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.

Rapid Analysis of ADP-Ribosylation Dynamics and Site-Specificity Using TLC-MALDI.

Poly(ADP-ribose) polymerases, PARPs, transfer ADP-ribose onto target proteins from nicotinamide adenine dinucleotide (NAD+). Current mass spectrometric analytical methods require proteolysis of target proteins, limiting the study of dynamic ADP-ribosylation on contiguous proteins. Herein, we present a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) method that facilitates multisite analysis of ADP-ribosylation. We observe divergent ADP-ribosylation dynamics for the catalytic domains of PARPs 14 and 15, with PARP15 modifying more sites on itself (+3-4 ADP-ribose) than the closely related PARP14 protein (+1-2 ADP-ribose)─despite similar numbers of potential modification sites. We identify, for the first time, a minimal peptide fragment (18 amino-acids) that is preferentially modified by PARP14. Finally, we demonstrate through mutagenesis and chemical treatment with hydroxylamine that PARPs 14/15 prefer acidic residues. Our results highlight the utility of MALDI-TOF in the analysis of PARP target modifications and in elucidating the biochemical mechanism governing PARP target selection.

Combining Chemical Genetics with Proximity-Dependent Labeling Reveals Cellular Targets of Poly(ADP-ribose) Polymerase 14 (PARP14).

Poly(ADP-ribose) polymerase 14 (PARP14) is a member of the PARP family of enzymes that transfer ADP-ribose from NAD+ to nucleophilic amino acids on target proteins, a process known as mono-ADP-ribosylation (MARylation). PARP14 is involved in normal immune function through the IL-4 signaling pathway and is a prosurvival factor in multiple myeloma and hepatocellular carcinoma. A mechanistic understanding of the physiological and pathophysiological roles of PARP14 has been limited by the dearth of PARP14-specific MARylation targets. Herein we engineered a PARP14 variant that uses an NAD+ analog that is orthogonal to wild-type PARPs for identifying PARP14-specific MARylation targets. Combining this chemical genetics approach with a BioID approach for proximity-dependent labeling of PARP14 interactors, we identified 114 PARP14-specific protein substrates, several of which are RNA regulatory proteins. One of these targets is PARP13, a protein known to play a role in regulating RNA stability. PARP14 MARylates PARP13 on several acidic amino acids. This study not only reveals crosstalk among PARP family members but also highlights the advantage of using disparate approaches for identifying the direct targets of individual PARP family members.

Identifying Family-Member-Specific Targets of Mono-ARTDs by Using a Chemical Genetics Approach.

ADP-ribosyltransferases (ARTD1-16) have emerged as major downstream effectors of NAD(+) signaling in the cell. Most ARTDs (ARTD7 and 8, 10-12, and 14-17) catalyze the transfer of a single unit of ADP-ribose from NAD(+) to target proteins, a process known as mono-ADP-ribosylation (MARylation). Progress in understanding the cellular functions of MARylation has been limited by the inability to identify the direct targets for individual mono-ARTDs. Here, we engineered mono-ARTDs to use an NAD(+) analog that is orthogonal to wild-type ARTDs. We profiled the MARylomes of ARTD10 and ARTD11 in vitro, identifying isoform-specific targets and revealing a potential role for ARTD11 in nuclear pore complex biology. We found that ARTD11 targeting is dependent on both its regulatory and catalytic domains, which has important implications for how ARTDs recognize their targets. We anticipate that our chemical genetic strategy will be generalizable to all mono-ARTD family members based on the similarity of the mono-ARTD catalytic domains.

Identifying Direct Protein Targets of Poly-ADP-Ribose Polymerases (PARPs) Using Engineered PARP Variants-Orthogonal Nicotinamide Adenine Dinucleotide (NAD+) Analog Pairs.

Poly-ADP-ribose polymerases (PARPs) comprise a family of 17 distinct enzymes that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor sites on protein targets. PARPs have been implicated in a number of essential signaling pathways regulating both normal cell function and pathophysiology. To understand the physiological role of each PARP family member in the cell we need to identify the direct targets for each unique PARP in a cellular context. PARP-family member-specific target identification is challenging because of their shared catalytic mechanism and functional redundancy. To address this challenge, we have engineered a PARP variant that efficiently uses an orthogonal NAD+ analog, an analog that endogenous PARPs cannot use, as a substrate for ADP-ribosylation. The protocols in this unit describe a general procedure for using engineered PARP variants-orthogonal NAD+ analog pairs for labeling and identifying the direct targets of the poly-subfamily of PARPs (PARPs 1-3, 5, and 6).

