Evaluating the Catalytic Efficiency of the Human Membrane-type 1 Matrix Metalloproteinase (MMP-14) Using AuNP-Peptide Conjugates.

Interactions of plasmonic nanocolloids such as gold nanoparticles and nanorods with proximal dye emitters result in efficient quenching of the dye photoluminescence (PL). This has become a popular strategy for developing analytical biosensors relying on this quenching process for signal transduction. Here, we report on the use of stable PEGylated gold nanoparticles, covalently coupled to dye-labeled peptides, as sensitive optically addressable sensors for determining the catalytic efficiency of the human matrix metalloproteinase-14 (MMP-14), a cancer biomarker. We exploit real-time dye PL recovery triggered by MMP-14 hydrolysis of the AuNP-peptide-dye to extract quantitative analysis of the proteolysis kinetics. Sub-nanomolar limit of detections for MMP-14 has been achieved using our hybrid bioconjugates. In addition, we have used theoretical considerations within a diffusion-collision framework to derive enzyme substrate hydrolysis and inhibition kinetics equations, which allowed us to describe the complexity and irregularity of enzymatic proteolysis of nanosurface-immobilized peptide substrates. Our findings offer a great strategy for the development of highly sensitive and stable biosensors for cancer detection and imaging.

Metalloproteinase disintegrins ADAM8 and ADAM19 are highly regulated in human primary brain tumors and their expression levels and activities are associated with invasiveness.

Patients with primary brain tumors have bleak prognoses and there is an urgent desire to identify new markers for sensitive diagnosis and new therapeutic targets for effective treatment. A family of proteins, the disintegrin and metalloproteinases (ADAMs or adamalysins), are cell surface and extracellular multidomain proteins implicated in cell-cell signaling, cell adhesion, and cell migration. Their putative biological and pathological roles make them candidates for promoting tumor growth and malignancy. We investigated the expression levels of 12 cerebrally expressed ADAM genes in human primary brain tumors (astrocytoma WHO grade I-III, glioblastoma WHO grade IV, oligoastrocytoma WHO grade II and III, oligodendroglioma WHO grade II and III, ependymoma WHO grade II and III, and primitive neuroectodermal tumor WHO grade IV) using real-time PCR. The mRNAs of the five ADAMs 8, 12, 15, 17, and 19 were significantly upregulated. The ADAM8 and ADAM19 proteins were mainly located in tumor cells and in some tumors in endothelia of blood vessels. In brain tumor tissue, ADAM8 and ADAM19 undergo activation by prodomain removal resulting in active proteases. By using specific peptide substrates for ADAM8 and ADAM19, respectively, we demonstrated that the proteases exert enhanced proteolytic activity in those tumor specimens with the highest expression levels. In addition, expression levels and the protease activities of ADAM8 and ADAM19 correlated with invasive activity of glioma cells, indicating that ADAM8 and ADAM19 may play a significant role in tumor invasion that may be detrimental to patients survival.

Expression of matrix metalloproteinase-26 (MMP-26) mRNA in mouse uterus during the estrous cycle and early pregnancy.

Matrix metalloproteinases (MMPs) and their tissue inhibitors play important roles in the remodeling of extracellular matrix (ECM). MMP-26, also called endometase or matrilysin-2, is a novel member of the MMP family. The present study was to investigate the temporal and spatial expression of MMP-26 mRNA in mouse uterus during the estrous cycle and early pregnancy by using in situ hybridization and semi-quantitative RT-PCR. In this study, MMP-26 mRNA was found to be localized to the luminal and glandular epithelium at proestrus and estrus, and the expression level was decreased significantly from metestrus to dioestrus. During pre-implantation period, MMP-26 mRNA was predominantly expressed in luminal and glandular epithelium at much higher level; whereas it switched to stroma during peri-implantation period, and also appeared in the blastocysts and the implantation sites. The results suggested that MMP-26 might play a role in the cycling changes of mouse uterus during the estrous cycle and embryo implantation.

Focal prostate basal cell layer disruptions and leukocyte infiltration are correlated events: A potential mechanism for basal cell layer disruptions and tumor invasion.

To assess the potential correlation between basal cell layer disruptions and leukocyte infiltration, consecutive sections of normal (n=5) and tumor (n=50) prostate tissues were double immunostained for cytokeratin 34betaE12 (CK 34betaE12) plus leukocyte common antigen, Ki-67, or proliferating cell nuclear antigen (PCNA). Of 2047 acini and ducts examined, 201 contained focal basal cell layer disruptions. Of those, 183 (91%) showed leukocyte infiltration, compared to 67 (33.3%) in 201 morphologically comparable structures with an intact basal cell layer (P<0.01). Basal cell layers adjacent to or surrounded by leukocytes were often attenuated or fragmented, and leukocytes were generally located at or near disruptions. Disrupted basal cell layers showed a markedly reduced proliferation rate, compared to their non-disrupted counterparts. Cells overlying focal basal cell layer disruptions often displayed distinct changes in the size, nuclear shape, density, and polarity, compared to those away from disruptions. A vast majority of proliferating tumor cells were located at or near basal cell layer disruptions. These findings suggest that focal basal cell layer disruptions and leukocyte infiltration are correlated events, representing a potential trigger factor for prostate tumor invasion.

Evidence for disulfide involvement in the regulation of intramolecular autolytic processing by human adamalysin19/ADAM19.

Human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19) is activated by furin-mediated cleavage of the prodomain followed by an autolytic processing within the cysteine-rich domain at Glu586-Ser587, which occurs intramolecularly, producing an NH2 terminal fragment (N-fragment) associated with its COOH-terminal fragment (C-fragment), most likely through disulfide bonds. When stable Madin-Darby canine kidney (MDCK) transfectants overexpressing soluble hADAM19 were treated with dithiothreitol (DTT) or with media at pH 6.5, 7.5, or 8.5, the secretion and folding of the enzyme were not affected. Autolytic processing was blocked by DTT and pH 6.5 media, which favor disulfide reduction, but was increased by pH 8.5 media, which promotes disulfide formation. Cys605, Cys633, Cys639, and Cys643 of the C-fragment appear to be partially responsible for the covalent association between the C-fragment and the N-fragment. A new autolytic processing site at Lys543-Val544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. Shed fragments were also detectable in the media from MDCK cells stably expressing the full-length Cys633Ser mutant. Ilomastat/GM6001 inhibited hADAM19 with an IC50 of 447 nM, but scarcely affected the shedding process. The cysteine-rich domain likely forms disulfide bonds to regulate the autolytic processing and shedding of hADAM19.