Matrix Metalloproteinase Cleavage Research Articles

Vascular-adhesion protein 1 in giant cell arteritis and polymyalgia rheumatica.

Vascular adhesion protein-1 (VAP-1) is a type 2 transmembrane sialoglycoprotein with oxidative deamination functionality, encoded by the amine oxidase copper-containing 3 (AOC3) gene. VAP-1 is widely expressed across various tissues, particularly in highly vascularized tissues and organs essential for lymphocyte circulation. In the vascular system, VAP-1 is predominantly found in vascular smooth muscle cells and endothelial cells, with higher expression levels in vascular smooth muscle cells. Under inflammatory conditions, VAP-1 rapidly translocates to the endothelial cell surface, facilitating leukocyte adhesion and migration through interactions with specific ligands, such as sialic acid-binding immunoglobulin-type lectins (Siglec)-9 on neutrophils and monocytes, and Siglec-10 on B cells, monocytes, and eosinophils. This interaction is crucial for leukocyte transmigration into inflamed tissues. Furthermore, VAP-1's enzymatic activity generates hydrogen peroxide and advanced glycation end-products, contributing to cytotoxic damage and vascular inflammation. In this context, the soluble form of VAP-1 (sVAP-1), produced by matrix metalloproteinase cleavage from its membrane-bound counterpart, also significantly influences leukocyte migration. This review aims to elucidate the multifaceted pathophysiological roles of VAP-1 in vascular inflammation, particularly in giant cell arteritis (GCA) and associated polymyalgia rheumatica (PMR). By exploring its involvement in immune cell adhesion, migration, and its enzymatic contributions to oxidative stress and tissue damage, we investigate the importance of VAP-1 in GCA. Additionally, we discuss recent advancements in imaging techniques targeting VAP-1, such as [68Ga]Ga-DOTA-Siglec-9 PET/CT, which have provided new insights into VAP-1's role in GCA and PMR. Overall, understanding VAP-1's comprehensive roles could pave the way for improved strategies in managing these conditions.

N-Terminomic Identification of Intracellular MMP-2 Substrates in Cardiac Tissue.

Proteases are enzymes that induce irreversible post-translational modifications by hydrolyzing amide bonds in proteins. One of these proteases is matrix metalloproteinase-2 (MMP-2), which has been shown to modulate extracellular matrix remodeling and intracellular proteolysis during myocardial injury. However, the substrates of MMP-2 in heart tissue are limited, and lesser known are the cleavage sites. Here, we used degradomics to investigate the substrates of intracellular MMP-2 in rat ventricular extracts. First, we designed a novel, constitutively active MMP-2 fusion protein (MMP-2-Fc) that we expressed and purified from mammalian cells. Using this protease, we proteolyzed ventricular extracts and used subtiligase-mediated N-terminomic labeling which identified 95 putative MMP-2-Fc proteolytic cleavage sites using mass spectrometry. The intracellular MMP-2 cleavage sites identified in heart tissue extracts were enriched for proteins primarily involved in metabolism, as well as the breakdown of fatty acids and amino acids. We further characterized the cleavage of three of these MMP-2-Fc substrates based on the gene ontology analysis. We first characterized the cleavage of sarco/endoplasmic reticulum calcium ATPase (SERCA2a), a known MMP-2 substrate in myocardial injury. We then characterized the cleavage of malate dehydrogenase (MDHM) and phosphoglycerate kinase 1 (PGK1), representing new cardiac tissue substrates. Our findings provide insights into the intracellular substrates of MMP-2 in cardiac cells, suggesting that MMP-2 activation plays a role in cardiac metabolism.

Engineered protein cages with enhanced extracellular drug release for elevated antitumor efficacy

Human heavy chain ferritin (HFn) protein cage has been explored as a nanocarrier for targeted anticancer drug delivery. Here, we introduced a matrix metalloproteinases (MMPs)-cleavable sequence into the DE loop of HFn, creating an MMP-responsive variant, MR-HFn, for localized and extracellular drug release. The crystal structure of MR-HFn revealed that the addition of the MMPs recognition sequence did not affect the self-assembly of HFn but presented a surface-exposed loop susceptible to MMPs cleavage. Biochemical analysis indicated that this engineered protein cage is responsive to MMPs, enabling the targeted release of encapsulated drugs. To evaluate the therapeutic potential of this engineered protein cage, monosubstituted β-carboxy phthalocyanine zinc (CPZ), a type of photosensitizer, was loaded inside this protein cage. The prepared CPZ@MR-HFn showed higher uptake and stronger phototoxicity in MMPs overexpressed tumor cells, as well as enhanced penetration into multicellular tumor spheroids compared with its counterpart CPZ@HFn in vitro. In vivo, CPZ@MR-HFn displayed a higher tumor inhibitory rate than CPZ@HFn under illumination. These results indicated that MR-HFn is a promising nanocarrier for anticancer drug delivery and the MMP-responsive strategy here can also be adapted for other stimuli.

