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Mass spectrometric approaches towards site‐specific characterization of the ADP‐ribosylated proteome
Author(s) -
Yu Yonghao
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.926.17
Subject(s) - parp1 , olaparib , dna damage , poly adp ribose polymerase , dna repair , proteome , adp ribosylation , chemistry , biology , computational biology , microbiology and biotechnology , biochemistry , dna , polymerase , enzyme , nad+ kinase
Poly‐ADP‐ribosylation (PARylation) is a protein posttranslational modification (PTM) that was first discovered in 1963. It is catalyzed by a family of enzymes called Poly‐ADP‐ribose polymerases (PARPs). In particular, PARP1 is a nuclear enzyme that is involved in mediating DNA damage response (DDR). During genotoxic stress, PARP1 is recruited to nicked DNA and is rapidly activated, resulting in the synthesis of a large number of PARylated proteins and initiation of the DNA repair mechanisms. The critical role of PARP1 in DDR also provides the rationale for developing PARP1 inhibitors for the treatment of cancer (e.g. olaparib has recently been approved to treat BRCA1/2 ‐mutated ovarian cancer). Despite the highly successful clinical development of PARP inhibitors, we know surprisingly little about the mechanism by which PARP1 and other PARPs signal downstream. Specifically, very few genuine PARP1 substrates have been identified. In addition, site‐localization of protein PARylation is also a daunting challenge, due to the labile and heterogeneous nature of this modification. The paucity of validated PARP1 effectors and their sites of modification has greatly hampered its functional study. Recently, we were able to develop an integrated mass spectrometric (MS) approach towards site‐specific characterization of the Asp‐ and Glu‐ADP‐ribosylated proteome. Using this technology, we (1) identified 1,048 unique, endogenously modified D/E‐ADP‐ribosylation sites on 340 proteins. (2) provided evidence that protein PARylation is involved in a surprisingly wide array of nuclear functions, including epigenetic regulation, transcription control, DNA damage repair and mRNA processing. (3) quantitatively characterized the D/E‐ADP‐ribosylated proteome, and, in so doing, identified many proteins whose PARylation is sensitive to clinically relevant PARP1 inhibitors, including Olaparib, A966492 and AG14361. Many of these proteins are novel substrates of PARP. (4) found that iniparib, a compound that was previously thought to be a covalent PARP inhibitor, does not inhibit PARP in intact cells. This robust technology has thus greatly expanded our understanding of PARP biology. Furthermore, the PARylated proteins identified in the abovementioned experiments have provided many potential hits to the entire PARP field that seed future functional studies of this important PTM. Support or Funding Information Welch Foundation (I‐1800)