Isozyme Selectivity among Triple Helix Cleaving Metalloproteinases

Srivastava, D.

Abstract

Several matrix metalloproteinases (MMPs) cleave triple helical peptides, prevalent in collagen fibrils, and the overexpression/activation of some such isozymes (""""""""triple helicases"""""""") cause pathogenic conditions such as arthritis, atherosclerosis, chronic obstructive pulmonary disease, ulcers, and various cancers among others. Hence, the detection and inhibition of those enzymes are of significant importance from the point of view of prevention and/or treatment of associated human diseases. The proposed research will employ our recently developed liposome-based methodologies for the isozyme selective detection and desensitization/inhibition of """"""""triple helicases"""""""", responsible for causing various human diseases. These objectives will be accomplished by formulating two-types of liposomes: (1) For isozyme selective detection, we will formulate liposomes with triple helical peptides on the surface and the signal amplification probes in the lumen, such that the enzymatic cleavage of the triple helical peptides would destabilize the liposomes and release their signal generating probes to facilitate the detection of the parent enzyme. (2) For isozyme selective desensitization/inhibition, we will formulate liposomes with """"""""active site"""""""" and """"""""surface affine"""""""" residues (akin to our """"""""two-prong"""""""" inhibitor designing approach) such that the lipid mobilit of liposomes would facilitate juxtaposition of the above residues to the complementary regions of the isozymes to selectively knock down the enzyme activity. To gain insight into the molecular basis of isozyme selectivity in achieving the above objectives, as well as fine-tuning our overall detection and desensitization protocols, we will delineate the energetic contributions of """"""""head-head"""""""" and """"""""tail-tail"""""""" interactions among different lipo-conjugates in stabilization/destabilizationof liposomes, attaining surface complementarity between liposomal head groups and triple helicase isozymes, and the release of the liposome's encapsulated probes to serve as the signal amplification system.
The Specific Aims of the proposed research are as follows: (1) Develop the liposome-based signal amplification strategy for the isozyme specific detection of triple helicases: We will formulate liposomes, harboring isozyme selective triple helical peptides on the surface and a small DNA fragment in the lumen. Depending on the amino acid sequences of the monomeric peptide units, compactness of resultant triple helical peptides on the liposomal surface, and the electrostatic surface complementarity between liposomes and the enzymes, individual triple helicase isozymes will selectively recognize and cleave their cognate triple helical peptides (resident on the liposomal surface), destabilize the liposomal assembly and release their encapsulated DNA fragment. The latter will be utilized as the signal amplification probe, and will be detected via the real time PCR method. Besides being robust, sensitive, and isozyme selective, our liposome based approach will selectively and uniquely detect the """"""""catalytically active"""""""" forms of triple helicase isozymes, and not the total isozymes as conventionally detected by the ELISA method. (2) Simulate the """"""""two-prong"""""""" ligand designing strategy on the liposome surface to serve as the isozyme selective inhibitors against triple helicases: We will simulate our """"""""two-prong"""""""" ligand designing strategy by incorporating both """"""""active site"""""""" and the """"""""enzyme's surface"""""""" directed binding groups on the liposome surface for selectively inhibiting one triple helicase isozyme in preference to the other. The active site directed ligand (a hydroxamate inhibitor) on the liposome surface will serve as a """"""""bait"""""""" to lure the binding of the enzyme in the form of an """"""""anchored complex"""""""". Subsequently, the liposomal lipid mobility will facilitate juxtaposition of the complementary charged residues (from the liposomal surface) to the enzyme's site. Once the stable complex between a triple helical isozyme and the liposomal assembly is formed, we will lock the mobility of the liposomal lipids (via photo-polymerization or bifunctional cross- linking) to generate the """"""""templated"""""""" liposomes, which will selectively inhibit its parent triple helicase isozyme in preference to the other triple helicase isozymes. (3) Ascertain the energetic contributions of """"""""head-head"""""""" and """"""""tail-tail"""""""" interaction in modulating the liposome-triple helicase interactions: By formulating liposomes with surface exposed triple helical peptides in the """"""""head"""""""" region and fluorescence probes at both """"""""head"""""""" and """"""""tail"""""""" regions, we will investigate the energetic contribution of """"""""head-head"""""""" and """"""""tail-tail"""""""" interactions on binding of triple helicases to liposomes and their destabilization via the cleavage of the triple helical peptides. The mechanistic insights gained from these studies will be utilized toward fine-tuning the liposome based detection and desensitization protocols for triple helicase isozymes. These objectives will be accomplished by employing the techniques of synthetic organic chemistry, molecular biology, electronic spectroscopy, kinetics and thermodynamics. The outcome of this """"""""basic"""""""" """"""""fundamental"""""""" research will find applications in detection and desensitization/inhibition of individual triple helicase isozymes toward therapeutic intervention in associated human diseases.

Public Health Relevance

The proposed research will employ our recently developed liposome-based methodologies for isozyme selective detection and inhibition of triple helix cleaving enzymes, which are responsible for causing various human diseases. The insight gained from this project will allow development of liposomes based diagnostics and therapeutic tools.