Abstract 6720: HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors

Abstract Introduction/Purpose: The Wagner lab has developed a protein nanoring based platform to direct human immune cells to combat solid tumors. These nanorings are bispecific, comprised of tumor-targeting and immune cell-targeting dihydrofolate reductase (DHFR2) fusion proteins. These protein nanorings direct immune cells to target and destroy cancer cells. For treating solid tumors, we aimed to target epidermal growth factor receptor (EGFR). However, targeting EGFR could lead to potential “on-target, off-tumor” effects due to expression of EGFR in healthy tissue. Matrix metalloproteinases are overexpressed in a variety of solid tumors. Therefore, to improve tumor specificity, we hypothesized that masking the immune cell targeting DHFR2 protein through a matrix metalloproteinase-2 (MMP-2) sensitive linker will prevent engagement and activation of immune cells outside the tumor microenvironment (TME). Once these protease-activatable nanorings enter the TME and bind to EGFR, cleavage by MMP-2 will lead to unmasking of the immune cell binder, thereby crosslinking and activating immune cells, causing targeted cancer cell lysis. Methods: We designed human histidine triad nucleotide binding protein-1 (HINT1) masked anti-immune cell targeting nanobody-DHFR2 fusion proteins. HINT1 was hypothesized to sterically block the engagement of immune cells outside the tumor. HINT1-anti-TCR-DHFR2 (TCR = T cell receptor) and HINT1-anti-CD16-DHFR2 were cloned, expressed, purified, and characterized. Their apparent binding affinities were determined using flow cytometry. The anti-tumor efficacy of these nanorings was evaluated against EGFR overexpressing A431 (human epidermoid carcinoma) cells. Results: HINT1-anti-TCR-DHFR2 and HINT1-anti-CD16-DHFR2 rings showed reduced binding to T and NK-92-CD16 cells, respectively. In vitro MMP-2 cleavage assay showed complete removal of the HINT1 mask within 2 hours. Western blot analysis showed that both proteins were stable in human serum up to 3 days. 2D cytotoxicity assays with protein nanorings assembled using anti-EGFR-DHFR2 and anti-TCR-DHFR2 or anti-CD16-DHFR2 with T and NK-92-CD16 cells respectively, showed a potent lysis of A431 cells. The EGFR-targeted lysis was diminished in the presence of HINT1 mask, thereby providing proof-of-concept for the steric masking of the anti-immune cell targeting nanobodies. Conclusion: Protease-activatable nanorings can selectively lyse EGFR+ tumor cells in vitro. Their antitumor efficacy and safety are currently being evaluated using a previously established in vivo mouse model. Therefore, HINT1-steric masking can serve as a simple and effective strategy to address off-tumor toxicities associated with bispecific immunotherapies. Citation Format: Abhishek Kulkarni, Debasmita Paul, Declan Dahlberg, Kostana Ligori, Freddys Rodriguez, Lakmal Rozumalski, Bruce Walcheck, Carston R. Wagner. HINT1 steric masked, protease-activatable bispecific protein nanorings direct human T and NK cells to eradicate EGFR+ solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6720.

A Targeted Exosome Therapeutic Confers Both CfDNA Scavenging and Macrophage Polarization for Ameliorating Rheumatoid Arthritis.

Only a minority of rheumatoid arthritis (RA) patients achieve disease remission, so the exploration of additional pathogenic factors and the development of new therapeutics are needed. Here, strong correlations among the cell-free DNA (cfDNA) level and the inflammatory response in clinical synovial fluid samples and RA disease activity are discovered. The important role of cfDNA in disease development in a collagen-induced arthritis (CIA) murine model is also demonstrated. Building on these findings, a novel therapeutic based on anti-inflammatory (M2) macrophage-derived exosomes as chassis, that are modified with both oligolysine and matrix metalloproteinase (MMP)-cleavable polyethylene glycol (PEG) on the membrane, is developed. After intravenous injection, PEG-enabled prolonged circulation and C─C motif chemokine ligand-directed accumulation together result in enrichment at inflamed joints. Following subsequent MMP cleavage, the positively charged oligolysine is exposed for cfDNA scavenging, while exosomes induce M2 polarization. By using a classical CIA murine model and a newly established CIA canine model, it is demonstrated that the rationally designed exosome therapeutic substantially suppresses inflammation in joints and provides strong chondroprotection and osteoprotection, revealing its potential for effective CIA amelioration.

