissue Damage and Mortality.

Mounting an effective immune response, while also protecting tissue integrity, is critical for host survival. We used a combined genomic and proteomic approach to investigate the role of extracellular matrix (ECM) proteolysis in achieving this balance in the lung during influenza virus infection. We identified the membrane-tethered matrix metalloprotease MT1-MMP as a prominent host-ECM-remodeling collagenase in influenza infection. Selective inhibition of MT1-MMP protected the tissue from infection-related structural and compositional tissue damage. MT1-MMP inhibition did not significantly alter the immune response or cytokine expression. The available flu therapeutic Oseltamivir did not prevent lung ECM damage and was less effective than anti-MT1-MMP in influenza virus Streptococcus pneumoniae coinfection paradigms. Combination therapy of Oseltamivir with anti-MT1-MMP showed a strong synergistic effect and resulted in complete recovery of infected mice. This study highlights the importance of tissue resilience in surviving infection and the potential of such host-pathogen therapy combinations for respiratory infections.

Inhibition mechanism of membrane metalloprotease by an exosite-swiveling conformational antibody.

Membrane type 1 metalloprotease (MT1-MMP) is a membrane-anchored, zinc-dependent protease. MT1-MMP is an important mediator of cell migration and invasion, and overexpression of this enzyme has been correlated with the malignancy of various tumor types. Therefore, modulators of MT1-MMP activity are proposed to possess therapeutic potential in numerous invasive diseases. Here we report the inhibition mode of MT1-MMP by LEM-2/15 antibody, which targets a surface epitope of MT1-MMP. Specifically, the crystal structures of Fab LEM-2/15 in complex with the MT1-MMP surface antigen suggest that conformational swiveling of the enzyme surface loop is required for effective binding and consequent inhibition of MT1-MMP activity on the cell membrane. This inhibition mechanism appears to be effective in controlling active MT1-MMP in endothelial cells and at the leading edge of migratory cancer cells.

SSNMR of biosilica-entrapped enzymes permits an easy assessment of preservation of native conformation in atomic detail.

Bioinspired silica coprecipitates of enzymes find a wide range of applications for analysis and catalysis in industrial and academic research. Here we used SSNMR as a proxy for the 3D structure of enzymes trapped in bioinspired silica. We show that it is easy to assess whether the enzymes retain their native conformation in atomic detail. We thus propose SSNMR as a rapid and reliable tool to analyze this kind of samples at high resolution.

Unraveling hidden regulatory sites in structurally homologous metalloproteases.

Monitoring enzymatic activity in vivo of individual homologous enzymes such as the matrix metalloproteinases (MMPs) by antagonist molecules is highly desired for defining physiological and pathophysiological pathways. However, the rational design of antagonists targeting enzyme catalytic moieties specific to one of the homologous enzymes often appears to be an extremely difficult task. This is mainly due to the high structural homology at the enzyme active sites shared by members of the protein family. Accordingly, controlling enzymatic activity via alternative allosteric sites has become an attractive proposition for drug design targeting individual homologous enzymes. Yet, the challenge remains to identify such regulatory alternative sites that are often hidden and scattered over different locations on the protein's surface. We have designed branched amphiphilic molecules exhibiting specific inhibitory activity towards individual members of the MMP family. These amphiphilic isomers share the same chemical nature, providing versatile nonspecific binding reactivity that allows to probe hidden regulatory residues on a given protein surface. Using the advantage provided by amphiphilic ligands, here we explore a new approach for determining hidden regulatory sites. This approach includes diverse experimental analysis, such as structural spectroscopic analyses, NMR, and protein crystallography combined with computational prediction of effector binding sites. We demonstrate how our approach works by analyzing members of the MMP family that possess a unique set of such sites. Our work provides a proof of principle for using ligand effectors to unravel hidden regulatory sites specific to members of the structurally homologous MMP family. This approach may be exploited for the design of novel molecular effectors and therapeutic agents affecting protein catalytic function via interactions with structure-specific regulatory sites.