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The accurate identification of mucinous pancreatic cystic lesions (PCLs) is paramount for cancer risk stratification. Cyst fluid carcinoembryonic antigen (CEA), the only routinely used test, requires high volumes and has low sensitivity. We aimed to compare the performance of two investigational small-volume biomarkers, glucose and the protease gastricsin, to CEA for PCL classification.We obtained cyst fluid samples from 81 patients with pathologically confirmed PCLs from four institutions between 2003 and 2016. Gastricsin activity was measured using an internally quenched fluorescent substrate. Glucose levels were measured with a standard glucometer. CEA levels were obtained from the medical record. Models using Classification and Regression Trees were created to predict mucinous status. Model performance was evaluated using nested cross-validation.Gastricsin activity, CEA, and glucose levels from patients with mucinous (n = 50) and nonmucinous (n = 31) PCLs were analyzed. Area under the curve (AUC) was similar for individual classifiers (gastricsin volume normalized [GVN] 0.88; gastricsin protein concentration normalized [GPN] 0.95; glucose 0.83; CEA 0.84). The combination of two classifiers did not significantly improve AUC, with CEA + GVN (0.88) performing similarly to CEA + GPN (0.95), GVN + glucose (0.87), GPN + glucose (0.95), and CEA + glucose (0.84). The three-analyte combination performed similarly to single and dual classifiers (GPN + glucose + CEA AUC 0.95; GVN + glucose + CEA AUC 0.87). After multiple comparison corrections, there were no significant differences between the individual, dual, and triple classifiers.Gastricsin and glucose performed similarly to CEA and required5% of the volume required for CEA; these classifiers may be useful in patients with limited cyst fluid. Future multicenter prospective studies are needed to validate and compare these novel small-volume biomarkers.

Proteins identified in cyst fluid samples.

Detailed description of peptide synthesis and proteomic search parameters for cyst fluid samples.

Figures supporting our finding of increased aspartyl protease activity in mucinous pancreatic cysts.

Purpose: Pancreatic cysts are estimated to be present in 2%–3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts.Experimental Design: We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 μL of cyst fluid.Results: We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity.Conclusions: Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions. Clin Cancer Res; 23(16); 4865–74. ©2017 AACR.

Background: The accurate identification of high-grade dysplasia (HGD) or invasive cancer (advanced neoplasia, AN) in pancreatic cystic lesions (PCLs) is needed to identify PCLs that warrant surgical intervention. Cytologic evaluation of cyst fluid is widely-used but has poor sensitivity. We previously found that activity of the lysosomal serine protease tripeptidyl peptidase 1 (TPP1) is associated with mucinous PCLs that harbor AN (AUC 0.72). We aimed to identify additional functional biomarkers that would improve performance of our classifier for dysplastic grade in mucinous PCLs. Methods: We used a combination of global protease activity profiling and shotgun proteomics to identify differentiating markers between PCLs with low-grade dysplasia (LGD, n=3) and HGD (n=3). Candidate biomarkers were validated in an independent cohort of 28 clinically-annotated mucinous PCLs (HGD n=12, LGD n=16), by measuring both protein concentration (using ELISA) and enzymatic activity (using internally-quenched fluorescent substrates). We used a nested cross-validation approach to iteratively split our cohort into training and validation sets to predict HGD. Model accuracy was evaluated using area under the curve (AUC), sensitivity, specificity, and accuracy. Results: We identified 8 proteins that were highly abundant in mucinous PCLs with HGD. Among these were the inflammatory enzymes myeloperoxidase (MPO) and neutrophil elastase (ELANE), suggesting that differential activity levels could be exploited that would minimize fluid requirements and cost. Among 28 mucinous PCLs, a composite score that included both activity and mass of MPO performed the best for HGD in mucinous cysts (AUC 0.955, sensitivity 98%, specificity 93%). ELANE activity (AUC 0.76, sensitivity 69%, specificity 59%) and TPP1 activity (AUC 0.765, sensitivity 71%, specificity 74%) had modest performance. Conclusions: Our functional biomarkers accurately classified HGD among mucinous cysts. Functional biomarkers that require low sample volumes ( Citation Format: Francesco Caiazza, Andre Lourenco, Patricia Conroy, Thomas Hoffmann, Sam L. Ivry, Tyler York, Gina Zhu, Audrey Mustoe, Anthony J. O'Donoghue, Charles S. Craik, Kimberly Kirkwood. Using functional biomarkers to accurately predict advanced neoplasia in pancreatic cystic lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2226.