Copper-pyridine-2,4,6-tricarboxylate metal organic framework nanoparticles based opto-electrochemical sensor for sensitive detection of matrix metalloproteinase‑2

We designed a multiple signal sensing probe based on highly luminescent nanosized Cu-PTC metal-organic framework (MOF). The as synthesized nano-MOF was characterized by various techniques to confirm its morphology, crystallinity and elemental composition. A positively charged matrix metalloproteinase-2 (MMP-2) substrate (a polypeptide) was designed and prepared to interact electrostatically with the negatively charged Cu-PTC MOF probe, resulting in decrease in fluorescence response of the probe. Subsequently, MMP-2 was specifically recognized by the MMP-2 substrate and cleavage of the MMP-2 substrate resulted in “turn-on” fluorescence response from 1 to 175 ng/mL of MMP-2 concentrations with a limit of detection (LOD) of 0.8 ng/mL. In addition, the developed Cu-PTC MOF also exhibited peroxidase-like activity, which was exploited in the electrochemical detection of MMP-2 subjected to the catalysis of 3,3,5,5-tetramethylbenzidine (TMB) in the presence of H2O2. The large surface area of the probe and unique catalytic activity toward TMB facilitated fabrication of an excellent sensing platform to detect MMP-2 with a linear range of 0.5–200 ng/mL with a LOD of 0.25 ng/mL. Various potential interfering biomolecules were evaluated and the stability of the constructed biosensor was assessed in optical and electrochemical modes. The reliability of the electrochemical detection mode was further executed by monitoring MMP-2 in biological samples (human serum and cell samples) and good recoveries were recorded.

Enzyme Regulating the Wettability of the Outer Surface of Nanochannels.

Functional probes not only at the inner wall but also at the outer surface of nanochannel systems could be used for the recognition and detection of biotargets. Despite the advancements, the current detection mechanisms are still mainly based on the surface charge variation. We proposed a strategy of using the variation of wettability on the outer surface of nanochannels for detecting a tumor marker, herein, exemplifying matrix metalloproteinase-2 (MMP-2). The outer surface of the nanochannels were modified with amphipathic peptide probe consisting of hydrophilic unit (CRRRR), MMP-2 cleavage unit (PLGLAG), and hydrophobic unit (Fn). After recognition of MMP-2, due to the release of hydrophobic unit, the hydrophilicity of the outer surface was expected to increase, thus leading to the increase of ion current. Furthermore, the number (n) of phenylalanine (F) in the hydrophobic unit was modulated from 2, 4, to 6. By lengthening the hydrophobic unit, the limit of detection for MMP-2 detection could reach 1 ng/mL (when n = 6) and improve by 50-fold (to n = 2). This nanochannel system was utilized to successfully detect the MMP-2 secreted from cells and demonstrated that the expression of MMP-2 was related to the cell cycle and exhibited the highest level in G1/S phase. This study proved that in addition to the surface charge, wettability regulation could also be utilized as a variation factor to broaden the design strategy of a probe on OS to achieve the detection of biotargets.

Loss of Mitochondrial Dynamics Proteins Mitofusin‐2 and Drp‐1 in Myocardial Ischemia‐Reperfusion Injury Is Prevented by Matrix Metalloproteinase‐2 Preferring Inhibitors

