Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects

Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes. Analyses of such ‘bypass Sec14p’ mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function. SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD). PLD function, while essential for meiosis, is dispensable for vegetative growth. Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed. This PLD involvement is essential in ‘bypass Sec14p’ mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner. PLD catalytic activity is necessary but not sufficient for ‘bypass Sec14p’, and yeast operating under ‘bypass Sec14p’ conditions are ethanol-sensitive. These data suggest that PLD supports ‘bypass Sec14p’ by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.

d-β-Hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease

The heroin analogue 1-methyl-4-phenylpyridinium, MPP+, both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Aβ1–42, is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-β-hydroxybutyrate protected cultured mesencephalic neurons from MPP+ toxicity and hippocampal neurons from Aβ1–42 toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

Homocysteine induces congenital defects of the heart and neural tube: Effect of folic acid

The biological basis or mechanism whereby folate supplementation protects against heart and neural tube defects is unknown. It has been hypothesized that the amino acid homocysteine may be the teratogenic agent, since serum homocysteine increases in folate depletion; however, this hypothesis has not been tested. In this study, avian embryos were treated directly with d,l-homocysteine or with l-homocysteine thiolactone, and a dose response was established. Of embryos treated with 50 μl of the teratogenic dose (200 mM d,l-homocysteine or 100 mM l-homocysteine thiolactone) on incubation days 0, 1, and 2 and harvested at 53 h (stage 14), 27% showed neural tube defects. To determine the effect of the teratogenic dose on the process of heart septation, embryos were treated during incubation days 2, 3, and 4; then they were harvested at day 9 following the completion of septation. Of surviving embryos, 23% showed ventricular septal defects, and 11% showed neural tube defects. A high percentage of the day 9 embryos also showed a ventral closure defect. The teratogenic dose was shown to raise serum homocysteine to over 150 nmol/ml, compared with a normal level of about 10 nmol/ml. Folate supplementation kept the rise in serum homocysteine to ≈45 nmol/ml, and prevented the teratogenic effect. These results support the hypothesis that homocysteine per se causes dysmorphogenesis of the heart and neural tube, as well as of the ventral wall.

Mutant LexA proteins with specific defects in autodigestion.

In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure. Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule. In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein. RecA acts indirectly as a coprotease to stimulate LexA autodigestion. We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity. Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage. The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage. To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction. Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.

Mutations in the small subunit of the Drosophila U2AF splicing factor cause lethality and developmental defects.

The essential eukaryotic pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein auxiliary factor) is required to specify the 3' splice at an early step in spliceosome assembly. U2AF binds site-specifically to the intron polypyrimidine tract and recruits U2 small nuclear ribonucleoprotein to the branch site. Human U2AF (hU2AF) is a heterodimer composed of a large (hU2AF65) and small (hU2AF35) subunit. Although these proteins associate in a tight complex, the biochemical requirement for U2AF activity can be satisfied solely by the large subunit. The requirement for the small subunit in splicing has remained enigmatic. No biochemical activity has been found for hU2AF35 and it has been implicated in splicing only indirectly by its interaction with known splicing factors. In the absence of a biochemical assay, we have taken a genetic approach to investigate the function of the small subunit in the fruit fly Drosophila melanogaster. A cDNA clone encoding the small subunit of Drosophila U2AF (dU2AF38) has been isolated and sequenced. The dU2AF38 protein is highly homologous to hU2AF35 containing a conserved central arginine- and serine-rich (RS) domain. A recessive P-element insertion mutation affecting dU2AF38 causes a reduction in viability and fertility and morphological bristle defects. Consistent with a general role in splicing, a null allele of dU2AF38 is fully penetrant recessive lethal, like null alleles of the Drosophila U2AF large subunit.

Human MutSalpha recognizes damaged DNA base pairs containing O6-methylguanine, O4-methylthymine, or the cisplatin-d(GpG) adduct.

Bacterial and mammalian mismatch repair systems have been implicated in the cellular response to certain types of DNA damage, and genetic defects in this pathway are known to confer resistance to the cytotoxic effects of DNA-methylating agents. Such observations suggest that in addition to their ability to recognize DNA base-pairing errors, members of the MutS family may also respond to genetic lesions produced by DNA damage. We show that the human mismatch recognition activity MutSalpha recognizes several types of DNA lesion including the 1,2-intrastrand d(GpG) crosslink produced by cis-diamminedichloroplatinum(II), as well as base pairs between O6-methylguanine and thymine or cytosine, or between O4-methylthymine and adenine. However, the protein fails to recognize 1,3-intrastrand adduct produced by trans-diamminedichloroplatinum(II) at a d(GpTpG) sequence. These observations imply direct involvement of the mismatch repair system in the cytotoxic effects of DNA-methylating agents and suggest that recognition of 1,2-intrastrand cis-diamminedichloroplatinum(II) adducts by MutSalpha may be involved in the cytotoxic action of this chemotherapeutic agent.

Rel-deficient T cells exhibit defects in production of interleukin 3 and granulocyte-macrophage colony-stimulating factor.

