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- Title
- Metalloprotease Profiling in Melanoma.
- Creator
- Giricz, Orsolya, Florida Atlantic University, Fields, Gregg B., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
- Abstract/Description
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The proteolytic activities of the ADAM (a disintegrin and metalloproteinase), ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and MMP (matrix metalloproteinase) protein families play important roles in normal and multiple pathological conditions. These metalloproteases have potential implications in the degradation of the extracellular matrix and in the processing of bioactive molecules. Under pathological conditions these proteases are involved in many diverse...
Show moreThe proteolytic activities of the ADAM (a disintegrin and metalloproteinase), ADAMTS (a disintegrin and metalloproteinases with thrombospondin motifs) and MMP (matrix metalloproteinase) protein families play important roles in normal and multiple pathological conditions. These metalloproteases have potential implications in the degradation of the extracellular matrix and in the processing of bioactive molecules. Under pathological conditions these proteases are involved in many diverse processes from tumor cell migration to cartilage destruction in rheumatoid arthritis. In the present study, the gene expression levels of six ADAMs, eight MMPs, and four ADAMTSs were analyzed by Real Time PCR. RNA was isolated from multiple normal fibroblast and metastatic melanoma cell lines, as well as the isogenic normal tissue and tumor samples. This method allowed for detected changes in mRNA expressiOn of the individual metalloproteainase genes to be compared between normal and metastatic states, and also between tissue and cultured cells. Substantial differences have been observed in the level of ADAM and MMP mRNA expression between tissue and cell lines. In general, the level of expression is several folds higher in cultured cells compared to the isogenic tissue they are derived from. Protein microarrays were utilized in order to evaluate the correlations between MMP and TIMP mRNA copy numbers and protein abundance in cell culture. In several cases, distinct differences were observed regarding the localization of the proteins examined. In order to determine if the metalloprotease genes that were elevated at the level of RNA expression produce functional proteins, the foundations of an in situ FRET assay have been established. This will greatly aid in a better understanding of the behavior of metallopeptidases in a cellular context.
Show less - Date Issued
- 2008
- PURL
- http://purl.flvc.org/fau/fd/FA00000859
- Subject Headings
- Tumor markers--Research, Metalloproteinases--Inhibitors, Melanoma--Research, Proteins--Synthesis, Genetic translation
- Format
- Document (PDF)
- Title
- MONITORING COLLAGENOLYSIS UTILIZING TRIPLE HELICAL PEPTIDE PROBES.
- Creator
- Tokmina-Roszyk, Michal, Fields, Gregg B., Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
- Abstract/Description
-
Collagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found...
Show moreCollagen is the major structural scaffold in the body and serves as barrier between tissues, and thus its turnover is tightly regulated. Collagen triple-helical structure renders it resistant to general proteolysis. Several proteases are capable of cleaving the triplehelical regions of collagen, including several mammalian matrix metalloproteinases (MMPs) and bacterial collagenases. MMP-mediated collagenolysis is associated with numerous diseases and some bacterial collagenases have found clinical application use due to their efficiency in the hydrolysis of the collagen triple-helix. A selective Förster resonance energy transfer triple-helical peptide (fTHP) probe for monitoring the activity of Clostridial collagenase has been developed. The fTHP [sequence: Gly-mep-Flp-(Glyvi Pro-Hyp)4-Gly-Lys(Mca)-Thr-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Lys(Dnp)-Ser-(Gly-Pro-Hyp)4-NH2] was stable at 37 °C and was efficiently hydrolyzed by bacterial collagenase (kcat/KM = 25,000 s -1 M-1) but not by clostripain, trypsin, neutral protease, thermolysin, or elastase. The bacterial collagenase fTHP assay can be utilized in applications where specific activity towards triple-helical collagen needs to be evaluated, such as isolation of cells from various tissues. An fTHP scaffold was also utilized to evaluate the sequence preferences of eight MMPs. Residues spanning from P3 to P11 investigated using a positional scanning synthetic combinatorial library. Deconvolution of the library data revealed distinct motifs for several MMPs and discrimination among closely related MMPs. The results of this study show that the P10 11 substrate play an important role in the collagenase-substrate interactions and that modifying these residues can drastically affect the affinity of MMPs towards THP substrates. The identified sequence preferences of MMPs 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.
Show less - Date Issued
- 2019
- PURL
- http://purl.flvc.org/fau/fd/FA00013422
- Subject Headings
- Collagen, Peptides, Matrix Metalloproteinases, Microbial Collagenase, Molecular Probes
- Format
- Document (PDF)
- Title
- Synthesis of Fluorogenic Probes Specific for Matrix Metalloproteinase 13.
- Creator
- Ibrahim, Mariam, Fields, Gregg B., Leventouri, Theodora, Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
- Abstract/Description
-
Matrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity...
Show moreMatrix Metalloproteinase-13 (MMP-13) belongs to a large family of proteolytic enzymes which are characterized by their ability to degrade the extracellular matrix components. MMP-13 appears to have a critical role in tumor invasion and metastasis. In this study, several fluorogenic probes specific for MMP-13 were designed and characterized. These synthesized probes could be modified with chelators to be applied for imaging MMP-13 in breast cancer and/or multiple myeloma models. The activity and selectivity of MMP-13 and other MMPs against these probes were studied through two approaches. It was found that these probes were cleaved by all MMPs, but MMP-13 showed the highest activity and selectivity towards these peptides.
Show less - Date Issued
- 2020
- PURL
- http://purl.flvc.org/fau/fd/FA00013507
- Subject Headings
- Matrix Metalloproteinases, Peptides, Fluorogenic probes
- Format
- Document (PDF)
- Title
- Targeted Drug Delivery Utilizing a Mini-Collagen Ligand Recognized by CD44/CSPG Melanoma Receptor.