ObjectiveMatrix metalloproteinase‐2 (MMP‐2) is a ubiquitous protease that cleaves several extracellular and intracellular proteins. MMP‐2 is activated intracellularly in response to enhanced oxidative stress resulting from myocardial ischemia/reperfusion (I/R) injury. Oxidative stress impairs mitochondrial function which is regulated by different proteins including mitofusin‐2 (Mfn‐2) and dynamin‐related protein‐1 (Drp‐1) which control mitochondrial dynamics involving fusion and fission, respectively. As both proteins are localized at the mitochondrial outer membrane and MMP‐2 is localized at the mitochondrial‐endoplasmic reticulum associated membrane we hypothesized that MMP‐2 may proteolyze Mfn‐2 and Drp‐1. We therefore investigated whether inhibition of MMP‐2 could protect against the loss of mitochondrial dynamics proteins during I/R injury.MethodsHearts isolated from 3 month old C57BL/6J mice were perfused according to Langendorff at constant pressure and subjected to I/R injury (30 min ischemia and 40 min reperfusion) in absence or presence of MMP‐2 preferring inhibitors ARP‐100 (10 µM) or ONO‐4817 (50 µM) or their vehicle (n=5 hearts per group). Effects on mechanical function were recorded. At the end of reperfusion, the hearts were homogenized and subcellular fractions were prepared. The degradation of troponin I (TnI), an intracellular MMP‐2 target, was measured in the cytosolic fraction as a marker of MMP‐2 activity. Levels of Mfn‐2 and Drp‐1 in the mitochondrial fraction were also measured using western blot. In silico analysis of potential MMP‐2 cleavage sites was performed using Procleave.ResultsARP‐100 or ONO‐4817 significantly increased left ventricular developed pressure compared to vehicle‐treated I/R hearts (% of pre‐ischemic baseline value at R40: IR+vehicle 25.5±3.2, IR+ARP 50.1±3.2, IR+ONO 57.3±1.6%, p<0.05). Similarly, both inhibitors significantly improved the rates of contraction and relaxation (+/‐dP/dt). TnI loss was increased in I/R hearts as shown by a significant reduction in TnI (~30 kDa) and the appearance of an ~22 kDa band. ARP‐100 or ONO‐4817 attenuated TnI loss, indicating MMP‐2 activation. Levels of Mfn‐2 and Drp‐1 in the mitochondrial fraction were significantly reduced in the I/R group and ARP‐100 or ONO‐4817 attenuated this reduction (p<0.05). Similar results were obtained using a different Mfn‐2 antibody that binds to its C‐terminus. In silico analysis of both mouse Mfn‐2 and Drp‐1 sequences showed several potential sites that could be targeted by MMP‐2.ConclusionsDuring myocardial I/R injury, MMP‐2 may affect mitochondrial dynamics by proteolysis of Mfn‐2 and Drp‐1. Inhibition of MMP‐2 activity could protect against cardiac contractile dysfunction in part by preserving these proteins affecting mitochondrial fusion and fission.

Mass-Encoded Suspension Array for Multiplex Detection of Matrix Metalloproteinase Activities.

This work designed a mass spectrometric biosensing strategy for the multiplex detection of matrix metalloproteinases (MMPs) with a mass-encoded suspension array. This array was fabricated as multiplex sensing probes by functionalizing magnetic beads with MMP-specific peptide-isobaric tags for relative and absolute quantification (iTRAQ) conjugates, which contained a hexahistidine tag for surface binding, a substrate region for MMP cleavage, and a coding region for the specific MMP. The integration of the multiplex coding ability of iTRAQ with ultrahigh performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and the proteolysis method for peptide digestion endowed the biosensing method with high throughput and ultrahigh sensitivity. This strategy could be conveniently performed by mixing the sample and the suspension array for enzymatic reactions and then digesting the uncleaved peptides with trypsin to release the coding regions for UPLC-MS/MS analysis. With MMP-2 and MMP-7 as analytes, the relative changes of peak area ratios of coding regions showed good linear responses in the ranges of 0.2-100 and 0.5-400 ng mL-1, with detection limits of 0.064 and 0.17 ng mL-1, respectively. The analysis of MMP activity in serum samples and its change responding to inhibitors demonstrated the specificity, practicability, and expansibility of the proposed strategy. This work paves a new avenue for the activity assays of multiplex enzymes and promotes the development of mass spectrometric biosensing.

