adp - ribosylation refers to the addition of one or more adp - ribose units onto proteins through the transfer of the adp - ribose group from nad to target proteins post - translationally . adp - ribose groups can be attached singly as mono(adp - ribose ) or in polymeric chains as poly(adp - ribose ) ( par ) by the enzymatically active members of the family of 17 human adp - ribosyltransferases , commonly known as parps ( 13 ) . in addition , mono(adp - ribosyl)ation can be facilitated by other enzymes including sirtuins , extracellular membrane - associated adp - ribosyltransferases and bacterial toxins ( 46 ) . of note , non - enzymatic addition of adp - ribose groups onto proteins was also reported in vitro ( 7,8 ) . adp - ribosylation can be added onto amino acids of diverse chemistry , including glutamate , aspartate , lysine , arginine and cysteine ( 9,10 ) . some of these adp - ribose conjugations have shown to be reversible and their removal is mediated by two broad classes of enzymes that cleave par or the bond between adp - ribose and its conjugated amino acid ( 11,12 ) . poly(adp - ribose ) glycohydrolase ( parg ) and adp - ribosylhydrolase 3 ( arh3 ) cleave the ribose  ribose bonds between adp - ribose subunits ( 11,12 ) . while adp - ribosylhydrolase arh1 removes single adp - ribose groups from conjugated arginine residues , adp - ribosylhydrolases macrod1 , macrod2 and targ1 remove the last remaining adp - ribose groups from poly(adp - ribosyl)ated substrates or single adp - ribose groups from mono(adp - ribosyl)ated proteins conjugated at acidic residues ( 11,12 ) . targ1 is unique for its additional ability to remove the whole par chain specifically at glutamate several techniques have been developed to enrich and identify adp - ribosylated substrates ( 14 ) . some techniques use antibodies or protein domains that bind adp - ribose to pull out the modification from cell lysates and use mass spectrometry to identify adp - ribosylated substrates ( 1517 ) . other techniques utilize protein microarrays and recombinant parps to identify parp - specific substrates ( 18,19 ) . adp - ribosylation has numerous and diverse effects on protein functions and cellular pathways , including dna damage , transcription , chromatin organization , stress responses , circadian rhythms , cell cycle regulation and rna metabolism ( 2,2024 ) . the addition of the modification can affect the substrate 's stability and activity , as well as serve as a scaffold to recruit other proteins non - covalently ( 20,2527 ) . adp - ribosylation activity spans a diverse range of organisms across different kingdoms , from viruses to bacteria to mammals , highlighting the importance of this modification in living organisms ( 28,29 ) . because of the diverse roles they play in the cell , adp - ribosylation and adp - ribosyltransferase / parp activity have been implicated in a spectrum of disease pathogenesis , such as cancer , inflammatory diseases and neurological disorders ( 30,31 ) . notably , parp inhibitors have already shown clinical benefits in multiple cancers , including olaparib ( lynparza ) that has already been approved by the food and drug administration ( fda ) in the united states and european medicines agency ( 32,33 ) . thus , understanding substrate specificity of parps and how protein function is affected by adp - ribosylation are of timely importance and have the potential to improve our understanding of the biology of diseases . though the modification was discovered over 50 years ago ( 34,35 ) , it was only until recently that several proteomics techniques have been developed to identify the adp - ribosylation sites ( 9,36 ) . such site information will provide initial insights to generate hypotheses for testing the function of adp - ribosylation in modified substrates . to facilitate researchers in appreciating the depth and breadth of adp - ribosylation 's role across the proteome , we have created adpribodb , a database of adp - ribosylated proteins curated from the literature between 1975 and july 2015 ( adpribodb.leunglab.org ) . adpribodb is freely accessible online and provides information for each entry on the protein name , gene symbol , uniprot i d , species / cell line of origin , experimental details and states the original figure / table from which the entry was curated . the database curation effort is ongoing and the website also allows single entries or bulk format upload for publication - associated data sets . it is our hope that adpribodb will offer insights into the adp - ribosylated proteome , and may reveal patterns of adp - ribosylation across substrate proteins , and highlight important interactions and networks , which will aid in our understanding of the myriad roles of adp - ribosylation played in cells . adpribodb was compiled through a manual curation of the literature accessed from pubmed , using the following 16 search terms : parsylation or  poly(adp - ribosyl)ation or  poly - adp - ribosylation or  poly(adp - ribosylation) or  parylated or  parsylated or  poly(adp)ribosylation or  marylation or  marsylation or  mono(adp - ribosyl)ation or  mono - adp - ribosylation or  mono(adp - ribosylation) or  marylated or  marsylated or this parameter was further limited for our search between january 1975 and july 2015 yielding 1456 articles . each journal article