Selective inhibition of PARP10 using a chemical genetics strategy.

The lack of inhibitors that are selective for individual poly-ADP-ribose polymerase (PARP) family members has limited our understanding of their roles in cells. Here, we describe a chemical genetics approach for generating selective inhibitors of an engineered variant of PARP10. We synthesized a series of C-7 substituted 3,4-dihydroisoquinolin-1(2H)-one (dq) analogues designed to selectively inhibit a mutant of PARP10 (LG-PARP10) that contains a unique pocket in its active site. A dq analogue containing a bromo at the C-7 position demonstrated a 10-fold selectivity for LG-PARP10 compared to its WT counterpart. This study provides a platform for the development of selective inhibitors of individual PARP family members that will be useful for decoding their cellular functions.

Engineering the substrate specificity of ADP-ribosyltransferases for identifying direct protein targets.

Adenosine diphosphate ribosyltransferases (ARTDs; ARTD1-17 in humans) are emerging as critical regulators of cell function in both normal physiology and disease. These enzymes transfer the ADP-ribose moiety from its substrate, nicotinamide adenine dinucleotide (NAD(+)), to amino acids of target proteins. The functional redundancy and overlapping target specificities among the 17 ARTDs in humans make the identification of direct targets of individual ARTD family members in a cellular context a formidable challenge. Here we describe the rational design of orthogonal NAD(+) analogue-engineered ARTD pairs for the identification of direct protein targets of individual ARTDs. Guided by initial inhibitor studies with nicotinamide analogues containing substituents at the C-5 position, we synthesized an orthogonal NAD(+) variant and found that it is used as a substrate for several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2 (PARP2) in nuclear extracts highlighted the semi-complementary, yet distinct, protein targeting. Using affinity purification followed by tandem mass spectrometry, we identified 42 direct ARTD1 targets and 301 direct ARTD2 targets. This represents a powerful new technique for identifying direct protein targets of individual ARTD family members, which will facilitate studies delineating the pathway from ARTD activation to a given cellular response.

Genome-wide characterization of the phosphate starvation response in Schizosaccharomyces pombe.

BACKGROUND: Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway, leading to expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway involving genetically identified positive (pho7+) and negative (csk1+) regulators. The genes induced by phosphate limitation and the molecular mechanism by which pho7+ and csk1+ function are unknown. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. RESULTS: We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response that contains the ScPHO5 (pho1+), ScPHO84 (SPBC8E4.01c), and ScGIT1 (SPBC1271.09) orthologs. We identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. CONCLUSIONS: We provide a global analysis of the transcriptional response to phosphate limitation in S. pombe. Our results elucidate the conserved core regulon induced in response to phosphate starvation in this ascomycete distantly related to S. cerevisiae and provide a better understanding of flexibility in environmental stress response networks.

Thermodynamic examination of trinucleotide bulged RNA in the context of HIV-1 TAR RNA.

RNA structures contain many bulges and loops that are expected to be sites for inter- and intra-molecular interactions. Nucleotides in the bulge are expected to influence the structure and recognition of RNA. The same stability is assigned to all trinucleotide bulged RNA in the current secondary structure prediction models. In this study thermal denaturation experiments were performed on four trinucleotide bulged RNA, in the context of HIV-1 TAR RNA, to determine whether the bulge sequence affects RNA stability and its divalent ion interactions. Cytosine-rich bulged RNA were more stable than uracil-rich bulged RNA in 1 M KCl. Interactions of divalent ions were more favorable with uracil-rich bulged RNA by approximately 2 kcal/mol over cytosine-rich bulged RNA. The UCU-TAR RNA (wild type) is stabilized by 1.7 kcal/mol in 9.5 mM Ca(2+) as compared with 1 M KCl, whereas no additional gain in stability is measured for CCC-TAR RNA. These results have implications for base substitution experiments traditionally employed to identify metal ion binding sites. To our knowledge, this is the first systematic study to quantify the effect of small sequence changes on RNA stability upon interactions with divalent ions.