Enzymes that modify the proteome, referred to as post-translational modifying (PTM) enzymes, are central regulators of cellular signaling. Determining the substrate specificity of PTM enzymes is a critical step in unraveling their biological functions both in normal physiological processes and in disease states. Advances in peptide chemistry over the last century have enabled the rapid generation of peptide libraries for querying substrate recognition by PTM enzymes. In this article, we highlight various peptide-based approaches for analysis of PTM enzyme substrate specificity. We focus on the application of these technologies to proteases and also discuss specific examples in which they have been used to uncover the substrate specificity of other types of PTM enzymes, such as kinases. In particular, we highlight our multiplex substrate profiling by mass spectrometry (MSP-MS) assay, which uses a rationally designed, physicochemically diverse library of tetradecapeptides. We show how this method has been applied to PTM enzymes to uncover biological function, and guide substrate and inhibitor design. We also briefly discuss how this technique can be combined with other methods to gain a systems-level understanding of PTM enzyme regulation and function.

Incidental detection of pancreatic cysts has increased dramatically over the last decade, but risk stratification and clinical management remain a challenge. Mucinous cysts are precursor lesions to pancreatic cancer, however, the majority are indolent. Current diagnostics cannot identify mucinous cysts that harbor cancer or reliably differentiate these lesions from nonmucinous cysts, which present minimal risk of malignant progression. We previously determined that activity of two aspartyl proteases was increased in mucinous cysts. Using a global protease activity profiling technology, termed multiplex substrate profiling by mass spectrometry (MSP-MS), we now show that aminopeptidase activity is also elevated in mucinous cysts. The serine aminopeptidase, tripeptidyl peptidase 1 (TPP1), was detected by proteomic analysis of cyst fluid samples and quantitation using targeted MS demonstrated that this protease was significantly more abundant in mucinous cysts. In a cohort of 110 cyst fluid samples, TPP1 activity was increased more than 3-fold in mucinous cysts relative to nonmucinous cysts. Moreover, TPP1 activity is primarily associated with mucinous cysts that harbor high-grade dysplasia or invasive carcinoma. Although only 59% accurate for differentiating these lesions, measurement of TPP1 activity may improve early detection and treatment of high-risk pancreatic cysts when used in conjunction with other promising biomarkers.

Single-copy loss-of-function mutations in the progranulin gene (PGRN) underlie the neurodegenerative disease frontotemporal lobar degeneration, while homozygous loss-of-function of PGRN results in the lysosomal storage disorder neuronal ceroid lipofuscinosis. Despite evidence that normal PGRN levels are critical for neuronal health, the function of this protein is not yet understood. Here, we show that PGRN stimulates the in vitro maturation of the lysosomal aspartyl protease cathepsin D (CTSD). CTSD is delivered to the endolysosomal system as an inactive precursor (proCTSD) and requires sequential cleavage steps via intermediate forms to achieve the mature state (matCTSD). In co-immunoprecipitation experiments, PGRN interacts predominantly with immature pro- and intermediate forms of CTSD. PGRN enhances in vitro conversion of proCTSD to matCTSD in a concentration-dependent manner. Differential scanning fluorimetry shows a destabilizing effect induced by PGRN on proCTSD folding (∆ Tm = − 1.7 °C at a 3:1 molar ratio). We propose a mechanism whereby PGRN binds to proCTSD, destabilizing the propeptide from the enzyme catalytic core and favoring conversion to mature forms of the enzyme. Further understanding of the role of PGRN in CTSD maturation will assist in the development of targeted therapies for neurodegenerative disease.