was assessed for the inclusion in adpribodb by at least two independent curators . diverse techniques have been applied to demonstrate that the entries are adp - ribosylated (  method ; see supplementary table s1 ) , including immunoprecipitation with anti - par antibodies from cells and autoradiography of in vitro modification of recombinant proteins by adp - ribosyltransferases with p - labeled nad . recent years have seen the application of mass spectrometry approaches for validation of protein modifications and site identification . for the sake of comprehensiveness , adpribodb includes all proteins identified from immunoprecipitation / affinity - based approaches as tentatively adp - ribosylated . the underlying caveat of these approaches using antibodies and adp - ribose binding domains is that it can not distinguish whether these proteins are non - covalently bound with adp - ribose groups or covalently attached to them . however , such distinction can now be made possible with the ability to identify the adp - ribosylation sites ( 9,36 ) . as a result , we include modification details based on mass spectrometry analyses in adpribodb whenever they are available . for example , cebpb was identified in an affinity - based proteomics study using the af1521 adp - ribose binding macrodomain ( 15 ) , but the adp - ribosylation site was subsequently identified in a global site mapping study ( 37 ) ( figure 1 ) . left : snapshots of adpribodb . right : the number of publications identifying adp - ribosylated protein substrates each year from january 1975 through july 2015 , totaling to 459 publications . literature references. if a modified protein was identified , the protein name , gene symbol and corresponding uniprot i d were included in the no uniprot ids and gene symbols were associated in the publication , the curator(s ) determined the best assignment of a uniprot i d based on all the information provided and noted that this entry as  self - assigned , along with any other comments in the  curator comments field . if multiple variants of the protein are available on uniprot , the first variant was chosen . several key experimental details were collected under the  experimental details section , including the origin of the cell line / tissue (  origin ) and the species they are derived from (  species ) . additionally , we noted whether the modification is induced / inhibited by certain drug treatment (  drug sensitivity ) . under the  modification details section , we noted whether the modification is regulated by certain adp - ribosyltransferases (  enzymes ) and whether the modification is mono(adp - ribosyl)ation or poly(adp - ribosyl)ation (  mono / poly ) . in addition , the adp - ribosylation sites and peptide sequences identified by mass spectrometry were included (  sequencing information ) . under the  literature references section , the figure / table number displaying the data for modification and an excerpt of the text describing the data data description. the pubmed i d and year of these publications were included to allow users to easily review or cite the primary sources for the identification of specific adp - ribosylated proteins . all entries , given a unique adpribodb identification number , were organized in a flat text file database . a user search interface was then created using xataface , a web application framework for implementing the open - source relational database management system mysql . adpribodb can be searched by protein name , gene symbol , uniprot i d , pubmed i d and enzymes ( e.g. parp1 ) . the rest of the webpages to explain the functioning of the database were created through weebly . manual curation of literature between january 1975 and july 2015 identified 12 428 entries , in which 8867 of them have protein sequencing data available . the current database entries are derived from 459 publications with experimental supports for parylated and/or marylated proteins . the database currently includes 2389 unique proteins and 573 of them are associated with sequencing information . though 93% of entries are derived from human , over 860 entries are from diverse organisms , which include not only the common model systems , such as mouse , rat , fruitfly and arabidopsis , but also quail , cow , chicken , sea urchin , octopus , various bacteria and viruses ( figure 2a ) . . the publications identifying the highest number of proteins are proteomics studies utilizing mass spectrometry . ( c ) only 79 proteins were overlapped between 10 large mass spectrometry proteomics analyses ( blue ) and the remaining 449 studies ( purple ) included in the database . the relative lack of overlap indicates that current proteomics approaches have yet to reach saturation in identifying adp - ribosylated substrates . ( d ) a representation of the number of times a specific protein is identified . however , 18 proteins were identified at least 10 times and are listed in table 1 . amongst 459 publications , 58% of them ( n = 264 ) identified single adp - ribosylated proteins , 5% ( n = 25 ) identified at least 10 substrates and 2% ( n = 10 ) from proteomics studies with more than 50 substrates identified ( figure 2b ) . among the 2389 unique proteins identified , 80% of adp - ribosylated substrates ( 1908 ) were uniquely identified in these 10 proteomics studies