Augmenting neurotransmitter release by enhancing the apparent Ca2+ affinity of synaptotagmin 1.

Synaptotagmin 1 likely acts as a Ca2+ sensor in neurotransmitter release by Ca2+-binding to its two C2 domains. This notion was strongly supported by the observation that a mutation in the C2A domain causes parallel decreases in the apparent Ca2+ affinity of synaptotagmin 1 and in the Ca2+ sensitivity of release. However, this study was based on a single loss-of-function mutation. We now show that tryptophan substitutions in the synaptotagmin 1 C2 domains act as gain-of-function mutations to increase the apparent Ca2+ affinity of synaptotagmin 1. The same substitutions, when introduced into synaptotagmin 1 expressed in neurons, enhance the Ca2+ sensitivity of release. Mutations in the two C2 domains lead to comparable and additive effects in release. Our results thus show that the apparent Ca2+ sensitivity of release is dictated by the apparent Ca2+ affinity of synaptotagmin 1 in both directions, and that Ca2+ binding to both C2 domains contributes to Ca2+ triggering of release.

Arfophilin is a common target of both class II and class III ADP-ribosylation factors.

Arfophilin was first identified as a target protein for GTP-ARF5. The N-terminus of ARF5 (amino acids 2-17), which is distinct from that of class I or class III ARFs, is essential for binding to the C-terminus of arfophilin (amino acids 612-756). This study using GST fusion proteins in pulldown experiments in CHO-K1 cell lysates showed that, unexpectedly, ARF6 also bound to full-length arfophilin or the C-terminus of arfophilin (amino acids 612-756) in a GTP-dependent manner. Studies with ARF1/ARF6 chimeras further showed that the amino acid sequence of residues 37-80 of ARF6, which is different from the corresponding sequences in class I and class II ARFs, was essential for binding to arfophilin. Both GTP-ARF5 and GTP-ARF6 bound to arfophilin in CHO-K1 cell lysates, while GTP-ARF1 did not bind. In contrast, all three forms of ARF bound to arfaptin 2, with ARF1 showing the strongest binding. Yeast two-hybrid studies with wild-type, dominant negative, and constitutively active forms of ARF1, -5, and -6 and with ARF1/ARF6 chimeras confirmed these results, except that constitutively active ARF6 was autoactivating. Our findings suggest that both class II and III ARFs may influence the same cellular pathways through arfophilin as a common downstream effector.

Differential binding of arfaptin 2/POR1 to ADP-ribosylation factors and Rac1.

We first identified arfaptin as a protein that bound to GTP-ARFs (especially ARF1). However, a second group reported that POR1, a truncated form of arfaptin, bound to GTP-Rac1. Therefore, we examined the possibility that arfaptin 2/POR1 was a common downstream effector for both ARF1 and Rac1. In this study, we found that constitutively active Rac1 or GTP-Rac1 showed negligible or no binding to arfaptin 2/POR1 in a yeast two-hybrid assay or a GST pull-down assay. However, wild-type or dominant negative Rac1 or Rac1 liganded to GDP showed strong binding. In contrast, constitutively active ARFs1, 5, and 6 showed binding, whereas the wild-type and dominant negative forms did not. Furthermore, the GTP-liganded ARFs bound arfaptin 2, whereas the GDP-bound forms showed little or no binding. Based on these observations, we suggest that arfaptin 2/POR1 is a target protein for GTP-ARFs and for GDP-Rac1, and that it may be involved in interactions between the Rac and ARF signaling pathways.

Identification of arfophilin, a target protein for GTP-bound class II ADP-ribosylation factors.

Yeast two-hybrid screening of a human kidney cDNA library using the GTP-bound form of a class II ADP-ribosylation factor (ARF5) identified a novel ARF5-binding protein with a calculated molecular mass of 82.4 kDa, which was named arfophilin. Northern hybridization analysis showed high level arfophilin mRNA expression in human heart and skeletal muscle. Arfophilin bound only to the active, GTP-bound form of ARF5 and did not bind to GTP-ARF3, which is a class I ARF. The N terminus of ARF5 (1-17 amino acids) was essential for binding to arfophilin. The GTP-bound form of ARF5 with amino acid residues in the N terminus mutated to those in ARF4 (another class II ARF) also bound to arfophilin, suggesting it is a target protein for GTP-bound forms of class II ARFs. The binding site for ARF on arfophilin was localized to the C terminus (residues 612-756), which contains putative coiled-coil structures. Recombinant arfophilin overexpressed in CHO-K1 cells was localized in the cytosol and translocated to a membrane fraction in association with GTP-bound ARF5. ARF5 containing the N terminus of ARF3 did not promote translocation indicating that class II ARFs are specific carriers for arfophilin.

Hepatic protein kinase C is not activated despite high intracellular 1,2-sn-diacylglycerol in obese Zucker rats.

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein X min(-1) vs. 780 pmol/mg protein X min(-1) in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.

Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes.

The mechanisms which drive initiated cells to progress to form carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 microM choline) as compared with controls (1% apoptotic at 48 h in 70 microM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFbeta1 and reactive oxygen species, since immunoneutralization of TGFbeta1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 microM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 microM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFbeta1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFbeta1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency.

Choline-deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline-deficiency are mediated by methyl-deficiency or by lack of choline moieties. SV40 immortalized CWSV-1 hepatocytes were cultivated in media that were choline-sufficient, choline-deficient, choline-deficient with methyl-donors (betaine or methionine), or choline-deficient with extra folate/vitamin B12. Choline-deficient CWSV-1 hepatocytes were not methyl-deficient as they had increased intracellular S-adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanol-amine, choline-deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl-supplementation in choline-deficiency enhanced intracellular methyl-group availability, but did not correct choline-deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl-supplemented, choline-deficient cells died by apoptosis. In a rat study, 2 weeks of a choline deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline-deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes.

Diet, apoptosis, and carcinogenesis.

It is known that long-term withdrawal of choline from the diet induces hepatocellular carcinomas in animal models in the absence of known carcinogens. We hypothesize that a choline deficient diet (CD) alters the balance of cell growth and cell death in hepatocytes and thus promotes the survival of clones of cells capable of malignant transformation. When grown in CD medium (5 microM or 0 microM choline) CWSV-1 rat hepatocytes immortalized with SV40 large T-antigen underwent p53-independent apoptosis (terminal dUTP end-labeling of fragmented DNA; laddering of DNA in agarose gel). CWSV-1 cells which were adapted to survive in 5 microM choline acquired resistance to CD-induced apoptosis and were able to form hepatocellular carcinomas in nude mice. These adapted CWSV-1 cells express higher amounts of both the 32 kDa membrane-bound and 6 kDa mature form of TGF alpha compared to cells made acutely CD. Control (70 microM choline) and adapted cells, but not acutely deficient hepatocytes, could be induced to undergo apoptosis by neutralization of secreted TGF alpha. Protein tyrosine phosphorylation is known to protect against apoptosis. We found decreased EGF receptor tyrosine phosphorylation in acutely choline deficient CWSV-1 cells. TGF beta 1 is an important growth-regulator in the liver. CWSV-1 cells express TGF beta 1 receptors and this peptide induced cell detachment and death in control and acutely deficient cells. Hepatocytes adapted to survive in low choline were also resistant to TGF beta 1, although TGF beta 1 receptors and protein could be detected in the cytoplasm of these cells. The non-essential nutrient choline is important in maintaining plasma membrane structure and function, and in intracellular signaling. Our results indicate that acute withdrawal of choline induces p53-independent programmed cell death in hepatocytes, whereas cells adapted to survive in low choline are resistant to this form of apoptosis, as well as to cell death induced by TGF beta 1. Our results also suggest that CD may induce alterations (mutations?) in growth factor signaling pathways which may enhance cell survival and malignant transformation.

Choline and hepatocarcinogenesis in the rat.

Rats fed a choline deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. This is the only nutritional deficiency that, in itself, causes cancer. We suggested that carcinogenesis is triggered, in part, because of abnormalities in cell signals which regulate cell proliferation and cell death. Because choline deficient rats develop fatty liver (choline is needed for hepatic secretion of certain lipoproteins), we examined whether an important lipid second messenger involved in proliferative signaling, 1,2-sn-diacylglycerol, accumulated in liver and resulted in the prolonged activation of protein kinase C. We observed that 1,2-sn-diacylglycerol accumulated in the plasma membrane from the non-tumor portion of livers of rats fed a choline deficient diet, and that unsaturated free fatty acids, another activator of protein kinase C, also accumulated in deficient livers. Protein kinase C in the hepatic plasma membrane and nucleus of choline deficient rats was elevated for months; this is the only model system which exhibits such prolonged activation of protein kinase C. Premalignant, abnormal hepatic foci were detected only in the deficient rats, and 15% of deficient rats (none of the controls) had hepatocellular carcinoma at 1 year on the diet. In rats, an early event in choline deficiency is an increase in the rate of cell death. In liver from choline deficient rats, we observed an increase in the numbers of liver cells with fragmented DNA (characteristic of programmed cell death; apoptosis). We used a cell culture model (immortalized rat hepatocytes) to study the effects of choline deficiency on apoptosis. Liver cells grown in a choline deficient medium became depleted of choline, accumulated triacylglycerol and 1,2-sn-diacylglycerol, and had increased DNA fragmentation and other morphologic and biochemical changes associated with apoptosis. This model has great potential as a tool for studying the underlying link between choline deficiency and the regulation of the balance between cell proliferation and cell death. We suggest that choline deficiency altered the cell proliferation signals mediated by protein kinase C within liver, and altered cell apoptosis. These changes in cell signaling may be the triggering events which result in hepatic carcinogenesis.