Computational structure-activity study directs synthesis of novel antitumor enkephalin analogs.

The capability of a Support Vector Machines QSAR model to predict the antiproliferative ability of small peptides was evaluated by screening a virtual library of enkephalin-like analogs modified by incorporation of the (R,S)-(1-adamantyl)glycine (Aaa) residue. From an initial set of 390 compounds, the peptides, Tyr-Aaa-Gly-Phe-Met (2), Tyr-Aaa-Gly-Phe-Phe (3), Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5) were selected, synthesized and their antitumor activity was tested and compared to that of Met-enkephalin (1). The antiproliferative activity correlated with the computational prediction and with the foldamer-forming ability of the studied peptides. The most active compounds were the hydrophobic peptides, Phe-Aaa-Gly-Phe-Phe (4) and Phe-Aaa-Gly-Phe-Met (5), having a greater propensity to adopt folded structures than the other peptides.

Organellar H(+)-ATPase--site directed mutagenesis and suppressor mutants.

The oligomeric state of the proteolipid subunit of V-ATPase from Saccharomyces cerevisiae was studied using hemagglutinine (HA) epitope-tag. Like with several other highly hydrophobic proteins, the proteolipid tends to aggregate in the presence of sodium dodecyl sulfate (SDS). We observed that the oligomeric state of the proteolipid predetermined its tendency for aggregation. Recently we discovered a novel V-ATPase subunit, denoted as M16 for the mammalian enzyme and Vma10p for the yeast enzyme, that is homologous to the b subunit of the membrane sector of F-ATPases. It is assumed that the structure of Vma10p resembles that of subunit b which is basically two anti parallel helices. We mutated the VMA10 gene to change charges on the protein in helices and to introduce helix braking instead of helix forming amino acids. The functionality of the mutated VMA10 was analyzed by growing the transformed yeast cells on a YPD medium buffered at pH 7.5. Two inactive site-directed mutants we used for obtaining second-site suppressors. Mutagenesis with EMS was utilized to get an equal chance of obtaining intra and extragene second-site suppressors. To our surprise the number of colonies that grew at pH 7.5 was too large to account for mutations in V-ATPase subunits. Apparently, mutations that are situated in genes that do not encode V-ATPase subunits could reverse the phenotype of V-ATPase null mutations resulting in growth at pH 7.5. The large number of colonies that grew at pH 7.5 after EMS treatment suggest a big complex with multiple subunits as a target for mutagenesis. The observed phenomenon is very intriguing. If the responsible protein complex is identified, it may shed light on an important and novel cell biology subject.

Negative control of heavy metal uptake by the Saccharomyces cerevisiae BSD2 gene.

We have previously shown that mutations in the Saccharomyces cerevisiae BSD2 gene suppress oxidative damage in cells lacking superoxide dismutase and also lead to hyperaccumulation of copper ions. We demonstrate here that bsd2 mutant cells additionally accumulate high levels of cadmium and cobalt. By biochemical fractionation and immunofluorescence microscopy, BSD2 exhibited localization to the endoplasmic reticulum, suggesting that BSD2 acts at a distance to inhibit metal uptake from the growth medium. This BSD2 control of ion transport occurs independently of the CTR1 and FET4 metal transport systems. Genetic suppressor analysis revealed that hyperaccumulation of copper and cadmium in bsd2 mutants is mediated through SMF1, previously shown to encode a plasma membrane transporter for manganese. A nonsense mutation removing the carboxyl-terminal hydrophobic domain of SMF1 was found to mimic a smf1 gene deletion by eliminating the copper and cadmium toxicity of bsd2 mutants and also by precluding the bsd2 suppression of superoxide dismutase deficiency. However, inactivation of SMF1 did not eliminate the elevated cobalt levels in bsd2 mutants. Instead, this cobalt accumulation was found to be specifically mediated through the SMF1 homologue, SMF2. Hence, BSD2 prevents metal hyperaccumulation by exerting negative control over the SMF1 and SMF2 metal transport systems.

Function of metal-ion homeostasis in the cell division cycle, mitochondrial protein processing, sensitivity to mycobacterial infection and brain function.

