After dilution with 3 volumes of distilled water, mitochondria were pelleted by centrifugation at 9,600 g at 4C for 15 min

After dilution with 3 volumes of distilled water, mitochondria were pelleted by centrifugation at 9,600 g at 4C for 15 min. in apoptosis was LY573636 (Tasisulam) first suggested by a study showing that ALG-3, a mouse homolog of the C-terminal fragment of PS2, rescues a T-cell hybridoma from Fas-induced apoptosis [8]. Interestingly, as in the case of PS2, the C-terminal fragment of PS1 was also found to inhibit Fas-induced apoptosis in Jurkat cells [9]. In an effort to understand the apoptotic regulatory effect of the C-terminal of PS1, we have identified a novel presenilin-associated protein (PSAP), which interacts with the C-terminal of PS1 [10]. Interestingly, in a subsequent study we found that PSAP is a pro-apoptotic protein that causes apoptosis when it is overexpressed [11]. This finding provides a direct molecular LY573636 (Tasisulam) link between presenilin and the apoptotic pathway and also places PSAP in an important position in apoptosis. Two major pathways lead to apoptotic cell death: namely, the cell surface death receptor-mediated pathway and the mitochondria-mediated pathway [12]. In the death receptor-mediated pathway, extracellular stimuli activate apoptotic cascades via binding and activation of cell surface death receptors. On the other hand, in the mitochondria-mediated pathway, apoptotic cascades are activated by small molecules, such as cytochrome c and apoptosis-inducing factor (AIF), released from the mitochondrial intermembrane space into the cytoplasm. In this Rabbit Polyclonal to MPHOSPH9 regard, it is notable that PSAP is localized in mitochondria and that overexpression of PSAP causes release of cytochrome c from mitochondria [11], suggesting that PSAP causes apoptosis through a mitochondria-mediated pathway. Since the report of the identification of PSAP, several groups have reported the cDNAs for PSAP from different species by directly depositing the DNA sequence information into the GenBank. Now the PSAP gene is also known as mitochondrial carrier homolog 1 (Mtch1). These DNA sequences revealed an important fact that some of the reported cDNAs are missing 51 nucleotides in the 3-half of the coding region, suggesting the presence of different splicing isoforms of mRNA for PSAP. This information prompted us to determine the structures of the gene and protein of PSAP and their relationship with the biological function of PSAP. In the current work, we report that PSAP is expressed in two isoforms as a result of alternative splicing and that both isoforms are apoptotic active. We sought to discover whether these potential isoforms are indeed expressed under physiological conditions and whether these isoforms may have different biological functions. In addition, we sought to determine the relationship between the structure and function of PSAP. 2. Materials and methods 2.1. Cell culture and transfection HEK293 cells were cultured in Dulbeccos modified Eagles medium (DMEM) supplemented LY573636 (Tasisulam) with 10% fetal bovine serum, 50 units/ml penicillin, and 50 g/ml streptomycin. Transfection was performed using LipofectAMINE 2000 transfection reagent (Invitrogen). PSAP-myc fusion protein expression plasmid was constructed as described previously [11]. All deletion and truncation mutants were constructed using the site-directed mutagenesis kit (Stratagene). 2.2. Mitochondria isolation and proteinase treatment Mitochondria were isolated as described previously [11]. Briefly, LY573636 (Tasisulam) HEK 293 cells were harvested and washed with phosphate buffered saline once and swelled with 10 volumes of Swell buffer A (20 mM Hepes, pH7.9, 1.5mM NaCl, 10mM KCl, 0.5 mM -mercaptoethanol) for 5 min on ice. After 500g centrifugation at 4C for 5 min, the cells were resuspended in 10 volumes of Swell buffer A and ruptured with 5 or 6 strokes with a Dounce Homogenizer using a tight-fitting pestle (pestle.