Matrix metalloproteinases (MMPs) are Zn(II)-dependent endopeptidases central to extracellular matrix remodeling and are therapeutic targets in cancer and arthritis. While the peptide-bond hydrolysis mechanism of MMPs is established, the subsequent catalytic steps governing product release and active-site regeneration, which regulate catalytic turnover, remain poorly understood. Here, we combine molecular dynamics (MD), well-tempered metadynamics (MetD), and hybrid quantum mechanics/molecular mechanics (QM/MM) simulations on the full MMP-1–triple-helical peptide (THP) complex to elucidate the mechanism of product release and collagen middle (M)-chain insertion, key steps in catalytic turnover. Our results show that intrinsic electric fields (IEFs) drive reaction-pathway selectivity for Zn(II)-coordinated carboxylate product dissociation, where the field projected along the Zn→COO⁻ axis stabilizes transition-state charge separation, lowering the ligand-exchange barrier for the two-water pathway and favoring formation of a five-coordinate (5C) Zn cat (II) state over the higher-energy single-water pathway leading to a four-coordinate (4C) state. Interdomain flexibility and loop motions promote solvent influx, enabling further product release. As the leading (L)-chain fragments dissociate, interactions redistribute to the M and trailing (T) chains, and Zn cat (II) relaxes from 5C to 4C. Insertion of the M-chain scissile bond becomes favorable only after L-chain displacement, establishing a sequential release–insertion mechanism. M-chain scissile carbonyl coordination proceeds with a lower barrier than the L-chain, as weaker IEFs stabilize C=O polarization, and thereby favor Zn cat (II) coordination. These findings highlight how IEFs, in concert with Zn cat (II) coordination changes, second-sphere interactions, and protein motions, regulate catalytic turnover in MMP-1, with implications for metalloprotease engineering and selective inhibitor design.

Spaceflight missions must limit biological contamination on both the outbound and return legs to comply with planetary protection requirements. Depending on the mission profile, contamination concerns may include the potential presence of bioactive molecules as defined by NASA’s Planetary Protection policies. Thus, the present study has examined the temperature and time requirements for sufficient inactivation/degradation of an infectious, heat-stable prion protein (Sup35NM), which serves as a model bioactive molecule. Bovine serum albumin was used to establish the method parameters and feasibility. Differential scanning calorimetry, Fourier transform infrared spectroscopy, analytical reversed-phase high-performance liquid chromatography, and mass spectrometry were utilized to analyze heat-treated samples, with non-treated samples serving as controls. Heat treatment at 400°C for 5 seconds was found to result in substantial decomposition of Sup35NM. In addition to the disruption of the protein backbone amide bonds, the side chain residues were also compromised. Fragments of molecular weight <4600 were observed by mass spectrometry but the impact of treatment on both the backbone and side chains of Sup35NM suggested that these fragments would not self-associate to create potentially pathogenic entities. The present methodology provided insight into the protein degradation process and can be applied to a variety of treatment strategies (e.g., any form of sterilization or inactivation) to ensure a lack of protein-based contamination of isolated extraterrestrial specimens.

Membrane type 1 matrix metalloproteinase/matrix metalloproteinase 14 (MT1-MMP-MMP-14) is associated with various stages of cancer, particularly metastasis. The intracellular cytoplasmic domain of MMP-14 has recently been recognized for the ability to regulate cellular behaviors, particularly under conditions of low pH and/or hypoxia, which can occur within the tumor microenvironment. The present work has focused on developing tools to evaluate changes that occur to the cytoplasmic domain of MMP-14 under the aforementioned conditions. Variants of the cytoplasmic domain containing posttranslational modifications have been synthesized and characterized, and flow cytometric analysis has been examined as an approach to quantify extracellular and intracellular MMP-14 in pancreatic cancer cell lines under normal and hypoxic conditions. Four variants of the MMP-14 cytoplasmic domain were synthesized, where the variants contained no modifications, phosphorylation, palmitoylation, or monoubiquitination. The structure of each variant was impacted when the pH was lowered. For quantification of intracellular MMP-14 flow cytometry was effective under both normoxic and hypoxic conditions. The synthesized cytoplasmic domain peptides can be used to raise antibodies for flow cytometric evaluation of the state of the cytoplasmic domain in cell spheroids under tumor microenvironment (low pH, hypoxic) conditions.

