RESUMO
Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.
Assuntos
ADP-Ribosilação , Proteínas de Transporte/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , ADP-Ribosilação/efeitos dos fármacos , Sequência de Aminoácidos , Anticorpos/metabolismo , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Técnicas de Visualização da Superfície Celular , Dano ao DNA , Glicosídeo Hidrolases/metabolismo , Histonas/metabolismo , Humanos , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Tirosina/metabolismoRESUMO
PARP1, an established anti-cancer target that regulates many cellular pathways, including DNA repair signaling, has been intensely studied for decades as a poly(ADP-ribosyl)transferase. Although recent studies have revealed the prevalence of mono-ADP-ribosylation upon DNA damage, it was unknown whether this signal plays an active role in the cell or is just a byproduct of poly-ADP-ribosylation. By engineering SpyTag-based modular antibodies for sensitive and flexible detection of mono-ADP-ribosylation, including fluorescence-based sensors for live-cell imaging, we demonstrate that serine mono-ADP-ribosylation constitutes a second wave of PARP1 signaling shaped by the cellular HPF1/PARP1 ratio. Multilevel chromatin proteomics reveals histone mono-ADP-ribosylation readers, including RNF114, a ubiquitin ligase recruited to DNA lesions through a zinc-finger domain, modulating the DNA damage response and telomere maintenance. Our work provides a technological framework for illuminating ADP-ribosylation in a wide range of applications and biological contexts and establishes mono-ADP-ribosylation by HPF1/PARP1 as an important information carrier for cell signaling.
Assuntos
ADP-Ribosilação , Histonas , Histonas/genética , Histonas/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Cromatina , Dano ao DNA , Anticorpos/genética , Transdução de SinaisRESUMO
Ester-linked post-translational modifications, including serine and threonine ubiquitination, have gained recognition as important cellular signals. However, their detection remains a significant challenge due to the chemical lability of the ester bond. This is the case even for long-known modifications, such as ADP-ribosylation on aspartate and glutamate, whose role in PARP1 signaling has recently been questioned. Here, we present easily implementable methods for preserving ester-linked modifications. When combined with a specific and sensitive modular antibody and mass spectrometry, these approaches reveal DNA damage-induced aspartate/glutamate mono-ADP-ribosylation. This previously elusive signal represents an initial wave of PARP1 signaling, contrasting with the more enduring nature of serine mono-ADP-ribosylation. Unexpectedly, we show that the poly-ADP-ribose hydrolase PARG is capable of reversing ester-linked mono-ADP-ribosylation in cells. Our methodology enables broad investigations of various ADP-ribosylation writers and, as illustrated here for noncanonical ubiquitination, it paves the way for exploring other emerging ester-linked modifications.
Assuntos
ADP-Ribosilação , Ácido Aspártico , Ésteres , Ácido Glutâmico , Poli(ADP-Ribose) Polimerase-1 , Processamento de Proteína Pós-Traducional , Poli(ADP-Ribose) Polimerase-1/metabolismo , Humanos , Ácido Aspártico/metabolismo , Ácido Glutâmico/metabolismo , Ésteres/química , Ésteres/metabolismo , Ubiquitinação , Dano ao DNA , Células HEK293 , Glicosídeo Hidrolases/metabolismo , Transdução de SinaisRESUMO
ADP-ribosylation (ADPr) is a widespread post-translational modification (PTM) spanning all kingdoms of life. It is employed by bacteria and viruses in their war against the host, and by eukaryotes to regulate many physiological processes, across almost all cellular compartments. PARP1, the founding member of the PARP family, is an early sensor of single- and double-strand breaks and catalyzes ADPr to mediate DNA damage repair. The recent discovery of Serine-ADPr and the PARP1 accessory factor HPF1 has brought a momentous change to the field. Bolstered by innovative ways to study ADPr, new and exciting research directions are rapidly emerging. In this review we explore our understanding of the HPF1/PARP1-mediated ADPr signaling pathway in DNA damage. We focus on the mechanistic steps leading to Serine-ADPr and its relevance in the DNA damage response. We discuss important technological advances that have enabled a nuanced study of Serine-ADPr, and conclude with an overview of the role of PARP inhibitors in cancer therapy.