- Creator
- Khan, David R., Florida Atlantic University, Fields, Gregg B., Charles E. Schmidt College of Science, Department of Chemistry and Biochemistry
- Abstract/Description
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Liposomes of varying lipid compositions are currently used as drug carriers. and great efforts have been made to target these vehicles to specific cellular receptors. Previous targeting components include peptides. proteins. antibodies. or vitamins. CD44/CSPG is among the receptors overexpressed in metastatic melanoma. and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical '·peptide-amphiphile .. (al(JV)1263-1277 PA) which binds CD44...
Show moreLiposomes of varying lipid compositions are currently used as drug carriers. and great efforts have been made to target these vehicles to specific cellular receptors. Previous targeting components include peptides. proteins. antibodies. or vitamins. CD44/CSPG is among the receptors overexpressed in metastatic melanoma. and the sequence to which it binds within the type IV collagen triple-helix has been identified. A triple-helical '·peptide-amphiphile .. (al(JV)1263-1277 PA) which binds CD44/CSPG has been constructed and incorporated into liposomes of differing lipid compositions. Liposomes containing distearoyl phosphatidylcholine (DSPC) as the major bilayer component. in combination with distearoyl phosphatidylglycerol (DSPG) and cholesterol. were more stable than analogous liposomes containing dipalmitoyl phosphatidylcholine (DPPC) instead of DSPC. The presence of the al(JV)1263-1277 PA conferred greater stability to the DPPC liposomal systems. and did not affect the stability of the DSPC liposomes. The addition of either PEG 750 or PEG 2000 (5 mol %) to DSPG/DSPC/Chol liposomes did not affect the stability of this system. Fluorophore delivery of rhodamine loaded liposomes to cells varymg 111 CD44/CSPG expresston was determined usmg fluorescence microscopy. The CD44/CSPG receptor content for two normal fibroblast cell lines, BJ and Hs895Sk, and a highly metastatic melanoma derived cell line, M 14#5. was determined using whole cell ELISA. The results of the ELISA showed varying levels of CD44 receptors amongst the cell lines. with M 14#5 cells having the most. A positive correlation was observed for cellular fluorophore delivery by the ai(IV)1263-1277 PA liposomes and CD44/CSPG receptor content. Conversely, non-targeted liposomes delivered minimal fluorophore to cells regardless of the CD44/CSPG receptor content. When cells were treated with exogeneous a I (IV) 1263-1277, prior to incubation with a I (IV) 1263 -1277 PA liposomes, a dose-dependent decrease in the amount of fluorophore delivered was observed with respect to increasing concentrations of exogeneous al(IV)1263-1277. In addition, we have found a positive correlation between the cytotoxic effect of ai(IV)l263-1277 PA targeted liposomes (with and without PEG) loaded with doxorubicin and the CD44/CSPG content amongst the three different cell lines. This trend was not observed with non-targeted liposomes. These findings provide the possibility of a novel drug carrier system to be used in future clinical applications against metastatic melanoma.
Show less - Date Issued
- 2007
- PURL
- http://purl.flvc.org/fau/fd/FA00000866
- Subject Headings
- Tumor suppressor proteins, Antioncogenes, Drug targeting, Melanoma--Treatment
- Format
- Document (PDF)
- Title
- THE ROLE OF MATRIX METALLOPROTEINASE-28 IN HEALTH AND DISEASE.
- Creator
- Tokmina-Roszyk, Dorota, Fields, Gregg B., Florida Atlantic University, Department of Chemistry and Biochemistry, Charles E. Schmidt College of Science
- Abstract/Description
-
Matrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating...
Show moreMatrix Metalloproteinase-28 (MMP-28) is the newest and least characterized member of MMP family. To date several potential substrate candidates for MMP-28 have been proposed but no in vivo substrates for this enzyme were confirmed. In the central nervous system (CNS) MMP-28 is believed to be important factor during myelination of the developing nervous system as well as during remyelination that follows neuronal injury. On the other hand, MMP-28 has been found in actively demyelinating lesions in both experimental autoimmune encephalopathy (EAE) and multiple sclerosis patients suggesting its possible role in pathological events associated with autoimmune neurodegenerative processes. In addition, MMP-28 has been linked to modulation of immune response and activation of macrophages which presents another role of this enzyme in autoimmune pathologies. In the study described herein, MMP-28 has been shown to affect myelin composition and appearance, mitochondrial protein content, and vesicular transport proteins. Moreover, the decrease in myelin basic protein quantity observed in healthy MMP-28KO animals affected the myelin staining intensity in various brain regions including corpus callous. Cellular energetic studies did not reveal differences in mitochondrial function in MMP-28KO animals and no difference in reactive oxygen species was observed. In the EAE model, MMP-28 deletion increased the occurrence of atypical form of EAE characterized by increased inflammation of arbor vitae of the brain. In addition, MMP-28 deletion decreased the inflammatory infiltrates present in brains obtained from EAE animals. Lastly, MMP-28 has been shown to affect cellular energetics and activation of bone marrow derived macrophages during the initial stages and after 24 h activation. In addition, MMP-28 deletion increased proinflammatory cytokines and receptors CD86 and iNOS found in M1 polarized macrophages.
Show less - Date Issued
- 2020
- PURL
- http://purl.flvc.org/fau/fd/FA00013601
- Subject Headings
- Matrix Metalloproteinases, Multiple sclerosis, Neurodegenerative disease
- Format
- Document (PDF)