A subset of rheumatoid arthritis (RA) is the production of autoantibodies, including antibodies to citrullinated proteins (ACPA) and antibodies to type II collagen (AC2A). Type II collagen (COL2) is the major protein in joint cartilage and is a target of arthritogenic autoantibodies. We could confirm that sera from RA patients react with both citrullinated and native triple-helical COL2 epitopes. We examined the modulation of COL2 processing by matrix metalloproteinase 13 (MMP-13), the main collagenase responsible for degradation of articular cartilage. Anti-COL2 antibodies (AC2A) targeting the C1 epitope (residues 359-363) partially inhibited intact COL2 and fragment hydrolysis, resulting in two distinct fragments in the 25-30 kDa range. The AC2A targeting the E10 epitope (residues 777-783, the region where MMP-13 initially cleaves COL2) partially inhibited intact COL2 and fragment hydrolysis, resulting in a distinct fragment of ∼30 kDa. The AC2As targeting the F4 epitope (residues 932-936) partially inhibited collagen fragment hydrolysis, resulting in four distinct fragments in the 20-30 kDa range. Sequencing of isolated fragments revealed 14 terminated cleavage sites. Citrullination of the COL2 cleavage site reduced MMP-13 efficiency, which should further exacerbate fragment production rather than complete digestion. The results indicated that, under normal maintenance, MMP-13 cleaves COL2 initially at the 775-776 bond, followed by further digestion of COL2 fragments. Citrullination slows the initial processing of COL2 by MMP-13. In concert, AC2As inhibit the action of MMP-13 at different stages, resulting in production of collagen fragments differing in composition encountered under normal circumstances. The abnormal COL2 fragments could activate the immune system to be more pathogenic or regulatory as well as modify chondrocyte functions, and thereby play a role in the initiation of RA.

The matrix metalloproteinase (MMP) family includes several membrane-bound enzymes. Membrane-type 5 matrix metalloproteinase (MT5-MMP) is unique amongst the MMP family in being primarily expressed in the brain and during development. It is proposed to contribute to synaptic plasticity and is implicated in several pathologies, including multiple cancers and Alzheimer’s disease. In cancer, MT5-MMP expression has been correlated to cancer progression, but a distinct mechanistic role has yet to be uncovered. In Alzheimer’s disease, MT5-MMP exhibits pro-amyloidogenic activity, functioning as an η-secretase that cleaves amyloid precursor protein (APP), ultimately generating two synaptotoxic fragments, Aη-α and Aη-β. Several intracellular binding partners for MT5-MMP have been identified, and of these, N4BP2L1, EIG121, BIN1, or TMX3 binding to MT5-MMP results in a significant increase in MT5-MMP η-secretase activity. Beyond direct effects on APP, MT5-MMP may also facilitate APP trafficking to endosomal/lysosomal compartments and enhance proinflammatory responses. Overall, the substrate profile of MT5-MMP has not been well defined, and selective inhibitors of MT5-MMP have not been described. These advances will be needed for further consideration of MT5-MMP as a therapeutic target in Alzheimer’s disease and other pathologies.

One of the pathological hallmarks of Alzheimer's disease (AD) is the presence of extracellular deposits of amyloid beta (Aβ) peptide. In addition to Aβ as the core component of the amyloid plaque, the amyloid precursor protein (APP) processing fragment Aβ was also found accumulated around the plaque. The APPη pathway, mainly mediated by membrane-type 5 matrix metalloproteinase (MT5-MMP), represents an important factor in AD pathogenesis. The proamyloidogenic features of MT5-MMP could result from interactions with APP when trafficking between organelles, so determination of the location within the cell of APPη cleavage and interacting proteins of MT5-MMP affecting this process will be of priority in understanding the role of MT5-MMP in AD. In the present study, MT5-MMP was found to be located in the nucleus, cytosol, and cytosolic subcellular granules of CHO cells that stably expressed wild-type human APP751. MT5-MMP fusion proteins were constructed that could localize enzyme production in the Golgi apparatus, endosome, ER, mitochondria, or plasma membrane. The fusion proteins significantly increased sAPPη when directed to the endosome, Golgi apparatus, plasma membrane, or mitochondria. Since the C-terminal region of MT5-MMP is responsible for its intracellular location and trafficking, this domain was used as the bait in a yeast two-hybrid screen to identify MT5-MMP protein partners in a human brain cDNA library. Identified binding partners included N4BP2L1, TMX3, EIG121, bridging Integrator 1 (BIN1), RUFY4, HTRA1, and TMEM199. The binding of N4BP2L1, EIG121, BIN1, or TMX3 to MT5-MMP resulted in the most significant increase in sAPPη production. Thus, the action of MT5-MMP on APP occurs in multiple locations within the cell and is facilitated by site-specific binding partners.