and 17% ( 402 ) from the rest of the 449 publications ( figure 2c ) . surprisingly , only 79 adp - ribosylated substrates were overlapped between these two sets , suggesting that we have yet to reach saturation in probing the adp - ribosylated proteome . consistently , > 71% ( 1708 out of 2389 unique proteins ) were identified only once ( figure 2d ) . adp - ribosylation of human parp1 ( p09874 ) has been the most comprehensively analyzed protein with 111 publications , followed by histones , other parps and their homologs in mouse and rat ( table 1 ) . other proteins identified in at least 10 publications , including tumor suppressor p53 and dna topoisomerase 1 , are also included in table 1 . the availability of this aggregate data potentially allows researchers to gain insights into global functions of adp - ribosylation . our previous informatics analyses indicated that adp - ribosylated substrates are significantly enriched with proteins that are enriched for low - complexity regions , which aid self - assembly of non - membraneous organelles ( 25 ) . using 457 human proteins that have sequencing information in adpribodb , we tested whether these substrates are enriched in particular subcellular compartments . adp - ribosylated substrates are significantly enriched in the proteome of non - membraneous rna granules , including the nucleolus ( 38 ) and stress granules ( 39 ) ( p - value = 1.85  10 and 5.00  10 respectively , fisher 's exact test ; figure 3a ) . these data are consistent with the identification of the adp - ribosyltransferases in these non - membraneous organelles parp1 and parp2 in the nucleolus ( 40 ) and parp5a , parp12 , parp13 , parp14 and parp15 in the stress granules ( 41 ) . consistent with a previous analysis ( 42 ) , both the proteomes of the nucleolus and stress granules are enriched for low complexity domain - containing proteins ( figure 3a ) . in contrast , similar analyses for n - glycosylated substrates are enriched in the endoplasmic reticulum , golgi , lysosome and membrane fractions and these substrates are not enriched for low complexity domain - containing proteins ( figure 3a and  b ) . such a statistically significant association between adp - ribosylated substrates and non - membraneous organelles is in agreement with our informatics - led hypothesis ( 25 ) and recent experimental evidence ( 43,44 ) indicating that par can help seed low complexity domain - containing proteins to form non - membraneous structures . ( a ) adp - ribosylated proteins are significantly enriched in non - membranous compartments , such as the nucleolus ( 38 ) and stress granules ( 39 ) , as determined by fisher 's exact test , with significant p - values highlighted in red . a similar analysis was performed with proteins that have experimentally validated n - glyosylation sites ( n - glyocsyldb ) deposited in dbptm ( 46 ) . as expected , n - glyocsylated proteins were found enriched in the golgi , endoplasmic reticulum , lysosomes and membrane fractions as defined by uniprotkb ( 51 ) . no enrichment of experimentally validated adp - ribosylation sites or n - glycosylation sites was found in the mitochondrial proteome ( 52 ) . ( b ) low complexity domain - containing proteins are significantly enriched in adpribodb in contrast to n - glycosyldb , suggesting a link between adp - ribosylation and low complexity domain - containing proteins . adpribodb is built with the anticipation of several major developments in the adp - ribosylation field first , given the development of various site identification methods ( 9,14,36 ) , we are expecting that a series of proteome - wide analyses will soon be available for a global survey of adp - ribosylated proteins in cells and tissues . the first generation of adpribodb will therefore help connect these newly acquired proteomics data with the existing literature between 1975 and 2015 . in addition , adp - ribosylation sites can be used to correlate with existing databases on other post - translational modifications , such as phosphorylation , acetylation and methylation ( 45,46 ) . second , 19% of adpribodb entries ( 2419 ) include the information regarding the enzyme responsible for the modification of specific substrates , where 69% ( 1659 ) are mediated by parp1 . however , we expect this trend will be changed soon with the coming - of - age technologies to investigate enzyme these include proteome arrays ( 18,19 ) , the development of analog - sensitive adp - ribosyltransferases ( 4749 ) as well as the improvement in performing genetic knockdown by rna interference and knockout using crispr in mammalian cell culture . finally , parp inhibition is actively used in clinical settings . therefore , future development of these parp - specific inhibitors for clinical use should couple with our basic scientific knowledge in how the endogenous adp - ribosylated proteome is affected ( 9,50 ) . as a result , drug sensitivity to report whether certain substrates / sites are sensitive to particular parp inhibitors ( 37 ) . with adpribodb , we aim to provide a one - stop portal for the community to search adp - ribosylation substrates and site information , where basic scientists and clinicians can use this cumulative information to understand basic biology of adp - ribosylation , generate hypotheses in examining adp - ribosylation functions