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1.  αvβ3-dependent cross-presentation of matrix metalloproteinase–2 by melanoma cells gives rise to a new tumor antigen 
A large array of antigens that are recognized by tumor-specific T cells has been identified and shown to be generated through various processes. We describe a new mechanism underlying T cell recognition of melanoma cells, which involves the generation of a major histocompatibility complex class I–restricted epitope after tumor-mediated uptake and processing of an extracellular protein—a process referred to as cross-presentation—which is believed to be restricted to immune cells. We show that melanoma cells cross-present, in an αvβ3-dependent manner, an antigen derived from secreted matrix metalloproteinase–2 (MMP-2) to human leukocyte antigen A*0201-restricted T cells. Because MMP-2 activity is critical for melanoma progression, the MMP-2 peptide should be cross-presented by most progressing melanomas and represents a unique antigen for vaccine therapy of these tumors.
doi:10.1084/jem.20042138
PMCID: PMC2212908  PMID: 15998788
2.  N-glycosylation enhances presentation of an MHC class I restricted epitope from tyrosinase1 
We recently demonstrated that the mechanism of processing of an HLA-A*0201-restricted peptide epitope, Tyr369(D), derived from the membrane protein tyrosinase, involves retrotranslocation of glycosylated molecules from the endoplasmic reticulum to the cytosol; removal of an N-linked carbohydrate from Asn371 by peptide N-glycanase; proteolysis by the proteasome and other proteases; and retransport of the resulting peptides into the endoplasmic reticulum for association with HLA-A*0201. Carbohydrate removal results in deamidation of Asn371 to Asp. The Asn-containing homolog of this peptide, Tyr369(N), is not presented by tyrosinase-expressing cells, and this has been presumed to be due to quantitative glycosylation of Asn371. While examining cytosolic intermediates that accumulated in human melanoma cells treated with proteasome inhibitors, we were surprised to find both molecules that had been deglycosylated by peptide N-glycanase and a large number of molecules that had not been previously glycosylated. The failure of Tyr369(N) to be processed and presented from these latter molecules may be partially due to a process of deamidation independent of glycosylation. However, we also established that proteasomes degrade tyrosinase molecules that are still glycosylated, giving rise to a set of discrete intermediates that are not observed when unglycosylated molecules are degraded. We propose that Tyr369(N) fails to be presented because unglycosylated tyrosinase is degraded rapidly and relatively non-selectively. In contrast, glycosylation alters the selectivity of tyrosinase processing by the proteasome, enhancing the production or survival of Tyr369(D).
doi:10.4049/jimmunol.0802902
PMCID: PMC2859980  PMID: 19342661
antigen presentation/processing; antigen/peptide/epitope; human; tumor immunity
3.  GILT: Shaping the MHC Class II-Restricted Peptidome and CD4+ T Cell-Mediated Immunity 
The MHC class II-restricted antigen processing pathway generates peptide:MHC complexes in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) reduces protein disulfide bonds in the endocytic compartment, thereby exposing buried epitopes for MHC class II binding and presentation. T cell hybridoma responses and elution of MHC class II bound peptides have identified GILT-dependent epitopes, GILT-independent epitopes, and epitopes that are more efficiently presented in the absence of GILT termed GILT-prevented epitopes. GILT-mediated alteration in the MHC class II-restricted peptidome modulates T cell development in the thymus and peripheral tolerance and influences the pathogenesis of autoimmunity. Recent studies suggest an emerging role for GILT in the response to pathogens and cancer survival.
doi:10.3389/fimmu.2013.00429
PMCID: PMC3885806  PMID: 24409178
MHC class II; antigen processing and presentation; GILT; autoimmunity; tumor immunity
4.  Evidence for the Presentation of Major Histocompatibility Complex Class I–restricted Epstein-Barr Virus Nuclear Antigen 1 Peptides to CD8+ T Lymphocytes 
The Epstein-Barr virus (EBV)-encoded nuclear antigen 1 (EBNA1) is expressed in all EBV-associated tumors, making it an important target for immunotherapy. However, evidence for major histocompatibility complex (MHC) class I–restricted EBNA1 peptides endogenously presented by EBV-transformed B and tumor cells remains elusive. Here we describe for the first time the identification of an endogenously processed human histocompatibility leukocyte antigen (HLA)-B8–restricted EBNA1 peptide that is recognized by CD8+ T cells. T cell recognition could be inhibited by the treatment of target cells with proteasome inhibitors that block the MHC class I antigen processing pathway, but not by an inhibitor (chloroquine) of MHC class II antigen processing. We also demonstrate that new protein synthesis is required for the generation of the HLA-B8 epitope for T cell recognition, suggesting that defective ribosomal products (DRiPs) are the major source of T cell epitopes. Experiments with protease inhibitors indicate that some serine proteases may participate in the degradation of EBNA1 DRiPs before they are further processed by proteasomes. These findings not only provide the first evidence of the presentation of an MHC class I–restricted EBNA1 epitope to CD8+ T cells, but also offer new insight into the molecular mechanisms involved in the processing and presentation of EBNA1.