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mmol l-1 EGTA, was isolated. The phenotype of the mutant is caused by a single amino acid change (Gly149 to Arg) in the essential yeast cell division cycle gene CDC1. The mutant could be suppressed by overexpression of the SMF1 gene, which codes for a plasma membrane Mn2+ transporter. We observed that the yeast SMF1 gene shares homology with the mouse Nramp gene. Nramp (Bcg) was cloned as a gene responsible for mouse resistance to infection with mycobacteria and is identical with the Ity and the Lsh genes conferring resistance to infection by Salmonella typhimurium and Leishmania donovani, respectively. Although the cloning of Nramp identified the gene responsible for the resistance of mice to mycobacteria, its function is unknown. We propose that the mammalian protein, like the yeast transporter, is a Mn2+ and/or Zn2+ transporter. Following the phagocytosis of a parasite into the phagosome, the macrophage produces reactive oxygen and/or nitrogen intermediates that are toxic for the internalized bacteria. The survival of the pathogen during the burst of macrophage respiratory activity is thought to be partly mediated by microbial superoxide dismutase (SOD), which contains Mn2+ or Fe2+ in its active centre. Nramp may transport Mn2+ from the extracellular milieu into the cytoplasm of a macrophage and, after the generation of the phagosome, remove Mn2+ from the organelle. Thus, the Mn(2+)-depletion of the phagosome microenvironment by the Nramp gene product may be a rate-limiting step in the metalloenzyme's production by the engulfed bacteria. This limitation will restrict the mycobacterial ability to produce active enzymes such as SOD and prevent the propagation of the ingested microorganisms. Conversely, an increased concentration of Mn2+ in the phagosome caused by a defective Nramp transporter (Bcgs) may promote the growth of the mycobacteria and render the organism sensitive to the pathogen. We use a similar approach to identify, clone and study other metal-ion transporters.

A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria.

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mM EGTA, was isolated by replica plating. The phenotype of the mutant is caused by a single amino acid change (Gly149 to Arg) in the essential yeast gene CDC1. The mutant could be suppressed by overexpression of the SMF1 gene, which was isolated as an extragenic high-copy suppressor. The SMF1 gene codes for a highly hydrophobic protein and its deletion renders the yeast cells sensitive to low manganese concentration. In accordance with this observation, the smf1 null mutant exhibits reduced Mn2+ uptake at micromolar concentrations. Using a specific antibody, we demonstrated that Smf1p is located in the yeast plasma membrane. These results suggest that Smf1p is involved in high-affinity Mn2+ uptake. This assumption was also tested by overexpressing the SMF1 gene in the temperature-sensitive mutant of the mitochondrial processing peptidase (MAS1). SMF1 overexpression as well as addition of 1 mM Mn2+ to the growth medium complemented this mutation. This also suggests that in vivo Mas1p is a manganese-dependent peptidase. The yeast Smf1p resembles a protein from Drosophila and mammalian macrophages. The latter was implicated in conferring resistance to mycobacteria. A connection between Mn2+ transport and resistance or sensitivity to mycobacteria is discussed.

A novel subunit of vacuolar H(+)-ATPase related to the b subunit of F-ATPases.

The subunit structure of the vacuolar H(+)-ATPase (V-ATPase) membrane sector is not entirely known. The proteolipid is the only subunit that has been implicated in the mechanism of energy transfer in the enzyme. We have identified a protein (M16) that co-purifies with the V-ATPase complex from bovine chromaffin granules. Information obtained from the amino acid sequence of a proteolytic fragment of M16 was used to clone a bovine adrenal cDNA encoding this protein. The cDNA encodes a hydrophilic protein of 118 amino acid residues with a calculated molecular mass of 13682Da. Amino acid sequence analysis revealed that M16 exhibits a significant homology to subunit b of F-ATPases. M16 is smaller than subunit b and contains no apparent transmembrane segment in its N terminus. The remainder of subunit b is related to M16 not only by its amino acid sequence but also in its predicted structure of helix-turn-helix. The structural and evolutionary implications of these findings are discussed.

The Saccharomyces cerevisiae VMA10 is an intron-containing gene encoding a novel 13-kDa subunit of vacuolar H(+)-ATPase.

The vacuolar H(+)-ATPase (V-ATPase) functions as a primary proton pump that generates an electrochemical gradient of protons across the membranes of several internal organelles. It is composed of distinct catalytic and membrane sectors, each containing several subunits. We identified a protein (M16) that copurifies with the V-ATPase complex from Saccharomyces cerevisiae and appears to be present at multiple copies/enzyme. Amino acid sequencing of its proteolytic products yielded three nonoverlapping peptide sequences matching an unidentified reading frame located on chromosome VIII. Sequence analysis of cDNA encoding M16 revealed that the gene encoding this protein (VMA10) is interrupted by a 162-nucleotide intron that begins after the ATG codon of the initiator methionine. The cDNA encodes an hydrophilic protein of 12,713 Da with a basic isoelectric point of pH 9. A delta vma10::URA3 null mutant exhibited growth characteristics typical of other vma disruptant mutants in genes encoding subunits of V-ATPase. The null mutant does not grow on medium buffered at pH 7.5. It fails to accumulate quinacrine into its vacuole, and subunits of the catalytic sector are not assembled onto the vacuolar membrane in the absence of M16. A cold inactivation experiment demonstrated that M16 is a subunit of the membrane sector of V-ATPase. M16 exhibits a significant sequence homology with subunit b of F-ATPase membrane sector.