and explain clinical benefits and side effects observed in patients at the molecular level . adpribodb was compiled through a manual curation of the literature accessed from pubmed , using the following 16 search terms : parsylation or  poly(adp - ribosyl)ation or  poly - adp - ribosylation or  poly(adp - ribosylation) or  parylated or  parsylated or  poly(adp)ribosylation or  marylation or  marsylation or  mono(adp - ribosyl)ation or  mono - adp - ribosylation or  mono(adp - ribosylation) or  marylated or  marsylated or this parameter was further limited for our search between january 1975 and july 2015 yielding 1456 articles . each journal article was assessed for the inclusion in adpribodb by at least two independent curators . diverse techniques have been applied to demonstrate that the entries are adp - ribosylated (  method ; see supplementary table s1 ) , including immunoprecipitation with anti - par antibodies from cells and autoradiography of in vitro modification of recombinant proteins by adp - ribosyltransferases with p - labeled nad . recent years have seen the application of mass spectrometry approaches for validation of protein modifications and site identification . for the sake of comprehensiveness , adpribodb includes all proteins identified from immunoprecipitation / affinity - based approaches as tentatively adp - ribosylated . the underlying caveat of these approaches using antibodies and adp - ribose binding domains is that it can not distinguish whether these proteins are non - covalently bound with adp - ribose groups or covalently attached to them . however , such distinction can now be made possible with the ability to identify the adp - ribosylation sites ( 9,36 ) . as a result , we include modification details based on mass spectrometry analyses in adpribodb whenever they are available . for example , cebpb was identified in an affinity - based proteomics study using the af1521 adp - ribose binding macrodomain ( 15 ) , but the adp - ribosylation site was subsequently identified in a global site mapping study ( 37 ) ( figure 1 ) . left : snapshots of adpribodb . right : the number of publications identifying adp - ribosylated protein substrates each year from january 1975 through july 2015 , totaling to 459 publications . literature references. if a modified protein was identified , the protein name , gene symbol and corresponding uniprot i d were included in the  identifiers section . in the circumstance in which a protein was identified but no uniprot ids and gene symbols were associated in the publication , the curator(s ) determined the best assignment of a uniprot i d based on all the information provided and noted that this entry as  self - assigned , along with any other comments in the  curator comments field . if multiple variants of the protein are available on uniprot , the first variant was chosen . several key experimental details were collected under the  experimental details section , including the origin of the cell line / tissue (  origin ) and the species they are derived from (  species ) . additionally , we noted whether the modification is induced / inhibited by certain drug treatment (  drug sensitivity ) . under the  modification details section , we noted whether the modification is regulated by certain adp - ribosyltransferases (  enzymes ) and whether the modification is mono(adp - ribosyl)ation or poly(adp - ribosyl)ation (  mono / poly ) . in addition , the adp - ribosylation sites and peptide sequences identified by mass spectrometry were included (  sequencing information ) . under the  literature references section , the figure / table number displaying the data for modification and an excerpt of the text describing the data data description. the pubmed i d and year of these publications were included to allow users to easily review or cite the primary sources for the identification of specific adp - ribosylated proteins . all entries , given a unique adpribodb identification number , were organized in a flat text file database . a user search interface was then created using xataface , a web application framework for implementing the open - source relational database management system mysql . adpribodb can be searched by protein name , gene symbol , uniprot i d , pubmed i d and enzymes ( e.g. parp1 ) . the rest of the webpages to explain the functioning of the database were created through weebly . manual curation of literature between january 1975 and july 2015 identified 12 428 entries , in which 8867 of them have protein sequencing data available . the current database entries are derived from 459 publications with experimental supports for parylated and/or marylated proteins . the database currently includes 2389 unique proteins and 573 of them are associated with sequencing information . though 93% of entries are derived from human , over 860 entries are from diverse organisms , which include not only the common model systems , such as mouse , rat , fruitfly and arabidopsis , but also quail , cow , chicken , sea urchin , octopus , various bacteria and viruses ( figure 2a ) . ( b ) a histogram displaying how many modified proteins were identified per publication . the publications identifying the highest number of proteins are proteomics studies utilizing mass spectrometry . ( c ) only 79 proteins were overlapped