Caspases have been strongly implicated to play an essential role in apoptosis. A critical question regarding the role(s) of these proteases is whether selective inhibition of an effector caspase(s) will prevent cell death. We have identified potent and selective non-peptide inhibitors of the effector caspases 3 and 7. The inhibition of apoptosis and maintenance of cell functionality with a caspase 3/7-selective inhibitor is demonstrated for the first time, and suggests that targeting these two caspases alone is sufficient for blocking apoptosis. Furthermore, an x-ray co-crystal structure of the complex between recombinant human caspase 3 and an isatin sulfonamide inhibitor has been solved to 2.8-Å resolution. In contrast to previously reported peptide-based caspase inhibitors, the isatin sulfonamides derive their selectivity for caspases 3 and 7 by interacting primarily with the S 2 subsite, and do not bind in the caspase primary aspartic acid binding pocket (S 1 ). These inhibitors blocked apoptosis in murine bone marrow neutrophils and human chondrocytes. Furthermore, in camptothecin-induced chondrocyte apoptosis, cell functionality as measured by type II collagen promoter activity is maintained, an activity considered essential for cartilage homeostasis. These data suggest that inhibiting chondrocyte cell death with a caspase 3/7-selective inhibitor may provide a novel therapeutic approach for the prevention and treatment of osteoarthritis, or other disease states characterized by excessive apoptosis.

Phosphatidylinositol (PI), once regarded as an obscure component of membranes, is now recognized as an important reservoir of second messenger precursors and as an anchor for membrane enzymes. PI‐specific phospholipase C (PI‐PLC) is the enzyme that cleaves PI, invoking numerous cellular responses. The crystal structure of PI‐PLC from Bacillus cereus (EC 3.1.4.10) has been solved at 2.6 A resolution and refined to a crystallographic R factor of 18.7%. The structure consists of an imperfect (beta alpha)8‐barrel similar to that first observed for triose phosphate isomerase and does not resemble any other known phospholipase structure. The active site of the enzyme has been identified by determining the structure of PI‐PLC in complex with its inhibitor, myo‐inositol, at 2.6 A resolution (R factor = 19.5%). This substrate‐like inhibitor interacts with a number of residues highly conserved among prokaryotic PI‐PLCs. Residues His32 and His82, which are also conserved between prokaryotic and eukaryotic PI‐PLCs, most likely act as general base and acid respectively in a catalytic mechanism analogous to that observed for ribonucleases.

The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed ∼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.

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The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V(1) sector catalyzes ATP hydrolysis and the V(0) sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V(0) assembly. We have discovered a fifth V(0) assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V(0) assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V(0) assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V(0)-Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V(0) subunits c, c', and c''. On assembly of the remaining three V(0) subunits (a, d, and e) into the V(0) complex, Voa1p dissociates from the now fully assembled V(0)-Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V(0) assembly in the ER, but then it dissociates before exit of the V(0)-Vma21p complex from the ER for transport to the Golgi compartment.