doi:10.1084/jem.20031219
PMCID: PMC2211826  PMID: 14769850
cancer vaccines; immunotherapy; MHC class I–restricted peptides; antigen presentation; CD8+ T cells
5.  GILT Accelerates Autoimmunity to the Melanoma Antigen Tyrosinase-Related Protein 1 
Melanocyte differentiation Ags, including tyrosinase-related protein (TRP) 1, are relevant to both autoimmune skin depigmentation (vitiligo) and tumor immunity, because they are expressed by both benign melanocytes and many malignant melanomas. Melanoma patients generate CD4+ T cells that specifically recognize these proteins. TRP1 contains internal disulfide bonds and is presented by MHC class II molecules. γ-IFN–inducible lysosomal thiol reductase (GILT) facilitates the generation of class II-binding peptides by the endocytic reduction of protein disulfide bonds. We show in this study that GILT is required for efficient MHC class II-restricted processing of a TRP1 epitope in vitro and accelerates the onset of vitiligo in TRP1-specific TCR transgenic mice. The presence of GILT confers a small increase in the percentage of autoreactive T cells with an effector memory phenotype that may contribute to earlier disease onset. The onset of vitiligo is associated with a greater increase in the percentage of autoreactive T cells with an effector memory phenotype. Given that many self and tumor Ags have disulfide bonds and are presented on MHC class II, GILT is likely to be important in the pathogenesis of other CD4+ T cell-mediated autoimmune diseases and for the development of effective cancer immunotherapy.
doi:10.4049/jimmunol.1000945
PMCID: PMC3060054  PMID: 20668223
6.  Detection of Functional Matrix Metalloproteinases by Zymography 
Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.
doi:10.3791/2445
PMCID: PMC3159606  PMID: 21085107
7.  Mechanistic Studies on the Triggered Release of Liposomal Contents by Matrix Metalloproteinase-9 
Journal of the American Chemical Society  2008;130(32):10633-10642.
Matrix metalloproteinases (MMPs) are a class of extracellular matrix degrading enzymes over-expressed in many cancers and contribute to the metastatic ability of the cancer cells. We have recently demonstrated that liposomal contents can be released when triggered by the enzyme MMP-9. Herein, we report our results on the mechanistic studies of the MMP-9 triggered release of the liposomal contents. We synthesized peptides containing the cleavage site for MMP-9 and conjugated them with fatty acids to prepare the corresponding lipopeptides. By employing Circular Dichroism spectroscopy, we demonstrate that the lipopeptides, when incorporated in liposomes, are de-mixed in the lipid bilayers and generate triple helical structures. MMP-9 cleaves the triple helical peptides, leading to the release of the liposomal contents. Other MMPs, which cannot hydrolyze triple helical peptides, failed to release the contents from the liposomes. We also observed that the rate and the extent of release of the liposomal contents depend on the mismatch between acyl chains of the synthesized lipopeptide and phospholipid components of the liposomes. Circular Dichroism spectroscopic studies imply that the observed differences in the release reflect the ability of the liposomal membrane to anneal the defects following the enzymatic cleavage of the liposome-incorporated lipopeptides.
doi:10.1021/ja801548g
PMCID: PMC2644422  PMID: 18642903
Triggered release; liposomes; matrix metalloproteinases; phase separation; triple helical lipopeptides
8.  Inhibition of endogenous dentin matrix metalloproteinases by ethylenediaminetetraacetic acid 
Journal of endodontics  2011;38(1):62-65.
Introduction
Endogenous dentin matrix metalloproteinases (MMPs) contribute to extracellular collagen matrix degradation in hybrid layers following adhesive dentin bonding procedures. Endodontic irrigants, including chlorhexidine (CHX) and ethylenediaminetetraacetic acid (EDTA) may help protect the hybrid layer from this process. The objective of the present study was to determine the exposure time necessary for EDTA to inactivate endogenous MMP activity in human dentin.
Methods
Dentin beams (2×1×3 mm) were prepared from mid-coronal dentin of extracted third molars. The beams were demineralized in 10 wt% phosphoric acid which also activated endogenous MMPs, and were divided into four experimental groups based on exposure time to 17% EDTA (0, 1, 2 or 5 min). A generic colorimetric MMP assay measured MMP activity via absorbance at 412 nm. Data were evaluated by Kruskal Wallis ANOVA, followed by Dunn’s pair-wise comparisons at α = 0.05.
