Site-specific characterization of the Asp- and Glu-ADP-ribosylated proteome
TL;DR: A method to characterize the human aspartic acid– and glutamic acid–ADP-ribosylated proteome was described and it was confirmed that iniparib had a negligible effect on PARP activity in intact cells.
Abstract: Poly(ADP-ribosyl)ation is catalyzed by a family of enzymes known as PARPs. We describe a method to characterize the human aspartic acid- and glutamic acid-ADP-ribosylated proteome. We identified 1,048 ADP-ribosylation sites on 340 proteins involved in a wide array of nuclear functions; among these were many previously unknown PARP downstream targets whose ADP-ribosylation was sensitive to PARP inhibitor treatment. We also confirmed that iniparib had a negligible effect on PARP activity in intact cells.
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TL;DR: In this overview for the special PTM issue of Molecular and Cellular Proteomics, it is taken stock of where MS-based proteomics stands in the large-scale analysis of protein modifications.
510 citations
TL;DR: New findings on the diverse roles of PARPs in chromatin regulation, transcription, RNA biology, and DNA repair have been complemented by recent advances that link ADP-ribosylation to stress responses, metabolism, viral infections, and cancer.
Abstract: The discovery of poly(ADP-ribose) >50 years ago opened a new field, leading the way for the discovery of the poly(ADP-ribose) polymerase (PARP) family of enzymes and the ADP-ribosylation reactions that they catalyze. Although the field was initially focused primarily on the biochemistry and molecular biology of PARP-1 in DNA damage detection and repair, the mechanistic and functional understanding of the role of PARPs in different biological processes has grown considerably of late. This has been accompanied by a shift of focus from enzymology to a search for substrates as well as the first attempts to determine the functional consequences of site-specific ADP-ribosylation on those substrates. Supporting these advances is a host of methodological approaches from chemical biology, proteomics, genomics, cell biology, and genetics that have propelled new discoveries in the field. New findings on the diverse roles of PARPs in chromatin regulation, transcription, RNA biology, and DNA repair have been complemented by recent advances that link ADP-ribosylation to stress responses, metabolism, viral infections, and cancer. These studies have begun to reveal the promising ways in which PARPs may be targeted therapeutically for the treatment of disease. In this review, we discuss these topics and relate them to the future directions of the field.
475 citations
Cites background from "Site-specific characterization of t..."
...Zhang et al. (2015) showed that the phenotypic outcomes of a STAT1 mutant that is hyperresponsive to interferon signaling depend on the PARP-9–DTX3L complex....
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...5E; Zhang et al. 2015)....
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...Activation of the PARP-9–DTX3L complex’s ubiquitin ligase activity also stimulated the degradation of the viral 3C proteases via the immunoproteasome (Zhang et al. 2015)....
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TL;DR: In this article, the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino-acid targets.
Abstract: The poly(adenosine diphosphate (ADP)-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD(+) as substrate. Based on the composition of three NAD(+) coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADPr) (PAR) or mono(ADPr) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino-acid targets. In addition, we identify cysteine as a novel amino-acid target for ADP-ribosylation on PARPs.
358 citations
TL;DR: It is concluded that at least one-third of unassigned spectra arise from peptides with substoichiometric modifications, and an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses is used.
Abstract: Fewer than half of all tandem mass spectrometry (MS/MS) spectra acquired in shotgun proteomics experiments are typically matched to a peptide with high confidence. Here we determine the identity of unassigned peptides using an ultra-tolerant Sequest database search that allows peptide matching even with modifications of unknown masses up to ± 500 Da. In a proteome-wide data set on HEK293 cells (9,513 proteins and 396,736 peptides), this approach matched an additional 184,000 modified peptides, which were linked to biological and chemical modifications representing 523 distinct mass bins, including phosphorylation, glycosylation and methylation. We localized all unknown modification masses to specific regions within a peptide. Known modifications were assigned to the correct amino acids with frequencies >90%. We conclude that at least one-third of unassigned spectra arise from peptides with substoichiometric modifications.