Features of vacuolar H(+)-ATPase revealed by yeast suppressor mutants.

The yeast Saccharomyces cerevisiae serves as an excellent model for the study of the structure and function of proteins. Numerous amino acid substitutions in the proteolipid subunit of yeast vacuolar H(+)-ATPase have been reported. Suppressed variants for several of the inactive mutants were selected after subjecting them to chemical or polymerase chain reaction mutagenesis and screening for second site suppressors. Suppressors for the mutation Gln90 to Lys change were intragenic and resulted from the changes: Ala14 to Val, Val74 to Ile, Ile89 to Leu, and Ile89 to Met. These residues are found on three different transmembrane segments but presumably at the same surface of the membrane. A new inactive proteolipid mutation was constructed by changing Val138 to Leu. This residue is situated in the middle of the fourth transmembrane segment, neighboring Glu137 which is the potential dicyclohexylcarbodiimide-binding site. The intragenic suppressor mutations for the above amino acid replacement resulted in changes of Val55 to Ala, Met59 to Val, or Ile130 to Thr. These residues are found in the second and fourth transmembrane segments, presumably on the same interface. It seems as if all those internal suppressor mutations compensate for the volume changes caused by the original displacement of the given amino acid. Five glycine residues, situated on the same face of the third transmembrane helix, were changed to valine and all these mutants were inactive. A suppressor mutation to one of those mutants (Gly101 to Val) was identified as substitution of Ile134 to Val. The structural and functional implications of these findings are discussed.

A novel accessory subunit for vacuolar H(+)-ATPase from chromaffin granules.

Three subunits, Ac115, Ac39, and the proteolipid, were positively identified in the membrane sectors of V-ATPases from different sources. We searched for organelle-specific protein in purified preparations of V-ATPase from bovine chromaffin granules. A diffused protein band at a position of about 45 kDa was identified in SDS-polyacrylamide gels of the above preparation. Following digestion with endopeptidase Glu-C (V-8), a polypeptide of about 10 kDa was isolated and subjected to amino acid sequencing. Hence, the cDNA encoding the protein Ac45 was cloned from a bovine adrenal medulla library. The cDNA sequence contains an open reading frame encoding a protein of 468 amino acids with a calculated molecular mass of 51,786 daltons. A potential signal sequence comprised of the first 35 amino acids and a potential transmembrane domain at the C terminus of the protein were identified. There exist seven potential glycosylation sites between the aforementioned protein motifs. Experiments with a specific antibody against Ac45 demonstrated that it is copurifying with the V-ATPase from chromaffin granules. Immunological cross-reactivity was observed with purified V-ATPase from bovine kidney microsomes but not from plasma membranes of epithelial cells. Cell-free expression of the protein from synthetic mRNA produced a single protein band at about 50 kDa on SDS gels. Upon inclusion of dog pancreas microsomes in the reaction mixture, a slow migrating band sensitive to peptide:N-glycosidase F was observed.

Transcriptional control of AAC3 gene encoding mitochondrial ADP/ATP translocator in Saccharomyces cerevisiae by oxygen, heme and ROX1 factor.

The AAC3 gene of Saccharomyces cerevisiae encodes a mitochondrial ADP/ATP translocator which is subject to oxygen repression. Evidence is presented here, that the repression of AAC3 expression is dependent upon heme and the ROX1 factor. The promoter region of the AAC3 gene was isolated, sequenced, and deletion analysis was performed using lacZ as a reporter gene to determine the cis-acting regions responsible for the regulation of AAC3 expression. The results of the deletion analysis show that the negative control of the AAC3 gene by oxygen and ROX1 factor is mediated by an upstream repression sequence consisting of a T-rich segment adjacent to the consensus elements that are present in the 5' flanking regions of several other yeast genes. An additional upstream repressor site was located within the AAC3 promoter which, however, is not related either to oxygen or to ROX1 factor. The data presented here delineate the main cellular factors and DNA sequences involved in the regulatory mechanism by which an essential function for anaerobic cells growth, ADP/ATP translocation, is ensured. In addition, they show that the AAC3 gene belongs to the family of yeast genes including TIF51B, COX5b, HEM13 and CYC7 that are negatively regulated by oxygen and heme.

Expression of chimeric proteins encoded by the fused BLV reverse transcriptase: beta-galactosidase in Escherichia coli.

The gene for bovine leukemia virus (BLV) reverse transcriptase was cloned in prokaryotic expression vector pUC8-2. After fusion of Escherichia coli lacZ gene to different parts of reverse transcriptase we detected expression of new proteins with molecular weights corresponding to the size of the hybrid genes. A coding region most probably responsible for about a hundred-fold decrease in expression of long fusion proteins has been identified. A few possible causes of this phenomenon were tested.