Dipeptidyl peptidase I (DPPI) is the sole activator in vivo of several granule-associated serine proteases of cytotoxic lymphocytes. In vitro, DPPI also activates mast cell chymases and tryptases. To determine whether DPPI is essential for their activation in vivo, we used enzyme histochemical and immunohistochemical approaches and solution-based activity assays to study these enzymes in tissues and bone marrow-derived mast cells (BMMCs) from DPPI ؉/؉ and DPPI ؊/؊ mice. We find that DPPI ؊/؊ mast cells contain normal amounts of immunoreactive chymases but no chymase activity, indicating that DPPI is essential for chymase activation and suggesting that DPPI ؊/؊ mice are functional chymase knockouts. The absence of DPPI and chymase activity does not affect the growth, granularity, or staining characteristics of BMMCs and, despite prior predictions, does not alter IgE-mediated exocytosis of histamine. In contrast, the level of active tryptase (mMCP-6) in DPPI ؊/؊ BMMCs is 25% that of DPPI ؉/؊ BMMCs. These findings indicate that DPPI is not essential for mMCP-6 activation but does influence the total amount of active mMCP-6 in mast cells and therefore may be an important, but not exclusive mechanism for tryptase activation.Granule-associated serine proteases of cytotoxic lymphocytes, neutrophils, and mast cells (i.e. granzymes, elastase, cathepsin G, and chymases) are structurally related (1). They have a two-amino acid propeptide (also referred to as the "activation dipeptide") and an isoleucine at the NH 2 terminus of the activated enzyme (2). The activation dipeptide maintains the protease in an inactive state. After removal of the dipeptide, the new NH 2 -terminal isoleucine moves from its position on the surface of the protease to a region in the interior of the enzyme, where it interacts with an aspartate residue near the catalytic center (3). These movements are thought to change the enzyme substrate binding cleft and catalytic apparatus to a catalytically competent conformation.Initial studies investigating the activation of the granuleassociated serine proteases focused on the cysteine protease dipeptidyl peptidase I (DPPI), 1 also known as cathepsin C (2, 4 -8). DPPI was a logical candidate activator of these proteases because it is found in the same cells and is a promiscuous exopeptidase that hydrolyzes most NH 2 -terminal dipeptides (9). Studies using the DPPI inhibitor Gly-Phe-diazomethyl ketone reported that DPPI inhibition reduces granzyme A, neutrophil elastase, and cathepsin G activity in the cells (2). However, because the inhibitor is not entirely specific for DPPI and does not eliminate the activity of the proteases in question, the possibility remained that another protease was responsible for their activation. This possibility was tested in a DPPI knockout mouse (10). Cytotoxic lymphocytes of these animals produce normal amounts of granzymes A and B, but both enzymes are inactive and present in their pro-forms. These findings suggest that DPPI is the sole activator of granzymes A and B in vivo...

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Genetic alpha-tryptase deficiency is common and varies strikingly between ethnic groups. Because beta-tryptases are implicated in allergic disorders, inherited differences in alpha/beta-genotype may affect disease susceptibility, severity and response to tryptase inhibitor therapy.

BackgroundPancreatic ductal adenocarcinoma has proven to be one of the most chemo-resistant among all solid organ malignancies. Several mechanisms of resistance have been described, though few reports of strategies to overcome this chemo-resistance have been successful in restoring sensitivity to the primary chemotherapy (gemcitabine) and enter the clinical treatment arena.MethodsWe examined the ability of cellular arginine depletion through treatment with PEG-ADI to alter in vitro and in vivo cytotoxicity of gemcitabine. The effect on levels of key regulators of gemcitabine efficacy (e.g. RRM2, hENT1, and dCK) were examined.ResultsCombination of PEG-ADI and gemcitabine substantially increases growth arrest, leading to increased tumor response in vivo. PEG-ADI is a strong inhibitor of the gemcitabine-induced overexpression of ribonucleotide reductase subunit M2 (RRM2) levels both in vivo and in vitro, which is associated with gemcitabine resistance. This mechanism is through the abrogation of the gemcitabine-mediated inhibitory effect on E2F-1 function, a transcriptional repressor of RRM2.ConclusionThe ability to alter gemcitabine resistance in a targeted manner by inducing metabolic stress holds great promise in the treatment of advanced pancreatic cancer.

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