358 citations
TL;DR: Dysregulation of these processes has been intimately associated with the development of diseases such as cancer and is thought to regulate chromatin structure and function by two mechanisms.
Abstract: 1.1. Nucleosome and Chromatin
In eukaryotic cells, chromosomal DNA is packaged into a compact structure, chromatin, with the use of four core histones (H2A, H2B, H3, and H4). The fundamental repeating unit of chromatin is the nucleosome, which is composed of an octamer of the core histones, around which ~147 base pairs of DNA are wrapped. Nucleosomes are in turn folded into progressively higher-order structures. Dynamic chromatin remodeling plays a critical role in regulating diverse DNA-based biological processes, such as transcription of RNA, DNA replication, and DNA repair, as well as chromosome condensation and segregation.1
The core histone proteins (not histone octamer) are small (10–20 kDa) and highly basic. They are predominantly globular except for their N-terminal “tails”, which are unstructured and protrude from the surface of the chromatin polymer. Amino acid sequence analysis shows that histone proteins are highly conserved in eukaryotic cells from yeast to human, implying that most amino acid residues, if not all, are likely to be important for structure or function. Indeed, studies among histone variants as well as mutational evidence in cancers suggest that a change of a single amino acid residue can lead to very different biological output and even disease, such as cancer.2
Histone post-translational modification (PTM), or histone mark, in combination with DNA modifications, histone variants, and ATP-dependent protein complex formation, is used by cells to dynamically modulate chromatin structure and function. Because PTMs alter the properties of the substrate amino acid residue, typically more significant than a mutation, they are likely to affect histone structure and therefore function.3 Indeed, PTMs are abundant in histones, especially at their N-terminal tails, and have roles in modulating chromatin dynamics and diverse DNA-templated biological processes (Figure 1).1 Dysregulation of these processes has been intimately associated with the development of diseases such as cancer.4
Figure 1
Structures of histone post-translational modifications.
1.2. Biological Mechanism of Histone PTMs
As of this writing, 20 types of histone PTMs had been reported: phosphorylation, acetylation, monomethylation, dimethylation, trimethylation, propionylation, butyrylation, crotonylation, 2-hydroxylisobutyrylation, malonylation, succinylation, glutarylation, formylation, hydroxylation, ubiquitination, SUMOylation, O-GlcNAcylation, ADP-ribosylation, proline isomerization, and citrullination (Figure 1).5 In more recent times, known PTM sites on histones have been identified either by sequence-specific antibodies or by mass spectrometry (MS) methods in an unbiased manner.6 The function and dynamic regulation of these PTMs have been the subject of extensive investigations over the past decade.
Histone PTMs are thought to regulate chromatin structure and function by two mechanisms.1a,b First, histone PTMs can directly modulate the packaging of chromatin by either altering the charge state of histones or through inter nucleosomal interactions, thereby regulating chromatin higher-order structure and the access of DNA-binding proteins, such as transcription factors. Additionally, histone PTMs can modify chromatin structure and function either by recruiting PTM-specific binding proteins (also called “readers”) and their associated binding partners (“effector proteins”) or by inhibiting the binding of a protein to the chromatin. PTM-induced changes in protein interactions between chromatin and its binding proteins are in turn translated into biological outcomes.7 Proteins are recruited to histone PTMs through direct binding to specific domains. For example, chromo, Tudor, PHD, MBT, PWWP, WD, ADD, zf-CW, BAH, and CHD domains are all known to bind methyllysine,8 while the bromodomain binds acetyllysine.9 Proteins containing these PTM-specific binding domains may recruit additional protein factors to execute their functions. Alternatively, they may carry enzymatic activities that can further modify chromatin structure and function.