Results
All exposure times resulted in significant inhibition (P<0.001) compared to unexposed controls. Specifically, percent inhibition for 1-, 2-, and 5-minute exposure times were 55.1±21.5%, 72.8±11.7%, and 74.7±19.7%, respectively.
Conclusions
17% EDTA significantly inhibits endogenous MMP activity of human dentin within 1–2 min. This may minimize hybrid layer degradation following resin bonding procedures in the root canal space.
doi:10.1016/j.joen.2011.09.005
PMCID: PMC3240849  PMID: 22152622
dentin; EDTA; hybrid layer; matrix metalloproteinases
9.  Membrane-Type-3 Matrix Metalloproteinase (MT3-MMP) Functions as a Matrix Composition-Dependent Effector of Melanoma Cell Invasion 
PLoS ONE  2011;6(12):e28325.
In primary human melanoma, the membrane-type matrix metalloproteinase, MT3-MMP, is overexpressed in the most aggressive nodular-type tumors. Unlike MT1-MMP and MT2-MMP, which promote cell invasion through basement membranes and collagen type I-rich tissues, the function of MT3-MMP in tumor progression remains unclear. Here, we demonstrate that MT3-MMP inhibits MT1-MMP-driven melanoma cell invasion in three-dimensional collagen, while yielding an altered, yet MT1-MMP-dependent, form of expansive growth behavior that phenocopies the formation of nodular cell colonies. In melanoma cell lines originating from advanced primary or metastatic lesions, endogenous MT3-MMP expression was associated with limited collagen-invasive potential. In the cell lines with highest MT3-MMP expression relative to MT1-MMP, collagen-invasive activity was increased following stable MT3-MMP gene silencing. Consistently, MT3-MMP overexpression in cells derived from less advanced superficially spreading melanoma lesions, or in the MT3-MMP knockdown cells, reduced MT1-MMP-dependent collagen invasion. Rather than altering MT1-MMP transcription, MT3-MMP interacted with MT1-MMP in membrane complexes and reduced its cell surface expression. By contrast, as a potent fibrinolytic enzyme, MT3-MMP induced efficient invasion of the cells in fibrin, a provisional matrix component frequently found at tumor-host tissue interfaces and perivascular spaces of melanoma. Since MT3-MMP was significantly upregulated in biopsies of human melanoma metastases, these results identify MT3-MMP as a matrix-dependent modifier of the invasive tumor cell functions during melanoma progression.
doi:10.1371/journal.pone.0028325
PMCID: PMC3229567  PMID: 22164270
10.  Efficient Identification of Novel Hla-A*0201–Presented Cytotoxic T Lymphocyte Epitopes in the Widely Expressed Tumor Antigen Prame by Proteasome-Mediated Digestion Analysis 
We report the efficient identification of four human histocompatibility leukocyte antigen (HLA)-A*0201–presented cytotoxic T lymphocyte (CTL) epitopes in the tumor-associated antigen PRAME using an improved “reverse immunology” strategy. Next to motif-based HLA-A*0201 binding prediction and actual binding and stability assays, analysis of in vitro proteasome-mediated digestions of polypeptides encompassing candidate epitopes was incorporated in the epitope prediction procedure. Proteasome cleavage pattern analysis, in particular determination of correct COOH-terminal cleavage of the putative epitope, allows a far more accurate and selective prediction of CTL epitopes. Only 4 of 19 high affinity HLA-A*0201 binding peptides (21%) were found to be efficiently generated by the proteasome in vitro. This approach avoids laborious CTL response inductions against high affinity binding peptides that are not processed and limits the number of peptides to be assayed for binding. CTL clones induced against the four identified epitopes (VLDGLDVLL, PRA100–108; SLYSFPEPEA, PRA142–151; ALYVDSLFFL, PRA300–309; and SLLQHLIGL, PRA425–433) lysed melanoma, renal cell carcinoma, lung carcinoma, and mammary carcinoma cell lines expressing PRAME and HLA-A*0201. This indicates that these epitopes are expressed on cancer cells of diverse histologic origin, making them attractive targets for immunotherapy of cancer.
PMCID: PMC2195886  PMID: 11136822
antigen presentation; antigen processing; cytotoxic T lymphocyte induction; human histocompatibility leukocyte antigen class I binding; tumor immunotherapy
11.  Fluorometric immunocapture assay for the specific measurement of matrix metalloproteinase-9 activity in biological samples: application to brain and plasma from rats with ischemic stroke 
Molecular Brain  2013;6:14.
Background
Matrix metalloproteinases are important factors in the molecular mechanisms leading to neuronal injury in many neurological disorders. Matrix metalloproteinase (MMP)-9 is up-regulated after cerebral ischemia and neuroinflammation and is actively involved in blood–brain barrier disruption. Current methods of measuring MMP-9 activity, such as gelatin-substrate zymography, are unspecific and arduous. Here we developed an immunocapture assay with high efficiency, specificity, and sensitivity for quantifying endogenously active as well as total MMP-9 activity.
