Fields, Gregg B.

Matrix metalloproteinase-9 MMP-9 is involved in the early stages of wound healing, including the
inflammatory reaction that follows myocardial infarction and neovascularization. However, its
overexpression in the infarct zone leads to deleterious effects. Understanding MMP-9 function and
modulation of its activity provides an opportunity to prevent excessive remodeling of the left ventricle.
To assess the role of MMP-9 in remodeling process we employed a broad search of in vivo substrates.
Based on comparative analysis of MMP-9 null and wild type mice, several peptides mimicking putative
substrates were synthesized. The cleavage sites in the substrates were identified using high
performance liquid chromatography and mass spectrometry. Peptide mapping studies revealed MMP-9
cleavage sites in several proteins, potential biomarkers of excessive remodeling. Specifically,
osteopontin, thrombospondin and C-terminal telopeptide regions of type I collagen were susceptible to
proteolysis by MMP-9. The best target for MMP-9 was fibronectin, which has multiple cleavage sites in
its sequence. In addition to in vivo substrate screening, a selective triple-helical peptide inhibitor MMP-
9i has been designed, synthesized, and utilized as an MMP-9 probe. The sequence of inhibitor was
derived from the known MMP-9 substrate type V collagen. In the MMP-9i construct, the G~V scissile
bond has been replaced with phosphinate moiety that mimics the transition state of hydrolysis but
cannot be cleaved. MMP-9i's effect on MMP-9 activity in serum was tested in a mouse model. The
administration of MMP-9i resulted in 30 loss of MMP-9 activity suggesting that MMP-9i can be utilized
to regulate activity of MMP-9 in vivo.

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 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.

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/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.

Melanoma starts on the surface of the skin where it is easily seen. It is curable
when detected early, but can be fatal if allowed to progress and spread. Melanoma can
spread downwards through the skin, ultimately reaching the blood and lymphatic vessels,
and metastasize. Thus, one goal is to detect melanoma early before it metastasizes. A high
throughput proteomics approach has been applied to better understand the processes that
underlie tumor formation and progression. Three studies were pursued: I) proteome
comparison of the matched primary WM-115 and metastatic WM-266-4 melanoma cell
lines; II) proteome comparison between the matched melanoma Hs 895.T and fibroblast
Hs 895Sk cell lines; and III) comprehensive proteome cataloging of two metastatic
melanoma cell lines Hs 895.T and SK-MEL-2. From these studies we identified proteins
that are involved in cellular functions such as metabolism, signal transduction, and DNA
binding, as well as structural and heat shock proteins. We hypothesized about a possible
oxidative stress pathway involved in melanoma progression, initiated the creation of a
melanoma proteome database, and also identified some proteins not previously studied in melanoma (such as cyclophilin A, ADP-ribosylation factor-1, 14-3-3 zeta ATP syntase, Rho
GTPase, Plastin T, galectin 1 and 3, annex in II, enolase 1, cofilin, RhoGDI, Rap 1,
G6PG, GAPDH, TKT, HK, and nuclear chloride channel protein). These results mark a
step forward in the development of a metstatic melanoma protein database, the
understanding of the chemical pathways that are involved in metastatic melanoma
development, and identification of possible new targets for inhibitor development.

In this present work we have examined the biophysical characterization of two peptides. One alpha-helical (SPARC) and the other triple-helical (collagen). We have compared the effect of lipidation on stabilizing of the alpha-helical and triple-helical peptides. For the first peptide, amino acids from the angiogenesis-inducing region of SPARC were incorporated into alpha-helical peptide sequence. A Tyr was placed between the alpha-helical sequence and the peptide to provide a chromophore; Lys-Ala-Glu-Ile-Glu-Ala-Leu-Lys-Ala-Glu-Ile-Glu-Ala-Leu-Lys-Ala-Tyr-Lys-His-Gly-Lys-NH 2 was the final sequence. For the second peptide, the sequence chosen to mimic the alpha2beta1 integrin binding site in type I collagen was (Gly-Pro-Hyp)4-Gly-Pro-Ala-Gly-Lys-Asp-Gly-Glu-Ala-Gly-Ala-Gln-(Gly-Pro-Hyp) 4-NH2. Next, each peptide was lipidated with a C-16 acid and was biophysically characterized to determine physiological compatibility. Techniques used included circular dichroism spectroscopy, gel electrophoresis, and Fourier transform infrared spectroscopy. Lastly, one of the peptide amphiphiles was examined as a biomaterial modifier.

Hydroxylysine is produced as a posttranslational modification mainly in collagens, the most abundant protein in mammals. Lysyl hydroxylase (LH) is the enzyme that catalyzes the formation of hydroxylysyl residues in collagen by hydroxylation of -X-Lys-Gly- sequences, for which it requires Fe 2+, 2-oxoglutarate, O2 and ascorbate. In order to study the hydroxylation reaction catalysed by LH, we have synthesized 4 different peptides [for example, GFP*GLP*GAKGE (P*=hydroxyproline) and the corresponding hydroxylated (hydroxylysine-containing) peptide] using Fmoc solid-phase methodology. Peptides have been characterized by HPLC, MALDI-TOF mass spectrometry and CD spectroscopy. A new method for efficient separation of lysine- from hydroxylysine-containing peptides by HPLC has been developed in both organic phase (1-anthroylnitrile as derivatizating reagent) and aqueous phase (dansyl chloride as derivatizating reagent). These reagents have been used to derivatize peptides prior to HPLC analysis. The products (di- and tetra-substituted lysine- and hydroxylysine-containing peptides) have been fully separated by HPLC and their structure confirmed by MALDI-TOF MS analysis. Efficient separation of derivatized peptides will allow for the convenient and rapid measurement of LH activity by HPLC methods.

The metastatic process involves tumor cell adhesion to basement membrane components, such as type IV collagen. A specific mitogen activated protein kinase cascade is activated by cell adhesion to type IV collagen. This activation causes the expression of proteolytic enzymes. These enzymes will then participate in compromising extracellular matrix components and enhance cell movement through them. To better understand tumor invasion of type IV collagen, we have constructed triple-helical peptide (THP) ligands for melanoma cell receptors, and used these ligands to determine if receptors such as CD44/CSPG and the alpha2beta1 integrin have unique matrix metalloproteinase (MMP) signaling pathways affected by the tyrosine kinase inhibitor genistein. MMP protein expression profiles were evaluated using the alpha2beta1 integrin ligand, and CD44/CSPG ligand. Results were indicative of specific activation sequences that tumor cells undergo upon binding to select regions of type IV collagen.