Using TLC-MALDI-TOF to Interrogate In Vitro Peptidyl Proximal Preferences of PARP14 and Glycohydrolase Specificity.

The transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human diphtheria toxin-like ADP-ribosyltransferases (ARTDs; previously referred to as poly-ADP-ribose polymerases or PARPs) and the removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about the sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the in vitro analysis of proximal factors that guide ARTD target selection. We identify a minimal 5-mer peptide sequence that is necessary and sufficient to drive glutamate/aspartate targeting using PARP14 while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is pH and temperature dependent, sequence independent, and occurs within hours. Finally, we use the ADPr-peptides to highlight differential activities within the glycohydrolase family and their sequence preferences. Our results highlight (1) the utility of MALDI-TOF in analyzing proximal ARTD-substrate interactions and (2) the importance of peptide sequences in governing ADPr transfer and removal.

Identification of a Novel PARP14 Site Motif and Glycohydrolase Specificity Using TLC-MALDI-TOF.

Transfer of ADP-ribose (ADPr) from nicotinamide adenine dinucleotide (NAD+) to target proteins is mediated by a class of human poly-ADP-ribose polymerases, PARPs, and removal of ADPr is catalyzed by a family of glycohydrolases. Although thousands of potential ADPr modification sites have been identified using high-throughput mass-spectrometry, relatively little is known about sequence specificity encoded near the modification site. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates the discovery and validation of ADPr site motifs. We identify a minimal 5-mer peptide sequence that is sufficient to drive PARP14 specific activity while highlighting the importance of the adjacent residues in PARP14 targeting. We measure the stability of the resultant ester bond and show that non-enzymatic removal is sequence independent and occurs within hours. Finally, we use the ADPr-peptide to highlight differential activities within the glycohydrolase family and their sequence specificities. Our results highlight: 1) the utility of MALDI-TOF in motif discovery and 2) the importance of peptide sequence in governing ADPr transfer and removal.

Application of the Extended Health Control Belief Model to Predict Hepatitis A and B Vaccinations.

BACKGROUND: Adult vaccination compliance rates vary according to sample and type of vaccine administered (influenza, pneumococcal). This study looked at vaccination of a community sample of low-income, minority adults. METHODS: Nurses offered free vaccination for hepatitis A and B in the form of the combined Twinrix vaccine to adults on a walk-in basis. In addition to dosing information, participants completed the Risk Behavior Assessment, the Coping Strategies Indicator and the Cardiovascular Risk Assessment. Skaff's extended Health Belief Model was used as the theoretical framework. Count regression was used to model receipt of one, two, or three doses. RESULTS: The majority of participants were male with a mean age of 40 years. The distribution of doses was: 173 individuals (27.6%) received one dose only, 261 (41.7%) received two doses, and 191 (30.5%) received three doses of vaccine. The multivariate count regression model including being male, having previously been told by a health care provider that one has syphilis, having severe negative emotions, and perceived social support were associated with participants' receiving fewer doses of hepatitis vaccine. A greater problem-solving score was associated with a higher number of vaccine doses received. CONCLUSION: Despite free vaccinations offered in an easily accessible community setting, the majority of participants failed to complete the hepatitis vaccine series. More effort is needed to get adult men to participate in hepatitis vaccination clinics. Additional research is necessary to understand barriers other than cost to adults receiving vaccination.