Results
A fluorescence resonance energy transfer (FRET) peptide-based immunocapture assay was developed that enables the accurate assessment of total and active forms of MMP-9 in complex biological samples. The FRET assay demonstrated correct and efficient binding of MMP-9 to a mouse monoclonal MMP-9 antibody and high specificity of the immunocapture antibody for MMP-9. Total and active levels of MMP-9 were measured in rat brain homogenates, plasma, human HT-1080 conditioned media, and RBE4 endothelial cell lysates. The FRET immunocapture assay yielded highly similar results for total MMP-9 activity when compared to gelatin-substrate zymography.
Conclusions
We suggest that the new FRET peptide-based immunocapture assay is a viable replacement of zymography for sensitive and high throughput quantification of MMP-9 activity in biological samples.
doi:10.1186/1756-6606-6-14
PMCID: PMC3620676  PMID: 23522154
Matrix metalloproteinase-9 activity; Fluorescence resonance energy transfer peptide; Immunocapture assay; Focal cerebral ischemia
12.  A monoclonal antibody interferes with TIMP-2 binding and incapacitates the MMP-2-activating function of multifunctional, pro-tumorigenic MMP-14/MT1–MMP 
Oncogenesis  2013;2(12):e80-.
Matrix metalloproteinases (MMPs) and, especially membrane type 1 (MT1)-MMP/MMP-14, are promising drug targets in malignancies. In contrast with multiple small-molecule and protein pan-inhibitors of MT1–MMP cleavage activity, the murine 9E8 monoclonal antibody targets the MMP-2-activating function of cellular MT1–MMP alone, rather than the general proteolytic activity and the pro-migratory function of MT1–MMP. Furthermore, the antibody does not interact in any detectable manner with other members of the membrane type (MT)-MMP family. The mechanism of this selectivity remained unknown. Using mutagenesis, binding and activity assays, and modeling in silico, we have demonstrated that the 9E8 antibody recognizes the MT-loop structure, an eight residue insertion that is specific for MT–MMPs and that is distant from the MT1–MMP active site. The binding of the 9E8 antibody to the MT-loop, however, prevents tissue inhibitor of metalloproteinases-2 (TIMP-2) association with MT1–MMP. As a result, the 9E8 antibody incapacitates the TIMP-2-dependent MMP-2-activating function alone rather than the general enzymatic activity of human MT1–MMP. The specific function of the 9E8 antibody we determined directly supports an essential, albeit paradoxical, role of the protein inhibitor (TIMP-2) in MMP-2 activation via a unique membrane-tethered mechanism. In this mechanism, the formation of a tri-molecular MT1–MMPTIMP-2MMP-2 complex is required for both the capture of the soluble MMP-2 proenzyme by cells and then its well-controlled conversion into the mature MMP-2 enzyme. In sum, understanding of the structural requirements for the 9E8 antibody specificity may pave the way for the focused design of the inhibitory antibodies against other individual MMPs.
doi:10.1038/oncsis.2013.44
PMCID: PMC3940861  PMID: 24296749
monoclonal antibody; TIMP-2; MT1–MMP; cancer; proteinase inhibitor
13.  Identification of Hepsin and Protein Disulfide Isomerase A3 as Targets of Gelatinolytic Action in Rat Ovarian Granulosa Cells During the Periovulatory Period1  
Biology of Reproduction  2011;85(4):858-866.
The matrix metalloproteinase (MMP) family is believed to play a role in the ovulatory process because MMP inhibitors block oocyte release. However, little is known about the mechanisms by which the MMPs affect ovulation. The present study investigated the degradomic actions of the gelatinases, MMP2 and MMP9, by identifying gelatinolytic targets in periovulatory granulosa cells. Granulosa cells were collected from immature rats 48 h after equine chorionic gonadotropin treatment and were cultured with human chorionic gonadotropin (hCG) in the absence or presence of a specific MMP2/9 inhibitor ((2R)-2-[(4-biphenylylsulfonyl)amino]-3-phenylpropionic acid) for an additional 24 h. The conditioned media was analyzed for gelatinolytic activity, progesterone, and peptide profiles. Gelatinolytic activity and progesterone were induced in response to hCG; however, there was no difference in progesterone between cells treated with or without the inhibitor. Peptide fragments of proteins altered in the presence of the gelatinase inhibitor were identified by two-dimensional gel electrophoresis and mass spectrometry. Protein disulfide isomerase A3 (PDIA3), which plays a role in protein folding, was identified as a peptide that decreased in the presence of inhibitor while the serine protease hepsin, was found to increase with inhibitor treatment. Subsequent experiments established that PDIA3 and hepsin were targets of MMP2/9 action by cleavage with MMP2 and Western blot analysis, respectively. Additionally, hepsin was identified as a gelatinolytic target in ovarian cancer cells. In the present study, proteomics has identified proteins that may be involved in novel ways in the complex cascades that are mediated by gelatinolytic MMPs during the periovulatory period.