Abstract Matrix metalloproteinases (MMP) are a family of proteolytic enzymes essential for various processes such as development and healing. Alternatively, the dysregulation of the gelatinases, MMP-2 and MMP-9, is implicated in immune cell migration and inflammation associated with multiple sclerosis (MS). MS is an autoimmune disease in which CD4+ T-cells play a key role both in humans and its animal model, experimental autoimmune encephalomyelitis (EAE). CD4+ T-cells are known to rely on both oxidative phosphorylation and aerobic glycolysis during various stages of activation. We have recently demonstrated that gelatinase inhibition can impact CD4+ T-cell activation and mitigate EAE progression. The present study utilized the Seahorse assay to characterize the cellular energetics of CD4+ T-cells in response to gelatinase inhibition. The resultant data suggests that gelatinases are important for both oxidative phosphorylation and the effective induction of glycolysis in CD4+ T-cells.

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.

The utilization of tumor spheroids and organoids has greatly facilitated mechanistic understanding of tumor growth and invasion and lead to more effective high-throughput analysis of potential chemotherapeutic agents. In spheroid and organoid systems, tumor invasion occurs in three dimensions and monitoring this behavior can be data intensive. Quantitative correlation of tumor invasion with protease activity can further exacerbate data storage issues. The present method utilizes the "Hit Pick" approach to provide quantitative analysis and correlation of tumor invasion and membrane type 1 matrix metalloproteinase (MT1-MMP) activity in a rapid fashion with greatly reduced data storage requirements compared with standard image analysis approaches. Inhibition of MT1-MMP activity in spheroids can also be monitored by the present approach.

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Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1 center dot THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's alpha-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

Human matrix metalloproteinase-1 (MMP-1) is a zinc(II)-dependent enzyme that catalyzes collagenolysis. Despite the availability of extensive experimental data, the mechanism of MMP-1-catalyzed collagenolysis remains poorly understood due to the lack of experimental structure of a catalytically productive enzyme-substrate complex of MMP-1. In this study, we apply molecular dynamics and combined quantum mechanics/molecular mechanics to reveal the reaction mechanism of MMP-1 based on a computationally modeled structure of the catalytically competent complex of MMP-1 that contains a large triple-helical peptide substrate. Our proposed mechanism involves the participation of an auxiliary (second) water molecule (wat2) in addition to the zinc(II)-coordinated water (wat1). The reaction initiates through a proton transfer to Glu219, followed by a nucleophilic attack by a zinc(II)-coordinated hydroxide anion nucleophile at the carbonyl carbon of the scissile bond, leading to the formation of a tetrahedral intermediate (IM2). The process continues with a hydrogen-bond rearrangement to facilitate proton transfer from wat2 to the amide nitrogen of the scissile bond and, finally, C-N bond cleavage. The calculations indicate that the rate-determining step is the water-mediated nucleophilic attack with an activation energy barrier of 22.3 kcal/mol. Furthermore, the calculations show that the hydrogen-bond rearrangement/proton-transfer step can proceed in a consecutive or concerted manner, depending on the conformation of the tetrahedral intermediate, with the consecutive mechanism being energetically preferable. Overall, the study reveals the crucial role of a second water molecule and the dynamics for effective MMP-1-catalyzed collagenolysis.

