Human fatty acid synthase: properties and molecular cloning.

Fatty acid synthase (FAS; EC 2.3.1.85) was purified to near homogeneity from a human hepatoma cell line, HepG2. The HepG2 FAS has a specific activity of 600 nmol of NADPH oxidized per min per mg, which is about half that of chicken liver FAS. All the partial activities of human FAS are comparable to those of other animal FASs, except for the beta-ketoacyl synthase, whose significantly lower activity is attributable to the low 4'-phosphopantetheine content of HepG2 FAS. We cloned the human brain FAS cDNA. The cDNA sequence has an open reading frame of 7512 bp that encodes 2504 amino acids (M(r), 272,516). The amino acid sequence of the human FAS has 79% and 63% identity, respectively, with the sequences of the rat and chicken enzymes. Northern analysis revealed that human FAS mRNA was about 9.3 kb in size and that its level varied among human tissues, with brain, lung, and liver tissues showing prominent expression. The nucleotide sequence of a segment of the HepG2 FAS cDNA (bases 2327-3964) was identical to that of the cDNA from normal human liver and brain tissues, except for a 53-bp sequence (bases 3892-3944) that does not alter the reading frame. This altered sequence is also present in HepG2 genomic DNA. The origin and significance of this sequence variance in the HepG2 FAS gene are unclear, but the variance apparently does not contribute to the lower activity of HepG2 FAS.

Amino-terminal alteration of the HLA-A*0201-restricted human immunodeficiency virus pol peptide increases complex stability and in vitro immunogenicity.

Initial studies suggested that major histocompatibility complex class I-restricted viral epitopes could be predicted by the presence of particular residues termed anchors. However, recent studies showed that nonanchor positions of the epitopes are also significant for class I binding and recognition by cytotoxic T lymphocytes (CTLs). We investigated if changing nonanchor amino acids could increase class I affinity, complex stability, and T-cell recognition of a natural viral epitope. This concept was tested by using the HLA-A 0201-restricted human immunodeficiency virus type 1 epitope from reverse transcriptase (pol). Position 1 (P1) amino acid substitutions were emphasized because P1 alterations may not alter the T-cell receptor interaction. The peptide with the P1 substitution of tyrosine for isoleucine (I1Y) showed a binding affinity for HLA-A 0201 similar to that of the wild-type pol peptide in a cell lysate assembly assay. Surprisingly, I1Y significantly increased the HLA-A 0201-peptide complex stability at the cell surface. I1Y sensitized HLA-A 0201-expressing target cells for wild-type pol-specific CTL lysis as well as wild-type pol. Peripheral blood lymphocytes from three HLA-A2 HIV-seropositive individuals were stimulated in vitro with I1Y and wild-type pol. I1Y stimulated a higher wild-type pol-specific CTL response than wild-type pol in all three donors. Thus, I1Y may be an "improved" epitope for use as a CTL-based human immunodeficiency virus vaccine component. The design of improved epitopes has important ramifications for prophylaxis and therapeutic vaccine development.

Free amino acids exhibit anthozoan "host factor" activity: they induce the release of photosynthate from symbiotic dinoflagellates in vitro.

Reef-building corals and other tropical anthozoans harbor endosymbiotic dinoflagellates. It is now recognized that the dinoflagellates are fundamental to the biology of their hosts, and their carbon and nitrogen metabolisms are linked in important ways. Unlike free living species, growth of symbiotic dinoflagellates is unbalanced and a substantial fraction of the carbon fixed daily by symbiont photosynthesis is released and used by the host for respiration and growth. Release of fixed carbon as low molecular weight compounds by freshly isolated symbiotic dinoflagellates is evoked by a factor (i.e., a chemical agent) present in a homogenate of host tissue. We have identified this "host factor" in the Hawaiian coral Pocillopora damicornis as a set of free amino acids. Synthetic amino acid mixtures, based on the measured free amino acid pools of P. damicornis tissues, not only elicit the selective release of 14C-labeled photosynthetic products from isolated symbiotic dinoflagellates but also enhance total 14CO2 fixation.

Identification of four acidic amino acids that constitute the catalytic center of the RuvC Holliday junction resolvase.

Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.

Periodicity of polar and nonpolar amino acids is the major determinant of secondary structure in self-assembling oligomeric peptides.

The tendency of a polypeptide chain to form alpha-helical or beta-strand secondary structure depends upon local and nonlocal effects. Local effects reflect the intrinsic propensities of the amino acid residues for particular secondary structures, while nonlocal effects reflect the positioning of the individual residues in the context of the entire amino acid sequence. In particular, the periodicity of polar and nonpolar residues specifies whether a given sequence is consistent with amphiphilic alpha-helices or beta-strands. The importance of intrinsic propensities was compared to that of polar/nonpolar periodicity by a direct competition. Synthetic peptides were designed using residues with intrinsic propensities that favored one or the other type of secondary structure. The polar/nonpolar periodicities of the peptides were designed either to be consistent with the secondary structure favored by the intrinsic propensities of the component residues or in other cases to oppose these intrinsic propensities. Characterization of the synthetic peptides demonstrated that in all cases the observed secondary structure correlates with the periodicity of the peptide sequence--even when this secondary structure differs from that predicted from the intrinsic propensities of the component amino acids. The observed secondary structures are concentration dependent, indicating that oligomerization of the amphiphilic peptides is responsible for the observed secondary structures. Thus, for self-assembling oligomeric peptides, the polar/nonpolar periodicity can overwhelm the intrinsic propensities of the amino acid residues and serves as the major determinant of peptide secondary structure.

The carboxyl-terminal domain of the p53 protein regulates sequence-specific DNA binding through its nonspecific nucleic acid-binding activity.

The murine p53 protein contains two nucleic acid-binding sites, a sequence-specific DNA-binding region localized between amino acid residues 102-290 and a nucleic acid-binding site without sequence specificity that has been localized to residues 364-390. Alternative splicing of mRNA generates two forms of this p53 protein. The normal, or majority, splice form (NSp53) retains its carboxyl-terminal sequence-nonspecific nucleic acid-binding site, which can negatively regulate the sequence-specific DNA-binding site. The alternative splice form of p53 (ASp53) replaces amino acid residues 364-390 with 17 different amino acids. This protein fails to bind nucleic acids nonspecifically and is constitutive for sequence-specific DNA binding. Thus, the binding of nucleic acids at the carboxyl terminus regulates sequence-specific DNA binding by p53. The implications of these findings for the activation of p53 transcriptional activity following DNA damage are discussed.