Elevated plasma Ninjurin-1 levels in atrial fibrillation is associated with atrial remodeling and thromboembolic risk

BackgroundNerve injury-induced protein 1 (Ninj1) is elevated in various inflammatory diseases. The soluble form of Ninj1 yield by matrix metalloproteinase cleavage is a secreted protein and inhibits cell adhesion and inflammation. However, the role of plasma Ninj1 in atrial fibrillation (AF) has not been reported. The present study aimed to investigate the correlation between plasma Ninj1 levels and AF.MethodsA total of 96 AF patients [age 66.00 (60.00, 72.00) years, male 56 (58.33%)] and 51 controls without AF [age 65.00 (55.00, 68.00) years, male 21 (41.18%)] were enrolled in this study. Plasma Ninj1 concentrations were detected using enzyme-linked immunosorbent assay. Also, the clinical characteristics, left atrial volume index (LAVI), CHA2DS2-VASc score, and HAS-BLED score were evaluated.ResultsPlasma Ninj1 levels were significantly higher in patients with AF than in controls (P < 0.001). Plasma Ninj1 levels were positively correlated with LAVI (P = 0.019) and CHA2DS2-VASc score (P = 0.024). Logistic regression analysis confirmed that the Ninj1 plasma levels were associated with AF (P = 0.009). The receiver operating characteristic analysis showed that plasma Ninj1 had a predictive value for AF (P < 0.001).ConclusionsPlasma Ninj1 levels were elevated in patients with AF, associated with left atrial enlargement and thromboembolic risk in AF.

Osteopontin: another piece in the systemic lupus erythematosus immunopathology puzzle.

Osteopontin (OPN) is a phosphoglycoprotein involved in bone remodelling, wound healing, cell adhesion, tissue remodelling, and immune response that is distributed widely in normal adult tissues. OPN biological activity is regulated by thrombin and matrix metalloproteinases (MMPs) cleavage, where the full-length (OPN-FL) protein and the cleaved OPN-N are associated with autoimmune diseases such as systemic lupus erythematosus (SLE). OPN overexpression has been associated with a predisposition to SLE and bad prognosis since OPN could mediate a sustained polyclonal B cell activation that besides to intracellular OPN (iOPN) form, promote the T follicular helper (TFH) cells and enhance anti-nuclear antibody production. Currently, the role of OPN in lupus nephritis (LN) has been reported and extensively studied; however, no data are available about the potential mechanism of OPN in neuropsychiatric SLE (NPSLE). In this review, we highlighted the contribution of OPN and iOPN in LN and NPSLE immunopathology.

Enzymatically releasable polyethylene glycol – host defense peptide conjugates with improved activity and biocompatibility

Host defense peptides (HDPs) have been the subject of great interest for the treatment of multidrug-resistant bacterial infections due to their multimodal activity and low induction of resistance. However, aggregation, toxicity, and short biological half-life have limited their applicability for clinical treatment. Many methods have been explored to alleviate these issues, such as polymer (e.g., polyethylene glycol (PEG)) conjugation, but these are often accompanied by reductions in the activity of the HDP. Here, we detail the design of a novel PEG-HDP conjugate incorporating an enzymatic cleavage sequence targeting matrix metalloproteinases (MMPs) that accumulate at sites of inflammation and infection. Addition of the cleavage sequence onto either the N- or the C-terminal region of the parent peptide (peptide 73, a derivative of the HDP aurein 2.2) was explored to determine the location for optimal antimicrobial activity following MMP cleavage; furthermore, the susceptibility of the peptide to MMP cleavage after conjugation to 2 kDa or 5 kDa PEG was examined. The top candidate, L73, utilized an N-terminal cleavage site that was subsequently conjugated to a 2 kDa PEG polymer. Both L73 and the conjugate exhibited no antimicrobial activity in vitro until cleaved by purified MMP, which liberated a peptide fragment with 16- or 63-fold improved activity, respectively, corresponding to a minimum inhibitory concentration (MIC) of 8 μg/mL, comparable to that of peptide 73 (4 μg/mL). Furthermore, PEG conjugation improved the blood compatibility and reduced the aggregation tendency of the HDP in vitro, indicating enhanced biocompatibility. When administered as a single subcutaneous dose (~3.6 mg, or a peptide concentration of 142 mg/kg) in a mouse abscess model of high-density methicillin-resistant Staphylococcus aureus (MRSA) infection, the conjugate displayed strong activity, reducing abscess size and bacterial load by 73.3% and 58-fold, respectively. This activity was completely lost when the cleavage site was rendered resistant to MMPs by the substitution of two d-amino acids, supporting the hypothesis that antimicrobial activity was dependent on cleavage by MMPs, which were shown here to increasingly accumulate at the abscess site up to 18 h post infection. Finally, the conjugate displayed biocompatibility in vivo, with no identifiable toxicity or aggregation.

The Product of Matrix Metalloproteinase Cleavage of Doxorubicin Conjugate for Anticancer Drug Delivery: Calorimetric, Spectroscopic, and Molecular Dynamics Studies on Peptide-Doxorubicin Binding to DNA.

