Upon DNA damage induction, DNA-dependent poly(ADP-ribose) polymerases (PARPs) synthesize an anionic poly(ADP-ribose) (pADPr) scaffold to which several proteins bind with the subsequent formation of pADPr-associated multiprotein complexes. We have used a combination of affinity-purification methods and proteomics approaches to isolate these complexes and assess protein dynamics with respect to pADPr metabolism. As a first approach, we developed a substrate trapping strategy by which we demonstrate that a catalytically inactive Poly(ADP-ribose) glycohydrolase (PARG) mutant can act as a physiologically selective bait for the isolation of specific pADPr-binding proteins through its macrodomain-like domain. In addition to antibody-mediated affinity-purification methods, we used a pADPr macrodomain affinity resin to recover pADPr-binding proteins and their complexes. Second, we designed a time course experiment to explore the changes in the composition of pADPr-containing multiprotein complexes in response to alkylating DNA damage-mediated PARP activation. Spectral count clustering based on GeLC-MS/MS analysis was complemented with further analyses using high precision quantitative proteomics through isobaric tag for relative and absolute quantitation (iTRAQ)- and Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics. Here, we present a valuable resource in the interpretation of systems biology of the DNA damage response network in the context of poly(ADP-ribosyl)ation and provide a basis for subsequent investigations of pADPr-binding protein candidates.

Background: Because systemic inflammation and endothelial dysfunction lead to heart failure with preserved ejection fraction, we characterized plasma levels of inflammatory and cardiac remodeling biomarkers in patients with Fabry disease ( FD ). Methods and Results: Plasma biomarkers were studied in multicenter cohorts of patients with FD (n=68) and healthy controls (n=40). Plasma levels of the following markers of inflammation and cardiac remodeling were determined: tumor necrosis factor ( TNF ), TNF receptor 1 ( TNFR 1) and 2 ( TNFR 2), interleukin‐6, matrix metalloprotease‐2 ( MMP ‐2), MMP ‐8, MMP ‐9, galectin‐1, galectin‐3, B‐type natriuretic peptide ( BNP ), midregional pro–atrial natriuretic peptide ( MR ‐pro ANP ), and globotriaosylsphingosine. Clinical profile, cardiac magnetic resonance imaging, and echocardiogram were reviewed and correlated with biomarkers. Patients with FD had elevated plasma levels of BNP , MR ‐pro ANP , MMP ‐2, MMP ‐9, TNF , TNFR 1, TNFR 2, interleukin‐6, galectin‐1, globotriaosylsphingosine, and analogues. Plasma TNFR 2, TNF , interleukin‐6, MMP ‐2, and globotriaosylsphingosine were elevated in FD patients with left ventricular hypertrophy, whereas diastolic dysfunction correlated with higher BNP , MR ‐pro ANP , and MMP ‐2 levels. Patients with late gadolinium enhancement on cardiac magnetic resonance imaging had greater levels of BNP , MR ‐pro ANP , TNFR 1, TNFR 2, and MMP ‐2. Plasma BNP , MR ‐pro ANP , MMP ‐2, MMP ‐8, TNF , TNFR 1, TNFR 2, galectin‐1, and galectin‐3 were elevated in patients with renal dysfunction. Patients undergoing enzyme replacement therapy who have more severe disease had higher MMP ‐2, TNF , TNFR 1, TNFR 2, and globotriaosylsphingosine analogue levels. Conclusions: Inflammatory and cardiac remodeling biomarkers are elevated in FD patients and correlate with disease progression. These features are consistent with a phenotype dominated by heart failure with preserved ejection fraction and suggest a key pathogenic role of systemic inflammation in FD .