ÌÇÐÄVlog

Our lab focuses on pediatric cancer research, specifically the biological roles of intracellular proteases, with particular emphasis on enzymes called cathepsins that are found in the endosomal-lysosomal system.

Originally, these proteases were discovered as enzymes with broad specificities that degrade proteins down to small peptides and amino acids. The amino acids produced can then be re-used for new protein synthesis.

The majority of the cathepsins were found in lysosomes, intracellular organelles that contain an array of hydrolytic enzymes. These organelles were viewed as terminal degradative compartments. This suggested that this group of enzymes were designed to be "the garbage disposal unit" of cells and at best could be viewed as "recycling centers." In recent years, we and others have begun to challenge these views.

The Role of Lysomal Proteases

Gene knock-outs of individual cathepsins do not block bulk protein turnover, but result in defective processing of unique proteins. It is now clear that the lysosomal proteases play critical roles in antigen processing and presentation, tumor cell invasion and bone remodeling.

Proteases in Discreet Intracellular Compartments

One project in the lab is to design novel inhibitor probes that target proteases in discreet intracellular compartments. We have designed inhibitors that can identify active proteases in living cells, allowing us to measure intracellular concentrations of active proteases without manipulating assay conditions.

More selective inhibitors have been synthesized by conjugating reagents to proteins that specifically target the endosomal compartments of cells. These inhibitors are being evaluated in more applied projects as potential agents that can selectively impair the immune response, tumor cell invasion and bone turnover.

Synthetic Protease Inhibitors

One project in the lab is to design novel inhibitor probes that target proteases in discreet intracellular compartments. We have designed inhibitors that can identify active proteases in living cells, allowing us to measure intracellular concentrations of active proteases without manipulating assay conditions.

More selective inhibitors have been synthesized by conjugating reagents to proteins that specifically target the endosomal compartments of cells. These inhibitors are being evaluated in more applied projects as potential agents that can selectively impair the immune response, tumor cell invasion and bone turnover.

Evolution of Placental Proteases

Background

The placenta is a unique organ found only in mammals within the animal kingdom. This organ is critical to mammalian development, providing a link between the maternal and fetal circulation. Unique proteolytic functions of the placenta include invasive implantation, hormone processing, regulation of the immune system, and remodeling of the growing organ. The organ has evolved relatively recently when compared with other animal organs. In two separate branches of mammalian species, new families of placenta-specific proteases have evolved by gene duplication. Sheep, pig, and cattle express a range of aspartic proteases, and rodents express a range of cysteine proteases. The hypothesis of this project is that gene duplications within these species have permitted the specialized development of placenta-specific proteases. These recently duplicated families of genes offer unique opportunities to determine the significance of gene duplications to mammalian development.

Lysosomal proteases perform a range of critical proteolytic functions that are distinct from their general roles in bulk protein turnover. In rodents, inhibitor studies have shown that lysosomal cysteine proteases play crucial roles in embryonic development. Subsequent animal studies that knocked-out cathepsins B and L, the presumed targets of these inhibitors, failed to corroborate these findings. The reason for these apparently contradictory results became clearer when it was discovered that the inhibitors react with additional targets in mouse placenta and that rodents have a unique family of placentally-expressed cathepsins (PECs). Molecular models of these PECs show that each enzyme has a restricted active-site cleft and each will probably have unique substrate specificities. The primary goal of this research is to obtain functional data to test the hypothesis that gene duplications give rise to a family of proteins with unique catalytic roles.

What We're Doing

The specific aims of this study are:

1. To fully characterize placentally expressed cathepsins P, 1, and 2. Cathepsins will be expressed in Pichia pastoris to obtain active enzyme to study substrate specificities. Specificity of active enzymes will be determined using substrate phage display and combinatorial peptide libraries. Properties of the placental proteases will be compared with mouse, rabbit and human cathepsin L.
2. To determine the cellular and sub-cellular distribution of cathepsins P, 1, and 2. Cathepsins function in lysosomes and endosomes and may have functions outside cells. Sorting of cathepsins will be examined in primary cultures of trophoblasts. In situ hybridization and immunolocalization will be used to show which cells synthesize and which cells contain the cathepsin proteins to determine their probable sites of action.
3. To determine the proteolytic roles of cathepsins P, 1 and 2 in trophoblast function. The effect of protease inhibitors and cathepsin RNA interference on trophoblast cell growth and invasion will be determined. Primary trophoblasts from normal and cathepsin L-deficient mice will be studied. The role of placental cathepsins in extracellular matrix remodeling later in gestation will also be examined.

Proteolytic Mechanisms of Tumor Cell Invasion

Background

When cancer cells become invasive, they spread to adjacent and distant normal organs and become much more difficult to treat. New treatment possibilities are now being explored that target the mechanisms of tumor cell invasion in order to stop the cancer from invading. Proteases are recognized as major mediators of the invasive process, and this project is aimed at defining mechanisms by which one family of proteases, called cathepsins, contribute to the invasive process. Correlative studies have already implicated cathepsins in tumor cell growth and invasion, but mechanisms by which these proteases become active against extracellular proteins are poorly understood. In this project, techniques to identify active proteases will be further improved and used to demonstrate the critical cellular locations of the cathepsins while the cancer cells are invading. These techniques will be used to identify tumors that are more likely to respond to inhibitors of cathepsins. The eventual goal is to treat cancers that demonstrably use cathepsins to invade normal organs with specific inhibitors of the cathepsins.

What We're Doing

It is clear that proteases perform a number of key roles in the invasive process of tumor cells. Identifying the critical proteases in any given type of cancer will aid the design of specific treatments to inhibit the invasive properties of cancers that use the proteases to invade normal tissues.

Matrix metalloproteases (MMPs) have emerged as one set of proteases important in tumor cell invasion. We hypothesize that cathepsins are a second group of proteases that make a significant contribution to the invasive process. Moreover, we hypothesize that regulation of the dynamic equilibrium of cathepsins between different cellular compartments will influence the ability of these proteases to facilitate invasion of tumor cells. In normal cells, 95% of the cathepsins are located in lysosomes, so redistribution of even a small portion of these proteases will have a major effect on the amount of cathepsin in the secondary location. Thus, aberrant regulation of the subcellular distribution of the cathepsins in cancer cells may result in delivery of these proteases to the extracellular milieu where they can destroy normal cellular matrices and enhance invasion.

The key to examining the effects of such redistributions is the development of techniques that selectively identify cathepsins at the secondary sites. We have developed techniques using fluorescent molecules that bind with high specificity to the active sites of cathepsins to identify the active proteases in cells.

To prove our hypothesis we propose to determine the effect of cathepsin inhibitors on tumor cell invasion, examine the cellular distribution of active cathepsins during the invasive process, and to experimentally manipulate the distribution of the cathepsins to modulate the invasive process. As a model system, we will focus on proteases in neuroblastoma cell lines and determine the relative contribution of MMPs and cathepsins to the invasive process.