Matrix metalloproteinases (MMPs) are extracellular matrix degradation factors, promoting cancer progression. Hence, they could provide an enzyme-assisted delivery of doxorubicin (DOX) in cancer treatment. In the current study, the intercalation process of DOX and tetrapeptide–DOX, the product of the MMPs’ cleavage of carrier-linked DOX, into dsDNA was investigated using stationary and time-resolved fluorescence spectroscopy, UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). The molecular dynamics (MD) simulations on the same tetrapeptide–DOX…DNA and DOX…DNA systems were also performed. The undertaken studies indicate that DOX and tetrapeptide–DOX can effectively bond with dsDNA through the intercalation mode; however, tetrapeptide–DOX forms less stable complexes than free DOX. Moreover, the obtained results demonstrate that the differences in DNA affinity of both forms of DOX can be attributed to different intercalation modes. Tetrapeptide–DOX shows a preference to intercalate into DNA through the major groove, whereas DOX does it through the minor one. In summary, we can conclude that the tetrapeptide–DOX intercalation to DNA is significant and that even the lack of non-specific proteases releasing DOX from the tetrapeptide conjugate, the presence of which is suggested by the literature for the efficient release of DOX, should not prevent the cytostatic action of the anthracycline.

Redirecting extracellular proteases to molecularly guide radiosensitizing drugs to tumors

Patients with advanced cancers are treated with combined radiotherapy and chemotherapy, however curability is poor and treatment side effects severe. Drugs sensitizing tumors to radiotherapy have been developed to improve cell kill, but tumor specificity remains challenging. To achieve tumor selectivity of small molecule radiosensitizers, we tested as a strategy active tumor targeting using peptide-based drug conjugates. We attached an inhibitor of the DNA damage response to antibody or cell penetrating peptides. Antibody drug conjugates honed in on tumor overexpressed cell surface receptors with high specificity but lacked efficacy when conjugated to the DNA damage checkpoint kinase inhibitor AZD7762. As an alternative approach, we synthesized activatable cell penetrating peptide scaffolds that accumulated within tumors based on matrix metalloproteinase cleavage. While matrix metalloproteinases are integral to tumor progression, they have proven therapeutically elusive. We harnessed these pro-tumorigenic extracellular proteases to spatially guide radiosensitizer drug delivery using cleavable activatable cell penetrating peptides. Here, we tested the potential of these two drug delivery platforms targeting distinct tumor compartments in combination with radiotherapy and demonstrate the advantages of protease triggered cell penetrating peptide scaffolds over antibody drug conjugates to deliver small molecule amine radiosensitizers.

Moonlighting matrix metalloproteinase substrates: Enhancement of proinflammatory functions of extracellular tyrosyl-tRNA synthetase upon cleavage

Tyrosyl-tRNA synthetase ligates tyrosine to its cognate tRNA in the cytoplasm, but it can also be secreted through a noncanonical pathway. We found that extracellular tyrosyl-tRNA synthetase (YRS) exhibited proinflammatory activities. In addition to acting as a monocyte/macrophage chemoattractant, YRS initiated signaling through Toll-like receptor 2 (TLR2) resulting in NF-κB activation and release of tumor necrosis factor α (TNFα) and multiple chemokines, including MIP-1α/β, CXCL8 (IL8), and CXCL1 (KC) from THP1 monocyte and peripheral blood mononuclear cell–derived macrophages. Furthermore, YRS up-regulated matrix metalloproteinase (MMP) activity in a TNFα-dependent manner in M0 macrophages. Because MMPs process a variety of intracellular proteins that also exhibit extracellular moonlighting functions, we profiled 10 MMPs for YRS cleavage and identified 55 cleavage sites by amino-terminal oriented mass spectrometry of substrates (ATOMS) positional proteomics and Edman degradation. Stable proteoforms resulted from cleavages near the start of the YRS C-terminal EMAPII domain. All of the MMPs tested cleaved at ADS386↓387LYV and VSG405↓406LVQ, generating 43- and 45-kDa fragments. The highest catalytic efficiency for YRS was demonstrated by MMP7, which is highly expressed by monocytes and macrophages, and by neutrophil-specific MMP8. MMP-cleaved YRS enhanced TLR2 signaling, increased TNFα secretion from macrophages, and amplified monocyte/macrophage chemotaxis compared with unprocessed YRS. The cleavage of YRS by MMP8, but not MMP7, was inhibited by tyrosine, a substrate of the YRS aminoacylation reaction. Overall, the proinflammatory activity of YRS is enhanced by MMP cleavage, which we suggest forms a feed-forward mechanism to promote inflammation.