Gelatinases from rat granulosa cells degrade hepsin and protein disulfide isomerase A3.
doi:10.1095/biolreprod.111.092072
PMCID: PMC3184295  PMID: 21734266
corpus luteum; hepsin; matrix metalloproteinase; ovulation; protein disulfide isomerase A3
14.  Downregulation of Matrix Metalloproteinase-2 (MMP-2) Utilizing Adenovirus-mediated Transfer of Small Interfering RNA (siRNA) in a Novel Spinal Metastatic Melanoma Model 
International journal of oncology  2008;32(3):557-564.
Matrix metalloproteinases (MMPs) comprise a class of secreted zinc-dependent endopeptidases implicated in the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix (ECM) and basement membrane. Matrix metalloproteinase-2 (MMP-2) has been detected in high levels and correlates with invasiveness in human melanoma. We have studied the effect of adenovirus-mediated transfer of small interfering RNA (siRNA) against MMP-2 in the human melanoma cell line A2058. The delivery of these double-stranded RNA molecules represents an efficient technology in silencing disease-causing genes with known sequences at the post-transcriptional level. siRNA against MMP-2 mRNA (Ad-MMP-2) was found to decrease MMP-2 protein expression and activity in melanoma cells as demonstrated by western blotting and gelatin zymography. Furthermore, infection of cells with Ad-MMP-2 inhibited cellular migration and invasion as indicated by spheroid and matrigel assays. We also observed dose-dependent suppression of vascular network formation in an angiogenesis assay. Finally, we developed a nude mouse spinal metastatic model to investigate the local effects of tumor metastasis. Intravenous tail vein injection with Ad-MMP-2 on days 5, 9, and 11 resulted in complete retention of neurological function as compared to control and scrambled vector (Ad-SV)-treated groups that showed complete paraplegia by day 14 ± 2 days. Hematoxylin and eosin staining revealed decreased tumor size in the Ad-MMP-2-treated animals. This novel experimental model revealed that adenoviral-mediated transfer of RNA interference against MMP-2 results in the retention of neurological function and significantly inhibited tumor growth.
PMCID: PMC2267753  PMID: 18292932
MMP-2; melanoma; invasion; spheroid migration; angiogenesis; metastatic model
15.  Intracellular events regulating cross-presentation 
Cross-presentation plays a fundamental role in the induction of CD8-T cell immunity. However, although more than three decades have passed since its discovery, surprisingly little is known about the exact mechanisms involved. Here we give an overview of the components involved at different stages of this process. First, antigens must be internalized into the cross-presenting cell. The involvement of different receptors, method of antigen uptake, and nature of the antigen can influence intracellular trafficking and access to the cross-presentation pathway. Once antigens access the endocytic system, different requirements for endosomal/phagosomal processing arise, such as proteolysis and reduction of disulfide bonds. The majority of cross-presented peptides are generated by proteasomal degradation. Therefore, antigens must cross a membrane barrier in a manner analogous to the fate of misfolded proteins in the endoplasmic reticulum (ER) that are retrotranslocated into the cytosol for degradation. Indeed, some components of the ER-associated degradation machinery have been implicated in cross-presentation. Further complicating the matter, endosomal and phagosomal compartments have been suggested as alternative sites to the ER for loading of peptides on major histocompatibility complex class I molecules. Finally, the antigen presenting cells involved, particularly dendritic cell subsets and their state of maturation, influence the efficiency of cross-presentation.
doi:10.3389/fimmu.2012.00138
PMCID: PMC3366438  PMID: 22675326
dendritic cell; MHC class I; endocytosis; phagocytosis
16.  Chemical Biology for Understanding Matrix Metalloproteinase Function 
The matrix metalloproteinase (MMP) family has long been associated with normal physiological processes such as embryonic implantation, tissue remodeling, organ development, and wound healing, as well as multiple aspects of cancer initiation and progression, osteoarthritis, inflammatory and vascular diseases, and neurodegenerative diseases. The development of chemically designed MMP probes has advanced our understanding of the roles of MMPs in disease in addition to shedding considerable light on the mechanisms of MMP action. The first generation of protease-activated agents has demonstrated proof of principle as well as providing impetus for in vivo applications. One common problem has been a lack of agent stability at nontargeted tissues and organs due to activation by multiple proteases. The present review considers how chemical biology has impacted the progress made in understanding the roles of MMPs in disease and the basic mechanisms of MMP action.