The c-rel protooncogene encodes a subunit of the NF-kappa B-like family of transcription factors. Mice lacking Rel are defective in mitogenic activation of B and T lymphocytes and display impaired humoral immunity. In an attempt to identify changes in gene expression that accompany the T-cell stimulation defects associated with the loss of Rel, we have examined the expression of cell surface activation markers and cytokine production in mitogen-stimulated Rel-/- T cells. The expression of cell surface markers including the interleukin 2 receptor alpha (IL-2R alpha) chain (CD25), CD69 and L-selectin (CD62) is normal in mitogen-activated Rel-/- T cells, but cytokine production is impaired. In Rel-/- splenic T cell cultures stimulated with phorbol 12-myristate 13-acetate and ionomycin, the levels of IL-3, IL-5, granulocyte- macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) were only 2- to 3-fold lower compared with normal T cells. In contrast, anti-CD3 and anti-CD28 stimulated Rel-/- T cells, which fail to proliferate, make little or no detectable cytokines. Exogenous IL-2, which restitutes the proliferative response of the anti-CD3- and anti-CD28-treated Rel-/- T cells, restores production of IL-5, TNF-alpha, and IFN-gamma, but not IL-3 and GM-CSF expression to approximately normal levels. In contrast to mitogen-activated Rel-/- T cells, lipopolysaccharide-stimulated Rel-/- macrophages produce higher than normal levels of GM-CSF. These findings establish that Rel can function as an activator or repressor of gene expression and is required by T lymphocytes for production of IL-3 and GM-CSF.

Fialuridine and its metabolites inhibit DNA polymerase gamma at sites of multiple adjacent analog incorporation, decrease mtDNA abundance, and cause mitochondrial structural defects in cultured hepatoblasts.

The thymidine analog fialuridine deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (FIAU) was toxic in trials for chronic hepatitis B infection. One mechanism postulated that defective mtDNA replication was mediated through inhibition of DNA polymerase-gamma (DNA pol-gamma), by FIAU triphosphate (FIALTP) or by triphosphates of FIAU metabolites. Inhibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl) -5-methyluracil (FAU), 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil triphosphate (TP) inhibition of DNA pol-gamma. dTMP incorporation by DNA pol-gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K1=0.015, 0.03, and 1.0 microM, respectively). By using oliginucleotide template-primers. DNA pol-gamma incorporated each analog into DNA opposite a single adenosine efficiently without effects on DNA chain elongation. Incorporation of multiple adjacent analogs at positions of consecutive adenosines dramatically impaired chain elongation by DNA pol-gamma. Effects of FIAU, FMAU, and FAU on HepG2 cell mmtDNA abundance and ultrastructure were determined. After 14 days, mtDNA decreased by 30% with 20 microM FIAU or 20 microM FMAU and decreased less than 10% with 100 microM FAU. FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets. Biochemical and cell biological findings suggest that FIAU and its metabolites inhibit mtDNA replication, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.

Neural tube defects and abnormal brain development in F52-deficient mice.

F52 is a myristoylated, alanine-rich substrate for protein kinase C. We have generated F52-deficient mice by the gene targeting technique. These mutant mice manifest severe neural tube defects that are not associated with other complex malformations, a phenotype reminiscent of common human neural tube defects. The neural tube defects observed include both exencephaly and spina bifida, and the phenotype exhibits partial penetrance with about 60% of homozygous embryos developing neural tube defects. Exencephaly is the prominent type of defect and leads to high prenatal lethality. Neural tube defects are observed in a smaller percentage of heterozygous embryos (about 10%). Abnormal brain development and tail formation occur in homozygous mutants and are likely to be secondary to the neural tube defects. Disruption of F52 in mice therefore identifies a gene whose mutation results in isolated neural tube defects and may provide an animal model for common human neural tube defects.

Equine severe combined immunodeficiency: a defect in V(D)J recombination and DNA-dependent protein kinase activity.

V(D)J rearrangement is the molecular mechanism by which an almost infinite array of specific immune receptors are generated. Defects in this process result in profound immunodeficiency as is the case in the C.B-17 SCID mouse or in RAG-1 (recombination-activating gene 1) or RAG-2 deficient mice. It has recently become clear that the V(D)J recombinase most likely consists of both lymphoid-specific factors and ubiquitously expressed components of the DNA double-strand break repair pathway. The deficit in SCID mice is in a factor that is required for both of these pathways. In this report, we show that the factor defective in the autosomal recessive severe combined immunodeficiency of Arabian foals is required for (i) V(D)J recombination, (ii) resistance to ionizing radiation, and (iii) DNA-dependent protein kinase activity.

The product of the ataxia-telangiectasia group D complementing gene, ATDC, interacts with a protein kinase C substrate and inhibitor.

Ataxia-telangiectasia (AT) is an autosomal recessive human genetic disease characterized by immunological, neurological, and developmental defects and an increased risk of cancer. Cells from individuals with AT show sensitivity to ionizing radiation, elevated recombination, cell cycle abnormalities, and aberrant cytoskeletal organization. The molecular basis of the defect is unknown. A candidate AT gene (ATDC) was isolated on the basis of its ability to complement the ionizing radiation sensitivity of AT group D fibroblasts. Whether ATDC is mutated in any AT patients is not known. We have found that the ATDC protein physically interacts with the intermediate-filament protein vimentin, which is a protein kinase C substrate and colocalizing protein, and with an inhibitor of protein kinase C, hPKCI-1. Indirect immunofluorescence analysis of cultured cells transfected with a plasmid encoding an epitope-tagged ATDC protein localizes the protein to vimentin filaments. We suggest that the ATDC and hPKCI-1 proteins may be components of a signal transduction pathway that is induced by ionizing radiation and mediated by protein kinase C.