Modular antibodies reveal DNA damage-induced mono-ADP-ribosylation as a second wave of PARP1 signaling.

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.

An HPF1/PARP1-Based Chemical Biology Strategy for Exploring ADP-Ribosylation.

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.

Interplay of Histone Marks with Serine ADP-Ribosylation.

Serine ADP-ribosylation (Ser-ADPr) is a recently discovered protein modification that is catalyzed by PARP1 and PARP2 when in complex with the eponymous histone PARylation factor 1 (HPF1). In addition to numerous other targets, core histone tails are primary acceptors of Ser-ADPr in the DNA damage response. Here, we show that specific canonical histone marks interfere with Ser-ADPr of neighboring residues and vice versa. Most notably, acetylation, but not methylation of H3K9, is mutually exclusive with ADPr of H3S10 in vitro and in vivo. We also broaden the O-linked ADPr spectrum by providing evidence for tyrosine ADPr on HPF1 and other proteins. Finally, we facilitate wider investigations into the interplay of histone marks with Ser-ADPr by introducing a simple approach for profiling posttranslationally modified peptides. Our findings implicate Ser-ADPr as a dynamic addition to the complex interplay of modifications that shape the histone code.

Nonlocalized Searching of HCD Data for Fast and Sensitive Identification of ADP-Ribosylated Peptides.

ADP-ribosylation is a technically challenging PTM which has just emerged into the field of PTM-specific proteomics. But this fragile modifier requires special treatment on both a data acquisition and data processing level: it is highly labile under higher-energy collisional dissociation (HCD), and the degree of lability can depend on the site it modifies. Its behavior thus violates some assumptions on which proteomics algorithms are based. Here we present nonlocalized ADPr searching: a simple principle for maximizing sensitivity toward ADP-ribosylation when searching conventional HCD data. By scoring the strong fragment ions generally observed in ADPr spectra rather than the weak and often absent localization-dependent ions, nonlocalized searches are more sensitive. They also run significantly faster, due to reduced search space, and require no assumptions about which amino acids can be modified. We illustrate implementation in three search systems: Morpheus, MaxQuant, and MASCOT, and we also present a means of rapidly finding and extracting ADP-ribosylated peptide spectra from large datasets for more focused searching. This approach both improves identification of ADP-ribosylated peptides and avoids mis-localization of the modification sites.

Specificity of reversible ADP-ribosylation and regulation of cellular processes.

Proper and timely regulation of cellular processes is fundamental to the overall health and viability of organisms across all kingdoms of life. Thus, organisms have evolved multiple highly dynamic and complex biochemical signaling cascades in order to adapt and survive diverse challenges. One such method of conferring rapid adaptation is the addition or removal of reversible modifications of different chemical groups onto macromolecules which in turn induce the appropriate downstream outcome. ADP-ribosylation, the addition of ADP-ribose (ADPr) groups, represents one of these highly conserved signaling chemicals. Herein we outline the writers, erasers and readers of ADP-ribosylation and dip into the multitude of cellular processes they have been implicated in. We also review what we currently know on how specificity of activity is ensured for this important modification.

Mitotic post-translational modifications of histones promote chromatin compaction in vitro.

How eukaryotic chromosomes are compacted during mitosis has been a leading question in cell biology since the nineteenth century. Non-histone proteins such as condensin complexes contribute to chromosome shaping, but appear not to be necessary for mitotic chromatin compaction. Histone modifications are known to affect chromatin structure. As histones undergo major changes in their post-translational modifications during mitotic entry, we speculated that the spectrum of cell-cycle-specific histone modifications might contribute to chromosome compaction during mitosis. To test this hypothesis, we isolated core histones from interphase and mitotic cells and reconstituted chromatin with them. We used mass spectrometry to show that key post-translational modifications remained intact during our isolation procedure. Light, atomic force and transmission electron microscopy analysis showed that chromatin assembled from mitotic histones has a much greater tendency to aggregate than chromatin assembled from interphase histones, even under low magnesium conditions where interphase chromatin remains as separate beads-on-a-string structures. These observations are consistent with the hypothesis that mitotic chromosome formation is a two-stage process with changes in the spectrum of histone post-translational modifications driving mitotic chromatin compaction, while the action of non-histone proteins such as condensin may then shape the condensed chromosomes into their classic mitotic morphology.