doi:10.1002/cbic.201200298
PMCID: PMC3951272  PMID: 22933318
activity-based protein profiling; cell-penetrating peptides; FRET; matrix metalloproteinases; optical imaging; triple-helical peptides
17.  Variability in Melanoma Metalloproteinase Expression Profiling 
The proteolytic activities of a disintegrin and metalloproteinase (ADAM); a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), and matrix metalloproteinase (MMP) families play important roles in normal and multiple pathological conditions. These metalloproteases have potential roles in the degradation of the ECM and in the processing of bioactive molecules. In the present study, RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples, and the gene expression levels of six ADAMs, eight MMPs, and four ADAMTSs were analyzed by real-time PCR. This approach allowed for detected changes in mRNA expression of the individual metalloproteinase genes to be compared between normal and metastatic states and also between tissue and cultured cells. Increased gene expression of several ADAM and MMP family members (MMP1, MMP8, MMP15, and ADAM15) occurred in melanoma tissue and was replicated in tissue cultures. In general, the level of ADAM and MMP mRNA expression was several-fold higher in cultured cells compared with the isogenic tissue from which they were derived. Passage-dependent expression patterns were observed for MMP8 and MMP9 in in-house-derived metastatic melanoma cell lines. This reiterates earlier suggestions that experiments using cells that have been maintained in culture should be interpreted with great care.
PMCID: PMC2977965  PMID: 21119930
ADAM; MMP; tissue
18.  T cell receptors used in cancer gene therapy cross-react with MART-1/Melan-A tumor antigens via distinct mechanisms1 
T cells engineered to express T cell receptors (TCRs) specific for tumor antigens can drive cancer regression. The first TCRs used in cancer gene therapy, DMF4 and DMF5, recognize two structurally distinct peptide epitopes of the melanoma-associated MART-1/Melan-A protein, both presented by the class I MHC protein HLA-A*0201. To help understand the mechanisms of TCR cross-reactivity and provide a foundation for the further development of immunotherapy, we determined the crystallographic structures of DMF4 and DMF5 in complex with both of the MART-1/Melan-A epitopes. The two TCRs use different mechanisms to accommodate the two ligands. Whereas DMF4 binds the two with a different orientation, altering its position over the peptide/MHC, DMF5 binds them both identically. The simpler mode of cross-reactivity by DMF5 is associated with higher affinity towards both ligands, consistent with the superior functional avidity of DMF5. More generally, the observation of two diverging mechanisms of cross-reactivity with the same antigens and the finding that TCR binding orientation can be determined by peptide alone extend our understanding of the mechanisms underlying TCR cross-reactivity.
doi:10.4049/jimmunol.1101268
PMCID: PMC3166883  PMID: 21795600
19.  The requirement for proteasome activity class I major histocompatibility complex antigen presentation is dictated by the length of preprocessed antigen 
The Journal of Experimental Medicine  1996;183(4):1545-1552.
Accumulating evidence has implicated the proteasome in the processing of protein along the major histocompatibility complex (MHC) class I presentation pathway. The availability of potent proteasome inhibitors provides an opportunity to examine the role of proteasome function in antigen presentation by MHC class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). We have investigated the processing and presenting of antigenic epitopes from influenza hemagglutinin in target cells treated with the inhibitor of proteasome activity MG132. In the absence of proteasome activity, the processing and presentation of the full- length hemagglutinin was abolished, suggesting the requirement for proteasome function in the processing and presentation of the hemagglutinin glycoprotein. Epitope-containing translation products as short as 21 amino acids when expressed in target cells required proteasome activity for processing and presentation of the hemagglutin epitope to CTLs. However, when endogenous peptides of 17 amino acids or shorter were expressed in target cells, the processing and presentation of epitopes contained in these peptides were insensitive to the proteasome inhibitor. Our results support the hypothesis that proteasome activity is required for the generation of peptides presented by MHC class I molecules and that the requirement for proteasome activity is dependent on the size of the translation product expressed in the target cell. The implications of these findings are discussed.
