The multifaceted roles of tumor-associated proteases and harnessing their activity for prodrug activation.

The role of proteases in cancer was originally thought to be limited to the breakdown of basement membranes and extracellular matrix (ECM), thereby promoting cancer cell invasion into surrounding normal tissues. It is now well understood that proteases play a much more complicated role in all stages of cancer progression and that not only tumor cells, but also stromal cells are an important source of proteases in the tumor microenvironment. Among all the proteolytic enzymes potentially associated with cancer, some proteases have taken on heightened importance due to their significant up-regulation and ability to participate at multiple stages of cancer progression and metastasis.

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors.

The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue.

Imaging active urokinase plasminogen activator in prostate cancer.

The increased proteolytic activity of membrane-bound and secreted proteases on the surface of cancer cells and in the transformed stroma is a common characteristic of aggressive metastatic prostate cancer. We describe here the development of an active site-specific probe for detecting a secreted peritumoral protease expressed by cancer cells and the surrounding tumor microenvironment. Using a human fragment antigen-binding phage display library, we identified a human antibody termed U33 that selectively inhibited the active form of the protease urokinase plasminogen activator (uPA, PLAU).

Tumor-specific activation of an EGFR-targeting probody enhances therapeutic index.

Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug strategy to create an epidermal growth factor receptor (EGFR)-directed Probody therapeutic-an antibody that remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab.

Global identification of peptidase specificity by multiplex substrate profiling.

We developed a simple and rapid multiplex substrate-profiling method to reveal the substrate specificity of any endo- or exopeptidase using liquid chromatography-tandem mass spectrometry sequencing. We generated a physicochemically diverse library of peptides by incorporating all combinations of neighbor and near-neighbor amino acid pairs into decapeptide sequences that are flanked by unique dipeptides at each terminus. Addition of a panel of evolutionarily diverse peptidases to a mixture of these tetradecapeptides generated information on prime and nonprime sites as well as on substrate specificity that matched or expanded upon known substrate motifs.

Inhibition of Granzyme B by PI-9 protects prostate cancer cells from apoptosis.

Background: In order for tumors to grow and proliferate, they must avoid recognition by immune cells and subsequent death by apoptosis. Granzyme B (GrB), a protease located in natural killer cells, initiates apoptosis in target cells. Inhibition of GrB by PI-9, its natural inhibitor, can prevent apoptosis.

Human and mouse granzyme M display divergent and species-specific substrate specificities.

Cytotoxic lymphocyte protease GrM (granzyme M) is a potent inducer of tumour cell death and a key regulator of inflammation. Although hGrM (human GrM) and mGrM (mouse GrM) display extensive sequence homology, the substrate specificity of mGrM remains unknown. In the present study, we show that hGrM and mGrM have diverged during evolution.

Monitoring of natural killer cell immunotherapy using noninvasive imaging modalities.

Cancer immunotherapies can be guided by cellular imaging techniques, which can identify the presence or absence of immune cell accumulation in the tumor tissue in vivo and in real time. This review summarizes various new and evolving imaging techniques employed for tracking and monitoring of adoptive natural killer cell immunotherapies.

Prediction of protease substrates using sequence and structure features.

Motivation: Granzyme B (GrB) and caspases cleave specific protein substrates to induce apoptosis in virally infected and neoplastic cells. While substrates for both types of proteases have been determined experimentally, there are many more yet to be discovered in humans and other metazoans. Here, we present a bioinformatics method based on support vector machine (SVM) learning that identifies sequence and structural features important for protease recognition of substrate peptides and then uses these features to predict novel substrates.

Antagonistic anti-urokinase plasminogen activator receptor (uPAR) antibodies significantly inhibit uPAR-mediated cellular signaling and migration.

Interactions between urokinase plasminogen activator receptor (uPAR) and its various ligands regulate tumor growth, invasion, and metastasis. Antibodies that bind specific uPAR epitopes may disrupt these interactions, thereby inhibiting these processes. Using a highly diverse and naïve human fragment of the antigen binding (Fab) phage display library, we identified 12 unique human Fabs that bind uPAR.

Continuous imaging of plasmon rulers in live cells reveals early-stage caspase-3 activation at the single-molecule level.

The use of plasmon coupling in metal nanoparticles has shown great potential for the optical characterization of many biological processes. Recently, we have demonstrated the use of "plasmon rulers" to observe conformational changes of single biomolecules in vitro. Plasmon rulers provide robust signals without photobleaching or blinking.

Optical imaging of cellular immunotherapy against prostate cancer.

The purpose of this study was to track fluorophore-labeled, tumor-targeted natural killer (NK) cells to human prostate cancer xenografts with optical imaging (OI). NK-92-scFv(MOC31)-zeta cells targeted to the epithelial cell adhesion molecule (EpCAM) antigen on prostate cancer cells and nontargeted NK-92 parental cells were labeled with the near-infrared dye DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine). The fluorescence, viability, and cytotoxicity of the labeled cells were evaluated.

Temperature dependence of myosin-II tail fragment assembly.

Dictyostelium myosin-II bipolar thick filament (BTF) assembly is heavily dependent on ionic strength and temperature and is reversible by the phosphorylation of just three threonines. Truncated tail fragments of Dictyostelium myosin-II are commonly used as models for BTF assembly, as they self-assemble into regular paracrystals that recapitulate the ionic strength and phosphorylation dependence of full-length Dictyostelium myosin-II BTF assembly. Here we show that Dictyostelium myosin-II tail fragment assembly is highly temperature dependent, similar to full-length Dictyostelium myosin-II.

Hip is a pro-survival substrate of granzyme B.

The extended substrate specificity of granzyme B (GrB) was used to identify substrates among the chaperone superfamily. This approach identified Hsp90 and Bag1-L as novel GrB substrates, and an additional GrB cleavage site was identified in the Hsc70/Hsp70-Interacting Protein, Hip. Hsp90, Bag1L, and Hip were validated as GrB substrates in vitro, and mutational analysis confirmed the additional cleavage site in Hip.

Dictyostelium myosin bipolar thick filament formation: importance of charge and specific domains of the myosin rod.

Myosin-II thick filament formation in Dictyostelium is an excellent system for investigating the phenomenon of self-assembly, as the myosin molecule itself contains all the information required to form a structure of defined size. Phosphorylation of only three threonine residues can dramatically change the assembly state of myosin-II. We show here that the C-terminal 68 kDa of the myosin-II tail (termed AD-Cterm) assembles in a regulated manner similar to full-length myosin-II and forms bipolar thick filament (BTF) structures when a green fluorescent protein (GFP) "head" is added to the N terminus.

The myosin coiled-coil is a truly elastic protein structure.

Coiled-coils occur in a variety of proteins involved in mechanical and structural tasks in the cell. Their mechanical properties are important in various contexts ranging from hair elasticity to synaptic fusion. Beyond their importance in biology, coiled-coils have also attracted interest as programmable protein sequences for the design of novel hydrogels and materials.