Junctophilin-2 is a target of matrix metalloproteinase-2 in myocardial ischemia-reperfusion injury.

Junctophilin-2 is a structural membrane protein that tethers T-tubules to the sarcoplasmic reticulum to allow for coordinated calcium-induced calcium release in cardiomyocytes. Defective excitation-contraction coupling in myocardial ischemia-reperfusion (IR) injury is associated with junctophilin-2 proteolysis. However, it remains unclear whether preventing junctophilin-2 proteolysis improves the recovery of cardiac contractile dysfunction in IR injury. Matrix metalloproteinase-2 (MMP-2) is a zinc and calcium-dependent protease that is activated by oxidative stress in myocardial IR injury and cleaves both intracellular and extracellular substrates. To determine whether junctophilin-2 is targeted by MMP-2, isolated rat hearts were perfused in working mode aerobically or subjected to IR injury with the selective MMP inhibitor ARP-100. IR injury impaired the recovery of cardiac contractile function which was associated with increased degradation of junctophilin-2 and damaged cardiac dyads. In IR hearts, ARP-100 improved the recovery of cardiac contractile function, attenuated junctophilin-2 proteolysis, and prevented ultrastructural damage to the dyad. MMP-2 was co-localized with junctophilin-2 in aerobic and IR hearts by immunoprecipitation and immunohistochemistry. In situ zymography showed that MMP activity was localized to the Z-disc and sarcomere in aerobic hearts and accumulated at sites where the striated JPH-2 staining was disrupted in IR hearts. In vitro proteolysis assays determined that junctophilin-2 is susceptible to proteolysis by MMP-2 and in silico analysis predicted multiple MMP-2 cleavage sites between the membrane occupation and recognition nexus repeats and within the divergent region of junctophilin-2. Degradation of junctophilin-2 by MMP-2 is an early consequence of myocardial IR injury which may initiate a cascade of sequelae leading to impaired contractile function.

Loss of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) in the Diabetic Retina: Role of Matrix Metalloproteinases.

PurposeTo test the hypothesis that high glucose and matrix metalloproteinases (MMPs) contribute to the diabetes-induced loss of platelet endothelial cell adhesion molecule-1 (PECAM-1) in the retinal microvasculature.MethodsPECAM-1 and MMP protein, activity, and interactions with PECAM-1 were assessed using western blotting, zymography, immunofluorescence, or coimmunoprecipitation assays. These assays were conducted using primary rat retinal microvascular endothelial cells (RRMECs) grown either in normal glucose (5 mM) or high glucose (25 mM) conditions and using retinas collected from streptozotocin-induced diabetic or control rats. The broad-spectrum MMP inhibitor GM6001 was administered in vivo and in vitro to ascertain the role of MMPs in the hyperglycemia-induced loss of PECAM-1.ResultsA dramatic decrease in PECAM-1 (western blotting, immunofluorescence) was observed in both the diabetic retina and in hyperglycemic RRMECs. The decrease in PECAM-1 was accompanied by a significant increase in the presence and activity of matrix metalloproteinase-2 (MMP-2) (but not matrix metalloproteinase-9 [MMP-9]) in the diabetic plasma (P < 0.05) and in hyperglycemic RRMECs (P < 0.05). Moreover, RRMEC PECAM-1 significantly decreased when treated with plasma collected from diabetic rats. Several MMP-2 cleavage sites on PECAM-1 were identified using in silico analysis. Moreover, PECAM-1/MMP-2 interactions were confirmed using coimmunoprecipitation. PECAM-1 was significantly decreased in RRMECs treated with MMP-2 (P < 0.05), but became comparable to controls with the MMP inhibitor GM6001 in both the diabetic retina and hyperglycemic RRMECs.ConclusionsThese results indicate a possible role of MMP-2 in hyperglycemia-induced PECAM-1 loss in retinal endothelial cells.