PMCID: PMC2192494  PMID: 8666912
20.  Proteomic Identification of In Vivo Substrates for Matrix Metalloproteinases 2 and 9 Reveals a Mechanism for Resolution of Inflammation1 
Clearance of allergic inflammatory cells from the lung through matrix metalloproteinases (MMPs) is necessary to prevent lethal asphyxiation, but mechanistic insight into this essential homeostatic process is lacking. In this study, we have used a proteomics approach to determine how MMPs promote egression of lung inflammatory cells through the airway. MMP2- and MMP9-dependent cleavage of individual Th2 chemokines modulated their chemotactic activity; however, the net effect of complementing bronchoalveolar lavage fluid of allergen-challenged MMP2−/−/MMP9−/− mice with active MMP2 and MMP9 was to markedly enhance its overall chemotactic activity. In the bronchoalveolar fluid of MMP2−/−/MMP9−/− allergic mice, we identified several chemotactic molecules that possessed putative MMP2 and MMP9 cleavage sites and were present as higher molecular mass species. In vitro cleavage assays and mass spectroscopy confirmed that three of the identified proteins, Ym1, S100A8, and S100A9, were substrates of MMP2, MMP9, or both. Function-blocking Abs to S100 proteins significantly altered allergic inflammatory cell migration into the alveolar space. Thus, an important effect of MMPs is to differentially modify chemotactic bioactivity through proteolytic processing of proteins present in the airway. These findings provide a molecular mechanism to explain the enhanced clearance of lung inflammatory cells through the airway and reveal a novel approach to target new therapies for asthma.
PMCID: PMC2580826  PMID: 17082650
21.  Association of a specific haplotype across the genes MMP1 and MMP3 with radiographic joint destruction in rheumatoid arthritis 
Arthritis Research & Therapy  2004;6(3):R199-R207.
The genetic background of rheumatoid arthritis (RA) is only partly understood, and several genes seem to be involved. The matrix metalloproteinases MMP1 (interstitial collagenase) and MMP3 (stromelysin 1) are thought to be important in destructive joint changes seen in RA. In the present study, functional relevant promoter polymorphisms of MMP1 and MMP3 were genotyped in 308 patients and in 110 controls, to test whether the polymorphisms contribute to the severity of the disease measured by radiographic progression of joint destruction. For comparison, the shared epitope of HLA DR4 and DR1 (SE) was determined by polymerase chain reaction. There was no association of MMP polymorphisms with susceptibility to RA. However, a strong linkage disequilibrium was observed between the 1G/2G (MMP1) and the 5A/6A (MMP3) polymorphisms (P << 10-6; linkage disequilibrium index D' = 0.46). In factorial regression, the degree of radiographic joint destruction correlated significantly with the 1G-5A haplotype (P = 0.0001) and the interaction term 'estimated number of 1G-5A haplotypes × duration of disease' (P = 0.0007). This association was phasic, indicating that possession of the 1G-5A haplotype has a protective effect over a period of about 15 years of RA, but might be associated with a more pronounced radiographic progression later on. Similar results were also found with the 1G allele of MMP1 alone (P = 0.015) and with the interaction term 'estimated number of 1G alleles × duration of disease' (P = 0.014). The correlation of SE with the Ratingen score was comparable (0.044). The regression model of MMP haplotypes explained 35% of the variance of the radiographic score, whereas the SE explained 29%. The 1G-5A haplotype across the closely linked MMP1 and MMP3 gene loci is a newly described genetic factor strongly associated with the progression of joint damage in RA. Our findings suggest that there are haplotypes in a MMP cluster region that modify the joint destruction in RA in a phasic manner.
doi:10.1186/ar1164
PMCID: PMC416441  PMID: 15142265
allelic polymorphism; matrix metalloproteinase; radiographic progression; rheumatoid arthritis
22.  Overlapping and independent contributions of MMP2 and MMP9 to lung allergic inflammatory cell egression through decreased CC chemokines 
The mechanisms that initiate allergic lung inflammation are relevant to expression of diseases such as asthma, but the factors underlying resolution of inflammation are equally important. Previously, we demonstrated the importance of matrix metalloproteinase 2 (MMP2) for airway egression of lung eosinophils, a critical anti-inflammatory mechanism without which mice are rendered highly susceptible to lethal asphyxiation. Here we show that leukocyte MMP9 is the dominant airway MMP controlling inflammatory cell egression. The allergic lung phenotype of MMP9−/− mice was similar to WT and was not altered by concomitant deletion of the MMP2 gene (double knockout; dko). However, inflammatory cells accumulated aberrantly in the lungs of allergen-challenged MMP9−/− and dko mice and fewer eosinophils and neutrophils were present in bronchoalveolar lavage. These aberrant cellular trafficking patterns were explained by disruption of transepithelial chemokine gradients, in MMP2−/− mice affecting only eotaxin (CCL11), but in MMP9−/− and dko mice involving eotaxin, MARC (CCL7), and TARC (CCL17). Thus, by establishing multiple transepithelial chemokine gradients, MMP9 is broadly implicated in the resolution of allergic inflammation, an essential protective mechanism that overlaps with a more limited role played by MMP2.
doi:10.1096/fj.03-1412fje
PMCID: PMC2771179  PMID: 15059974
chemotaxis; cytokines; gelatinase B; matrix metalloproteinase-9
23.  Portable flanking sequences modulate CTL epitope processing 
The Journal of Clinical Investigation  2007;117(11):3563-3575.