The mechanism of ethylene polymerization by the widely used Phillips catalyst remains controversial. In this work, we compare initiation, propagation, and termination pathways computationally using small chromasiloxane cluster models for several previously proposed and new mechanisms. Where possible, we consider complete catalytic cycles and compare predicted kinetics, active site abundances, and polymer molecular weights to known properties of the Phillips catalyst. Prohibitively high activation barriers for propagation rule out previously proposed chromacycle ring expansion and Green–Rooney (alternating alkylidene/chromacycle) mechanisms. A new oxachromacycle ring expansion mechanism has a plausible propagation barrier, but initiation is prohibitively slow. On sites with adjacent bridging hydroxyls, either ≡Si(OH)CrII-alkyl or ≡Si(OH)CrIII-alkyl, initiated by proton transfer from ethylene, chain growth by a Cossee–Arlman-type mechanism is fast. However, the initiation step is uphill and extremely slow, ...

Purpose: Pancreatic cysts are estimated to be present in 2%–3% of the adult population. Unfortunately, current diagnostics do not accurately distinguish benign cysts from those that can progress into invasive cancer. Misregulated pericellular proteolysis is a hallmark of malignancy, and therefore, we used a global approach to discover protease activities that differentiate benign nonmucinous cysts from premalignant mucinous cysts. Experimental Design: We employed an unbiased and global protease profiling approach to discover protease activities in 23 cyst fluid samples. The distinguishing activities of select proteases was confirmed in 110 samples using specific fluorogenic substrates and required less than 5 μL of cyst fluid. Results: We determined that the activities of the aspartyl proteases gastricsin and cathepsin E are highly increased in fluid from mucinous cysts. IHC analysis revealed that gastricsin expression was associated with regions of low-grade dysplasia, whereas cathepsin E expression was independent of dysplasia grade. Gastricsin activity differentiated mucinous from nonmucinous cysts with a specificity of 100% and a sensitivity of 93%, whereas cathepsin E activity was 92% specific and 70% sensitive. Gastricsin significantly outperformed the most widely used molecular biomarker, carcinoembryonic antigen (CEA), which demonstrated 94% specificity and 65% sensitivity. Combined analysis of gastricsin and CEA resulted in a near perfect classifier with 100% specificity and 98% sensitivity. Conclusions: Quantitation of gastricsin and cathepsin E activities accurately distinguished mucinous from nonmucinous pancreatic cysts and has the potential to replace current diagnostics for analysis of these highly prevalent lesions. Clin Cancer Res; 23(16); 4865–74. ©2017 AACR.

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. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites., Author Summary Certain parasitic flatworms pose serious threats to human health. Understanding more about the biology of flatworms can lead to new and better drug targets. However, due to the complex life cycles of parasitic flatworms, it has been suggested that a non-parasitic flatworm, Schmidtea mediterranea, be used to study flatworm biology. This worm can be used as a model to study processes that all flatworms have in common; these findings can be applied back to parasitic flatworms. Digestion occurs in all worms and relies on the activity of protease enzymes to break down protein. The authors were able to induce regurgitation in S. mediterranea and study the contents of the worm gut. These worms have several types of proteases present, although it seems that only two types, called cysteine and aspartyl proteases, are important in digestion in live worms. Furthermore, one specific cysteine protease, cathepsin B, is responsible for the bulk of the digestive capabilities in S. mediterranea. Cysteine proteases also appear to be involved in the ability of S. mediterranea to regenerate damaged body tissues. Understanding protease activity in S. mediterranea can provide insight as to the roles of these enzymes in medically relevant parasitic flatworms.