Serine ADP-ribosylation reversal by the hydrolase ARH3.

ADP-ribosylation (ADPr) is a posttranslational modification (PTM) of proteins that controls many cellular processes, including DNA repair, transcription, chromatin regulation and mitosis. A number of proteins catalyse the transfer and hydrolysis of ADPr, and also specify how and when the modification is conjugated to the targets. We recently discovered a new form of ADPr that is attached to serine residues in target proteins (Ser-ADPr) and showed that this PTM is specifically made by PARP1/HPF1 and PARP2/HPF1 complexes. In this work, we found by quantitative proteomics that histone Ser-ADPr is reversible in cells during response to DNA damage. By screening for the hydrolase that is responsible for the reversal of Ser-ADPr, we identified ARH3/ADPRHL2 as capable of efficiently and specifically removing Ser-ADPr of histones and other proteins. We further showed that Ser-ADPr is a major PTM in cells after DNA damage and that this signalling is dependent on ARH3.

cAMP-dependent cell differentiation triggered by activated CRHR1 in hippocampal neuronal cells.

Corticotropin-releasing hormone receptor 1 (CRHR1) activates the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). Both cAMP sources were shown to be required for the phosphorylation of ERK1/2 triggered by activated G protein coupled receptor (GPCR) CRHR1 in neuronal and neuroendocrine contexts. Here, we show that activated CRHR1 promotes growth arrest and neurite elongation in neuronal hippocampal cells (HT22-CRHR1 cells). By characterising CRHR1 signalling mechanisms involved in the neuritogenic effect, we demonstrate that neurite outgrowth in HT22-CRHR1 cells takes place by a sAC-dependent, ERK1/2-independent signalling cascade. Both tmACs and sAC are involved in corticotropin-releasing hormone (CRH)-mediated CREB phosphorylation and c-fos induction, but only sAC-generated cAMP pools are critical for the neuritogenic effect of CRH, further highlighting the engagement of two sources of cAMP downstream of the activation of a GPCR, and reinforcing the notion that restricted cAMP microdomains may regulate independent cellular processes.

Serine ADP-Ribosylation Depends on HPF1.

ADP-ribosylation (ADPr) regulates important patho-physiological processes through its attachment to different amino acids in proteins. Recently, by precision mapping on all possible amino acid residues, we identified histone serine ADPr marks in the DNA damage response. However, the biochemical basis underlying this serine modification remained unknown. Here we report that serine ADPr is strictly dependent on histone PARylation factor 1 (HPF1), a recently identified regulator of PARP-1. Quantitative proteomics revealed that serine ADPr does not occur in cells lacking HPF1. Moreover, adding HPF1 to in vitro PARP-1/PARP-2 reactions is necessary and sufficient for serine-specific ADPr of histones and PARP-1 itself. Three endogenous serine ADPr sites are located on the PARP-1 automodification domain. Further identification of serine ADPr on HMG proteins and hundreds of other targets indicates that serine ADPr is a widespread modification. We propose that O-linked protein ADPr is the key signal in PARP-1/PARP-2-dependent processes that govern genome stability.

Serine is a new target residue for endogenous ADP-ribosylation on histones.

ADP-ribosylation (ADPr) is a biologically and clinically important post-translational modification, but little is known about the amino acids it targets on cellular proteins. Here we present a proteomic approach for direct in vivo identification and quantification of ADPr sites on histones. We have identified 12 unique ADPr sites in human osteosarcoma cells and report serine ADPr as a new type of histone mark that responds to DNA damage.