Association of annexin V with mitochondria

AbstractAnnexin V is an intracellular protein recently shown to be localized to nucleoli and cytosol. In this study we show that cytosolic annexin V is associated with mitochondria. To assess the nature of the annexin V-mitochondrial interaction, an annexin V binding activity was partially purified from placental cytosol by annexin V-affinity chromatography. Five polypeptides in the eluate appeared to be associated with annexin V, with a predominant species of 27 kDa. Antibodies to the 27 kDa polypeptide recognised mitochondria but not nucleoli. We conclude that annexin V interacts with a 27 kDa nutochondrial polypeptide that is possibly part of a larger complex

The major human and mouse granzymes are structurally and functionally divergent

Approximately 2% of mammalian genes encode proteases. Comparative genomics reveals that those involved in immunity and reproduction show the most interspecies diversity and evidence of positive selection during evolution. This is particularly true of granzymes, the cytotoxic proteases of natural killer cells and CD8+ T cells. There are 5 granzyme genes in humans and 10 in mice, and it is suggested that granzymes evolve to meet species-specific immune challenge through gene duplication and more subtle alterations to substrate specificity. We show that mouse and human granzyme B have distinct structural and functional characteristics. Specifically, mouse granzyme B is 30 times less cytotoxic than human granzyme B and does not require Bid for killing but regains cytotoxicity on engineering of its active site cleft. We also show that mouse granzyme A is considerably more cytotoxic than human granzyme A. These results demonstrate that even “orthologous” granzymes have species-specific functions, having evolved in distinct environments that pose different challenges

Cationic Sites on Granzyme B Contribute to Cytotoxicity by Promoting Its Uptake into Target Cells

Granzyme B (GrB) is a key effector of cytotoxic lymphocyte-mediated cell death. It is delivered to target cells bound to the proteoglycan serglycin, but how it crosses the plasma membrane and accesses substrates in the cytoplasm is poorly understood. Here we identify two cationic sequences on GrB that facilitate its binding and uptake. Mutation of cationic sequence 1 (cs1) prevents accumulation of GrB in a distinctive intracellular compartment and reduces cytotoxicity 20-fold. Mutation of cs2 reduces accumulation in this intracellular compartment and cytotoxicity two- to threefold. We also show that GrB-mediated cytotoxicity is abrogated by heparin and that target cells deficient in cell surface sulfate or glycosaminoglycans resist GrB. However, heparin does not completely prevent GrB internalization and chondroitin 4-sulfate does not inhibit cytotoxicity, suggesting that glycosaminoglycans are not essential GrB receptors. We propose that GrB enters cells by nonselective adsorptive pinocytosis, exchanging from chondroitin sulfate on serglycin to anionic components of the cell surface. In this electrostatic “exchange-adsorption” model, cs1 and cs2 participate in binding of GrB to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm

A Novel Serpin Regulatory Mechanism

exterior, yard gate, July-Sept. 198