Extended Cleavage Specificities of Rabbit and Guinea Pig Mast Cell Chymases: Two Highly Specific Leu-Ases

Serine proteases constitute the major protein content of mast cell (MC) secretory granules. These proteases can generally be subdivided into chymases and tryptases based on their primary cleavage specificity. Here, we presented the extended cleavage specificities of a rabbit β-chymase and a guinea pig α-chymase. Analyses by phage display screening and a panel of recombinant substrates showed a marked similarity in catalytic activity between the enzymes, both being strict Leu-ases (cleaving on the carboxyl side of Leu). Amino acid sequence alignment of a panel of mammalian chymotryptic MC proteases and 3D structural modeling identified an unusual residue in the rabbit enzyme at position 216 (Thr instead of more common Gly), which is most likely critical for the Leu-ase specificity. Almost all mammals studied, except rabbit and guinea pig, express classical chymotryptic enzymes with similarly extended specificities, indicating an important role of chymase in MC biology. The rabbit and guinea pig are the only two mammalian species currently known to lack a classical MC chymase. Key questions are now how this major difference affects their MC function, and if genes of other loci can rescue the loss of a chymotryptic activity in MCs of these two species.

Identification of Novel Inhibitors That Specifically Target the Immunoproteasome, and Selectively Induce Apoptosis in Multiple Myeloma and Other Immunoproteasome-Expressing Model Systems.

Inhibition of the proteasome, a multi-catalytic proteinase complex responsible for regulated intracellular proteolysis, with bortezomib (VELCADE®) is a rational strategy against relapsed/refractory multiple myeloma, and studies are ongoing to further define the efficacy of this agent in other settings. However, bortezomib is associated with several toxicities, such as peripheral neuropathy, which can limit the ability of patients to receive maximal doses, possibly compromising anti-tumor efficacy. Novel proteasome inhibitors with an improved toxicity profile and improved anti-tumor activity are therefore needed. Most cells contain the constitutive version of the proteasome with three catalytic subunits, designated X, Y, and Z. Exposure of these cells to cytokines, such as γ-interferon, causes replacement of these subunits with three others, referred to as low molecular weight proteins (LMP)−2, −7, and −10. This LMP-containing proteasome, known as the immunoproteasome because it plays a role in generating antigens as part of the immune response, is also the major proteasome isoform in hematopoietic-derived cells even in the absence of exogenous cytokines. Using purified preparations of the XYZ and LMP proteasomes, we identified a panel of peptidyl-aldehydes that preferentially inhibit the immunoproteasome with relative sparing of the constitutive isoform. Among these, the most immunoproteasome specific inhibitors (IPSI) included compounds 001 and 006. IPSI-001 demonstrated a Ki of 1.03 μM against the chymotryptic activity of the LMP proteasome but only a 105 μM Ki against the XYZ proteasome, while IPSI-006 demonstrated Kis of 8.4 μM and 460 μM, respectively. In contrast, other commonly used peptidyl aldehydes, such as MG-132 and the aldehyde version of bortezomib, showed no ability to discriminate between these two proteasomes. Using radiolabeled 3,4-dichloroisocoumarin as a probe, IPSIs were shown to bind only to LMP-2 of the immunoproteasome, but not to any subunits of the constitutive proteasome in vitro. Studies of cell lines expressing immunoproteasome subunits showed IPSI-001 inhibited the chymotryptic activity of the LMP proteasome, with relative sparing of this activity in XYZ proteasome-expressing cell lines. IPSI-001 was also able to induce apoptosis in LMP-proteasome-expressing cells, including IM-9 lymphoblastoid cells and ANBL-6 interleukin-(IL)-6-dependent and RPMI-8226 IL-6-independent multiple myeloma cells, while relatively sparing XYZ-proteasome-expressing cell lines. Bortezomib, in contrast, induced apoptosis indiscriminately in both LMP- and XYZ-proteasome expressing cells. Taken together, these studies suggest that immunoproteasome specific inhibitors represent a novel class of drugs with activity against hematologic malignancies that may have less toxicity by virtue of their ability to spare the proteasome in most other tissues in the body, including in neural tissues. These properties could lead to an improved therapeutic index and anti-tumor efficacy, providing a rationale for development of these agents into clinically relevant drug candidates.

The Chymase, Mouse Mast Cell Protease 4, Constitutes the Major Chymotrypsin-like Activity in Peritoneum and Ear Tissue. A Role for Mouse Mast Cell Protease 4 in Thrombin Regulation and Fibronectin Turnover

To gain insight into the biological role of mast cell chymase we have generated a mouse strain with a targeted deletion in the gene for mast cell protease 4 (mMCP-4), the mouse chymase that has the closest relationship to the human chymase in terms of tissue localization and functional properties. The inactivation of mMCP-4 did not affect the storage of other mast cell proteases and did not affect the number of mast cells or the mast cell morphology. However, mMCP-4 inactivation resulted in complete loss of chymotryptic activity in the peritoneum and in ear tissue, indicating that mMCP-4 is the main source of stored chymotrypsin-like protease activity at these sites. The mMCP-4 null cells showed markedly impaired ability to perform inactivating cleavages of thrombin, indicating a role for mMCP-4 in regulating the extravascular coagulation system. Further, a role for mMCP-4 in connective tissue remodeling was suggested by the inability of mMCP-4 null peritoneal cells to process endogenous fibronectin.

Molecular cloning and functional analysis of three subunits of yeast proteasome.

The genes encoding three subunits of Saccharomyces cerevisiae proteasome were cloned and sequenced. The deduced amino acid sequences were homologous not only to each other (30 to 40% identity) but also to those of rat and Drosophila proteasomes (25 to 65% identity). However, none of these sequences showed any similarity to any other known sequences, including various proteases, suggesting that these proteasome subunits may constitute a unique gene family. Gene disruption analyses revealed that two of the three subunits (subunits Y7 and Y8) are essential for growth, indicating that the proteasome and its individual subunits play an indispensable role in fundamental biological processes. On the other hand, subunit Y13 is not essential; haploid cells with a disrupted Y13 gene can proliferate, although the doubling time is longer than that of cells with nondisrupted genes. In addition, biochemical analysis revealed that proteasome prepared from the Y13 disrupted cells contains tryptic and chymotryptic activities equivalent to those of nondisrupted cells, indicating that the Y13 subunit is not essential for tryptic or chymotryptic activity. However, the chymotryptic activity of the Y13 disrupted cells is not dependent on sodium dodecyl sulfate (SDS), an activator of proteasome, since nearly full activity was observed in the absence of SDS. Thus, the activity in proteasome of the Y13 disrupted cells might result in unregulated intracellular proteolysis, thus leading to the prolonged cell cycle. These results indicate that cloned proteasome subunits having similar sequences to the yeast Y13 subunit are structural, but not catalytic, components of proteasome. It is also suggested that two subunits (Y7 and Y8) might occupy positions essential to proteasome structure or activity, whereas subunit Y13 is in a nonessential but important position.