Histone marks are known to be critical in regulation of diverse DNA-templated biological processes.1 Interestingly, some of these histone PTMs correlate with transcriptional activation or repression, depending on the types and the locations of the PTMs.1b,10 To execute DNA-templated processes, histone PTMs coordinate the unraveling of chromatin to carry out specific functions. For example, histone lysine acetylation (Kac) typically correlates with transcriptional activation, while lysine deacetylation correlates with transcriptional repression.1b,11 Lysine methylation (Kme) is implicated in both gene activation (H3K4, H3K36, and H3K79) and transcriptional repression (H3K9, H3K27, and H4K20).12 As examples, some monomethylation (e.g., H3K9me1 and H3K27me1) is involved in transcriptional activation, while trimethylation at the same sites (H3K9me3 and H3K27me3) is linked to repression.13 Likewise, some other histone PTMs also correlate with DNA repair (e.g., H2AS129 phosphorylation and H4S1 phosphorylation)14 and replication (e.g., acetylation).15 Dysregulation of each step of histone PTMs, including adding the histone marks by a “writer”, removing the histone mark by an “eraser”, and misinterpretation by a “reader” protein, has shown to be associated with disease, such as cancer.4a,e
These histone PTMs are proposed to contribute a “histone code” or “histone language” that dictates the functions of the proteins in gene expression and chromatin dynamics.1a,c,d Addition and removal of histone PTMs are regulated by diverse groups of enzymes that were initially identified in the past decade, but still are being discovered in recent times. These enzymes are responsible for adding (“writing”) or removing (“erasing”) the histone PTM “code”. The resulting histone marks are in turn translated into biological outputs by different mechanisms.
Chromatin dynamics are mainly controlled by ATP-dependent chromatin remodeling enzymes/complexes and histone PTMs.16 The “histone code” can facilitate the recruitment of diverse chromatin remodeling enzymes to regulate chromatin dynamics. Conversely, chromatin remodeling enzymes can also influence the histone PTMs.17 For example, an ATP-dependent nucleosome remodeling complex, nucleosome remodelling and deacetylation complex (NuRD), can facilitate the deacetylation of the target histones.18
Some histone PTMs, if not all, are inheritable during cell division and correlate with gene expression. Therefore, histone PTMs are linked with epigenetic phenomena and are generally considered to be a major type of epigenetic marks.19
297 citations
References
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TL;DR: BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis, illustrating how different pathways cooperate to repair damage.
Abstract: BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.
5,650 citations
TL;DR: The data suggest that the "typical" phosphoprotein is widely expressed yet displays variable, often tissue-specific phosphorylation that tunes protein activity to the specific needs of each tissue, and is offered as an online resource for the biological research community.
1,520 citations
TL;DR: The human ubiquitin-modified proteome is characterized using a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion and it is demonstrated that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitIn ligases.
1,463 citations
TL;DR: What is known about the structures and functions of the family ofPARP enzymes are reviewed, and a series of questions that should be addressed are outlined to guide the rational development of PARP inhibitors as anticancer agents.
Abstract: Recent findings have thrust poly(ADP-ribose) polymerases (PARPs) into the limelight as potential chemotherapeutic targets. To provide a framework for understanding these recent observations, we review what is known about the structures and functions of the family of PARP enzymes, and then outline a series of questions that should be addressed to guide the rational development of PARP inhibitors as anticancer agents.
1,200 citations
TL;DR: This method, in addition to shedding light on the consensus sequences of identified and as yet unidentified kinases and modular protein domains, may also eventually be used as a tool to determine potential phosphorylation sites in proteins of interest.
Abstract: With the recent exponential increase in protein phosphorylation sites identified by mass spectrometry, a unique opportunity has arisen to understand the motifs surrounding such sites. Here we present an algorithm designed to extract motifs from large data sets of naturally occurring phosphorylation sites. The methodology relies on the intrinsic alignment of phospho-residues and the extraction of motifs through iterative comparison to a dynamic statistical background. Results show the identification of dozens of novel and known phosphorylation motifs from recently published serine, threonine and tyrosine phosphorylation studies. When applied to a linguistic data set to test the versatility of the approach, the algorithm successfully extracted hundreds of language motifs. This method, in addition to shedding light on the consensus sequences of identified and as yet unidentified kinases and modular protein domains, may also eventually be used as a tool to determine potential phosphorylation sites in proteins of interest.
839 citations