Biomedical subjects

Joseph A Vetro

Publications and source records attributed to Joseph A Vetro.

5 recordsLinked to original sources

PubMed2005

Evidence of a dominant negative mutant of yeast methionine aminopeptidase type 2 in Saccharomyces cerevisiae.

Eukaryotic methionine aminopeptidase type 2 (MetAP2, MetAP2 gene (MAP2)), together with eukaryotic MetAP1, cotranslationally hydrolyzes initiator methionine from nascent polypeptides when the side chain of the second residue is small and uncharged. In this report, we took advantage of the yeast (Saccharomyces cerevisiae) map1 null strain's reliance on MetAP2 activity for the growth and viability to provide evidence of the first dominant negative mutant of eukaryotic MetAP2. Replacement of the conserved His(174) with alanine within the C-terminal catalytic domain of yeast MetAP2 eliminated detectable catalytic activity against a peptide substrate in vitro. Overexpression of MetAP2 (H174A) under the strong GPD promoter in a yeast map1 null strain was lethal, whereas overexpression under the weaker GAL1 promoter slightly inhibited map1 null growth. Deletion mutants further revealed that the N-terminal region of MetAP2 (residues 2-57) is essential but not sufficient for MetAP2 (H174A) to fully interfere with map1 null growth. Together, these results indicate that catalytically inactive MetAP2 is a dominant negative mutant that requires its N-terminal region to interfere with wild-type MetAP2 function.

Aminopeptidases↗

PubMed2005

Structure/function relationships of polyamidoamine/DNA dendrimers as gene delivery vehicles.

PAMAM dendrimers are members of a class of polyamine polymers that demonstrate significant gene delivery ability. In this study, a selection of PAMAM dendrimers, spanning a range of sizes (generations 2, 4, 7, and 9) and transfection efficiencies, are characterized by various biophysical methods to search for structural properties that correlate with transfection. Measurements of colloidal properties (size and zeta potential) as a function of charge ratio reveal that highly transfecting dendrimer/DNA complexes have size/zeta potential values between 4 and 8. Circular dichroism (CD) and FTIR spectroscopy of complexes confirm the DNA component remains in B form when associated with all dendrimer generations up to a 5:1 charge ratio (+/-). Isothermal titration calorimetry and differential scanning calorimetry detect changes that are related to polymer structure and charge ratio but do not directly correlate with transfection efficiency. Despite DNA structural and stability changes detected by CD, FTIR, DSC, and ITC that are similar to those seen with other cationic delivery vehicles [e.g., cationic lipids, peptoids/lipitoids, peptides, polyethyleneimines (PEIs), etc.], clear correlations with transfection activity are not readily apparent. This may be due, at least in part, to the heterogeneity of the complexes.

Animals↗

PubMed2003

Structure/function analysis of peptoid/lipitoid:DNA complexes.

Previous transfection studies of cationic peptoid polymers (N-substituted polyglycines) and cationic lipitoid polymers (peptoid-phospholipid conjugates) have shown that only the polymers which possessed a repeating (cationic, hydrophobic, hydrophobic) substituent sequence are efficient in gene transfer in vitro. To determine if there is a physical attribute of peptoid and lipitoid complexes that correlates with efficient gene transfection, biophysical, and transfection measurements were performed with polymer:DNA complexes containing each of seven structurally diverse peptoid polymers and two lipitoids that possess different hydrophobic substituents. These measurements revealed that the biophysical properties of these complexes (size, zeta-potential, ethidium bromide exclusion) varied with polymer structure and complex (+/-) charge ratio but were not directly predictive of transfection efficiency. Unique alterations in the circular dichroism spectra of DNA were observed in complexes containing several of the peptoids and both lipitoids, although FTIR spectroscopy demonstrated that the DNA remained in the B-form. The lack of correlations between the physical properties and the transfection activities of these polyplexes suggests that a further subpopulation examination of these complexes by these methods may reveal hidden structure-activity relationships.

Animals↗

PubMed2002

The specificity in vivo of two distinct methionine aminopeptidases in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the essential function of amino-terminal methionine removal is provided cotranslationally by two methionine aminopeptidases (MetAP1 and MetAP2). To examine the individual processing efficiency of each MetAP in vivo, we measured the degree of N-terminal methionine cleavage from a series of mutated glutathione-S-transferase (GST) proteins isolated from yeast wild-type, a map1 deletion strain, a map2 deletion strain, and a map1 deletion strain overexpressing the MAP2 gene. We found that MetAP1 plays the major role in N-terminal methionine removal in yeast. Both MetAPs were less efficient when the second residue was Val, and MetAP2 was less efficient than MetAP1 when the second residue was Gly, Cys, or Thr. These findings indicate that MetAP1 and MetAP2 exhibit different cleavage efficiencies against the same substrates in vivo. Interestingly, although methionine is considered a stabilizing N-terminal residue, we found that retention of the initiator methionine on the Met-Ala-GST mutant protein drastically reduced its half-life in vivo.

Amino Acid Substitution↗

PubMed2002

Yeast methionine aminopeptidase type 1 is ribosome-associated and requires its N-terminal zinc finger domain for normal function in vivo.

Methionine aminopeptidase type 1 (MetAP1) cotranslationally removes N-terminal methionine from nascent polypeptides, when the second residue in the primary structure is small and uncharged. Eukaryotic MetAP1 has an N-terminal zinc finger domain not found in prokaryotic MetAPs. We hypothesized that the zinc finger domain mediates the association of MetAP1 with the ribosomes and have reported genetic evidence that it is important for the normal function of MetAP1 in vivo. In this study, the intracellular role of the zinc finger domain in yeast MetAP1 function was examined. Wild-type MetAP1 expressed in a yeast map1 null strain removed 100% of N-terminal methionine from a reporter protein, while zinc finger mutants removed only 31-35%. Ribosome profiles of map1 null expressing wild-type MetAP1 or one of three zinc finger mutants were compared. Wild-type MetAP1 was found to be an 80S translational complex-associated protein that primarily associates with the 60S subunit. Deletion of the zinc finger domain did not significantly alter the ribosome profile distribution of MetAP1. In contrast, single point mutations in the first or second zinc finger motif disrupted association of MetAP1 with the 60S subunit and the 80S translational complex. Together, these results indicate that the zinc finger domain is essential for the normal processing function of MetAP1 in vivo and suggest that it may be important for the proper functional alignment of MetAP1 on the ribosomes.

Amino Acid Sequence↗