Structure and proteolytic susceptibility of the inhibitory C-terminal tail of cardiac troponin I

BackgroundCardiac troponin I (cTnI) has two flexible tails that control the cardiac cycle. The C-terminal tail, cTnI135–209, binds actin to shut off cardiac muscle contraction, whereas the competing calcium-dependent binding of the switch region, cTnI146–158, by cardiac troponin C (cTnC) triggers contraction. The N-terminal tail, cTnI1–37, regulates the calcium affinity of cTnC. cTnI is known to be susceptible to proteolytic cleavage by matrix metalloproteinase-2 (MMP-2) and calpain, two intracellular proteases implicated in ischemia-reperfusion injury. MethodsSoluble fragments of cTnI containing its N- and C-terminal tails, cTnI1–77 and cTnI135–209, were highly expressed and purified from E. coli. We performed in vitro proteolysis studies of both constructs using liquid chromatography-mass spectrometry and solution NMR studies of the C-terminal tail. ResultscTnI135–209 is intrinsically disordered, though it contains three regions with helical propensity (including the switch region) that acquire more structure upon actin binding. We identified three precise MMP-2 cleavage sites at cTnI P17-I18, A156-L157, and G199-M200. In contrast, calpain-2 has numerous cleavage sites throughout Y25-T30 and A152-A160. The critical cTnI switch region is targeted by both proteases. ConclusionsBoth N-terminal and C-terminal tails of cTnI are susceptible to cleavage by MMP-2 and calpain-2. Binding to cTnC or actin confers some protection to proteolysis, which can be understood in terms of their interactions as probed by NMR studies. General significancecTnI is an important marker of intracellular proteolysis in cardiomyocytes, given its many protease-specific cut sites, high natural abundance, indispensable functional role, and clinical use as gold standard biomarker of myocardial injury.

Dissecting MMP P10′ and P11′ subsite sequence preferences, utilizing a positional scanning, combinatorial triple-helical peptide library

Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that remodel the extracellular matrix environment and mitigate outside-in signaling. Loss of regulation of MMP activity plays a role in numerous pathological states. In particular, aberrant collagenolysis affects tumor invasion and metastasis, osteoarthritis, and cardiovascular and neurodegenerative diseases. To evaluate the collagen sequence preferences of MMPs, a positional scanning synthetic combinatorial library was synthesized herein and was used to investigate the P10' and P11' substrate subsites. The scaffold for the library was a triple-helical peptide mimic of the MMP cleavage site in types I-III collagen. A FRET-based enzyme activity assay was used to evaluate the sequence preferences of eight MMPs. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. On the basis of the screening results, several individual peptides were designed and evaluated. A triple-helical substrate incorporating Asp-Lys in the P10'-P11' subsites offered selectivity between MMP-14 and MMP-15, whereas Asp-Lys or Trp-Lys in these subsites discriminated between MMP-2 and MMP-9. Future screening of additional subsite positions will enable the design of selective triple-helical MMP probes that could be used for monitoring in vivo enzyme activity and enzyme-facilitated drug delivery. Furthermore, selective substrates could serve as the basis for the design of specific triple-helical peptide inhibitors targeting only those MMPs that play a detrimental role in a disease of interest.

Sustained and Bioresponsive Two-Stage Delivery of Therapeutic miRNA via Polyplex Micelle-Loaded Injectable Hydrogels for Inhibition of Intervertebral Disc Fibrosis.

Intervertebral disc degeneration (IDD) is frequently caused by gradual pathological changes inside intervertebral discs (IVDs) and progressive fibrosis. MicroRNA-29 (miR-29) family possesses potent fibrosis suppression capability, but their application for treatment of chronic IDD is limited due to lack of suitable local delivery systems. In this report, given various overexpressed matrix metalloproteinases (MMPs) during IDD, injectable MMP-degradable hydrogels encapsulating MMP-responsive polyplex micelles are developed for sustained and bioresponsive delivery of miR-29a into nucleus pulposus cells via a two-stage process. Cationic block copolymers are designed to complex miR-29a, and subsequently mixed with the poly(ethylene glycol) (PEG) gelation precursors and MMP-cleavable peptide cross-linkers for in situ formation of polyplex micelle-encapsulated hydrogels in the diseased IVDs. In the presence of MMPs, the polyplex micelles are first released by MMP cleavage of the hydrogels, and subsequently, MMPs-responsive detachment of PEG shells from polyplex micelles contributes to efficient cellular uptake and endosomal escape. MiR-29a is demonstrated to effectively silence the expression of MMP-2, inhibit the fibrosis process, and reverse IDD in animal models through blocking the β-catenin translocation pathway from the cytoplasm to the nucleus. This two-stage bioresponsive local miRNA delivery system represents a novel and promising strategy for the treatment of chronic IDD.