Graphical Abstract The Front Cover show the initially formed and the catalytically productive conformations of MMP-1 complex with the Triple Helical Peptide (THP), the free energy profile connecting them as well as the coordination geometry of the catalytic zinc (II). The background shows the collagen macromolecule. More information can be found in the Research Article by Tatyana G. Karabencheva-Christova and co-workers.

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use.

Chronic inflammation is now recognized as one of the major risk factors and molecular hallmarks of chronic prostatitis, benign prostatic hyperplasia (BPH), and prostate tumorigenesis. However, the molecular mechanisms by which chronic inflammation signaling contributes to the pathogenesis of these prostate diseases are poorly understood. Previous efforts to therapeutically target the upstream (e.g., TLRs and IL1-Rs) and downstream (e.g., NF-κB subunits and cytokines) inflammatory signaling molecules in people with these conditions have been clinically ambiguous and unsatisfactory, hence fostering the recent paradigm shift towards unraveling and understanding the functional roles and clinical significance of the novel and relatively underexplored inflammatory molecules and pathways that could become potential therapeutic targets in managing prostatic diseases. In this review article, we exclusively discuss the causal and molecular drivers of prostatitis, BPH, and prostate tumorigenesis, as well as the potential impacts of microbiome dysbiosis and chronic inflammation in promoting prostate pathologies. We specifically focus on the importance of some of the underexplored druggable inflammatory molecules, by discussing how their aberrant signaling could promote prostate cancer (PCa) stemness, neuroendocrine differentiation, castration resistance, metabolic reprogramming, and immunosuppression. The potential contribution of the IL1R-TLR-IRAK-NF-κBs signaling molecules and NLR/inflammasomes in prostate pathologies, as well as the prospective benefits of selectively targeting the midstream molecules in the various inflammatory cascades, are also discussed. Though this review concentrates more on PCa, we envision that the information could be applied to other prostate diseases. In conclusion, we have underlined the molecular mechanisms and signaling pathways that may need to be targeted and/or further investigated to better understand the association between chronic inflammation and prostate diseases.

Rheumatoid arthritis (RA) involves several classes of pathogenic autoantibodies, some of which react with type-II collagen (COL2) in articular cartilage. We previously described a subset of COL2 antibodies targeting the F4 epitope (ERGLKGHRGFT) that could be regulatory. Here, using phage display, we developed recombinant antibodies against this epitope and examined the underlying mechanism of action. One of these antibodies, R69-4, protected against cartilage antibody- and collagen-induced arthritis in mice, but not autoimmune disease models independent of arthritogenic autoantibodies. R69-4 was further shown to cross-react with a large range of proteins within the inflamed synovial fluid, such as the complement protein C1q. Complexed R69-4 inhibited neutrophil FCGR3 signaling, thereby impairing downstream IL-1β secretion and neutrophil self-orchestrated recruitment. Likewise, human isotypes of R69-4 protected against arthritis with comparable efficiency. We conclude that R69-4 abrogates autoantibody-mediated arthritis mainly by hindering FCGR3 signaling, highlighting its potential clinical utility in acute RA.

The Front Cover shows the multilevel molecular dynamics (MD), quantum mechanics/molecular mechanics (QM/MM) and quantum mechanics (QM) study on matrix metalloproteinase-1 (MMP-1). More information can be found in the Article by Tatyana G. Karabencheva-Christova and co-workers.

Starting from an already known MMP-13 inhibitor, 1, we pursued an SAR-approach focusing on optimizing interactions close to the Zn2+ binding site of the enzyme. We found the oxetane containing compound 32 (MMP-13 IC50 = 42 nM), which exhibited complete inhibition of collagenolysis in in vitro studies and an excellent selectivity profile among the MMP family. Interestingly, docking studies propose that the oxetane ring in 32 is oriented towards the Zn2+ ion for chelating the metal ion. Chelating properties of MMP13-inhibitors are often connected with non-selectivity within the enzyme family. Compound 32 demonstrates a rare example where the selectivity can be explained via combinatorial effects of interactions within the S1 ' loop and a chelating effect of the oxetane moiety. Furthermore, in vivo pharmacokinetic studies were performed demonstrating a concentration of 1.97 mu M of 32 within the synovial fluid of the rat knee joint, which makes the compound a promising lead compound for further optimization and development for osteoarthritis.