Peptide presentation is critical for immune recognition of pathogen-infected cells by CD8+ T lymphocytes. Although a limited number of immunodominant peptide epitopes are consistently observed in diseases such as HIV-1 infection, the relationship between immunodominance and antigen processing in humans is largely unknown. Here, we have demonstrated that endogenous processing and presentation of a human immunodominant HIV-1 epitope is more efficient than that of a subdominant epitope. Furthermore, we have shown that the regions flanking the immunodominant epitope constitute a portable motif that increases the production and antigenicity of otherwise subdominant epitopes. We used a novel in vitro degradation assay involving cytosolic extracts as well as endogenous intracellular processing assays to examine 2 well-characterized HIV-1 Gag overlapping epitopes presented by the same HLA class I allele, one of which is consistently immunodominant and the other subdominant in infected persons. The kinetics and products of degradation of HIV-1 Gag favored the production of peptides encompassing the immunodominant epitope and destruction of the subdominant one. Notably, cytosolic digestion experiments revealed flanking residues proximal to the immunodominant epitope that increased the production and antigenicity of otherwise subdominant epitopes. Furthermore, specific point mutations in these portable flanking sequences modulated the production and antigenicity of epitopes. Such portable epitope processing determinants provide what we believe is a novel approach to optimizing CTL responses elicited by vaccine vectors.
doi:10.1172/JCI32047
PMCID: PMC2045603  PMID: 17975674
24.  Matrix Metalloproteinases 2 and 9 Are Dispensable for Pancreatic Islet Formation and Function In Vivo 
Diabetes  2005;54(3):694-701.
Pancreatic islet formation is a highly regulated process that is initiated at the end of gestation in rodents. Endocrine precursor cells first form within the epithelium of duct-like structures and then delaminate from the epithelium, migrate, and cluster during the early stages of islet formation. The molecular mechanisms that regulate endocrine cell migration and islet formation are not well understood. Cell culture studies suggest that matrix metalloproteinases (MMPs) 2 and 9 are required for islet formation. To address whether MMP2 and MMP9 function are essential for endocrine cell migration and islet formation in vivo, we analyzed pancreas development in MMP2/MMP9 double-deficient mice. Our results show that islet architecture and function are unperturbed in these knockout mice, demonstrating that both MMP2 and MMP9 functions are dispensable for islet formation and endocrine cell differentiation. Our studies also show that a number of other MMPs are expressed at the time islet formation is initiated. This observation suggests that other MMPs may substitute for MMP2 and MMP9 loss in pancreatic tissue. However, islet formation is unaffected in transgenic mice with modified tissue inhibitor of metalloproteinase-1 (TIMP1) levels, suggesting that MMP activity may contribute little to islet morphogenesis in vivo.
PMCID: PMC2771170  PMID: 15734845
25.  MT4-(MMP17) and MT6-MMP (MMP25), A unique set of membrane-anchored matrix metalloproteinases: properties and expression in cancer 
Cancer metastasis reviews  2008;27(2):289-302.
The process of cancer progression involves the action of multiple proteolytic systems, among which the family of matrix metalloproteinases (MMPs) play a pivotal role. The MMPs evolved to accomplish their proteolytic tasks in multiple cellular and tissue microenvironments including lipid rafts by incorporation and deletions of specific structural domains. The membrane type-MMPs (MT-MMPs) incorporated membrane anchoring domains that display these proteases at the cell surface, and thus they are optimal pericellular proteolytic machines. Two members of the MT-MMP subfamily, MMP-17 (MT4-MMP) and MMP-25 (MT6-MMP), are anchored to the plasma membrane via a glycosyl-phosphatidyl inositol (GPI) anchor, which confers these enzymes a unique set of regulatory and functional mechanisms that separates them from the rest of the MMP family. Discovered almost a decade ago, the body of work on GPI-MT-MMPs today is still surprisingly limited when compared to other MT-MMPs. However, new evidence shows that the GPI-MT-MMPs are highly expressed in human cancer, where they are associated with progression. Accumulating biochemical and functional evidence also highlights their distinct properties. In this review, we summarize the structural, biochemical, and biological properties of GPI-MT-MMPs and present an overview of their expression and role in cancer. We further discuss the potential implications of GPI-anchoring for enzyme function. Finally, we comment on the new scientific challenges that lie ahead to better understand the function and role in cancer of these intriguing but yet unique MMPs.
doi:10.1007/s10555-008-9129-8
PMCID: PMC3903121  PMID: 18286233
Matrix metalloproteinases; Glycosyl-phosphatidyl inositol; Membrane anchor; Proteolysis; Cancer; Lipid raft; Protease inhibitors; Plasma membrane; Extracellular matrix

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