between 10 large mass spectrometry proteomics analyses ( blue ) and the remaining 449 studies ( purple ) included in the database . the relative lack of overlap indicates that current proteomics approaches have yet to reach saturation in identifying adp - ribosylated substrates . ( d ) a representation of the number of times a specific protein is identified . however , 18 proteins were identified at least 10 times and are listed in table 1 . amongst 459 publications , 58% of them ( n = 264 ) identified single adp - ribosylated proteins , 5% ( n = 25 ) identified at least 10 substrates and 2% ( n = 10 ) from proteomics studies with more than 50 substrates identified ( figure 2b ) . among the 2389 unique proteins identified , 80% of adp - ribosylated substrates ( 1908 ) were uniquely identified in these 10 proteomics studies and 17% ( 402 ) from the rest of the 449 publications ( figure 2c ) . surprisingly , only 79 adp - ribosylated substrates were overlapped between these two sets , suggesting that we have yet to reach saturation in probing the adp - ribosylated proteome . consistently , > 71% ( 1708 out of 2389 unique proteins ) were identified only once ( figure 2d ) . adp - ribosylation of human parp1 ( p09874 ) has been the most comprehensively analyzed protein with 111 publications , followed by histones , other parps and their homologs in mouse and rat ( table 1 ) . other proteins identified in at least 10 publications , including tumor suppressor p53 and dna topoisomerase 1 , are also included in table 1 . the availability of this aggregate data potentially allows researchers to gain insights into global functions of adp - ribosylation . our previous informatics analyses indicated that adp - ribosylated substrates are significantly enriched with proteins that are enriched for low - complexity regions , which aid self - assembly of non - membraneous organelles ( 25 ) . using 457 human proteins that have sequencing information in adpribodb , we tested whether these substrates are enriched in particular subcellular compartments . adp - ribosylated substrates are significantly enriched in the proteome of non - membraneous rna granules , including the nucleolus ( 38 ) and stress granules ( 39 ) ( p - value = 1.85  10 and 5.00  10 respectively , fisher 's exact test ; figure 3a ) . these data are consistent with the identification of the adp - ribosyltransferases in these non - membraneous organelles  parp1 and parp2 in the nucleolus ( 40 ) and parp5a , parp12 , parp13 , parp14 and parp15 in the stress granules ( 41 ) . consistent with a previous analysis ( 42 ) , both the proteomes of the nucleolus and stress granules are enriched for low complexity domain - containing proteins ( figure 3a ) . in contrast , similar analyses for n - glycosylated substrates are enriched in the endoplasmic reticulum , golgi , lysosome and membrane fractions and these substrates are not enriched for low complexity domain - containing proteins ( figure 3a and  b ) . such a statistically significant association between adp - ribosylated substrates and non - membraneous organelles is in agreement with our informatics - led hypothesis ( 25 ) and recent experimental evidence ( 43,44 ) indicating that par can help seed low complexity domain - containing proteins to form non - membraneous structures . ( a ) adp - ribosylated proteins are significantly enriched in non - membranous compartments , such as the nucleolus ( 38 ) and stress granules ( 39 ) , as determined by fisher 's exact test , with significant p - values highlighted in red . a similar analysis was performed with proteins that have experimentally validated n - glyosylation sites ( n - glyocsyldb ) deposited in dbptm ( 46 ) . as expected , n - glyocsylated proteins were found enriched in the golgi , endoplasmic reticulum , lysosomes and membrane fractions as defined by uniprotkb ( 51 ) . no enrichment of experimentally validated adp - ribosylation sites or n - glycosylation sites was found in the mitochondrial proteome ( 52 ) . ( b ) low complexity domain - containing proteins are significantly enriched in adpribodb in contrast to n - glycosyldb , suggesting a link between adp - ribosylation and low complexity domain - containing proteins . adpribodb is built with the anticipation of several major developments in the adp - ribosylation field site identification , enzyme  substrate specificity and substrate / site sensitivity to parp inhibitors . first , given the development of various site identification methods ( 9,14,36 ) , we are expecting that a series of proteome - wide analyses will soon be available for a global survey of adp - ribosylated proteins in cells and tissues . the first generation of adpribodb will therefore help connect these newly acquired proteomics data with the existing literature between 1975 and 2015 . in addition , adp - ribosylation sites can be used to correlate with existing databases on other post - translational modifications , such as phosphorylation , acetylation and methylation ( 45,46 ) . second , 19% of adpribodb entries ( 2419 ) include the information regarding the enzyme responsible for the modification of specific substrates , where 69% ( 1659 ) are mediated by parp1 . however , we expect this trend will be changed soon with the coming - of - age technologies to investigate enzyme these include proteome arrays ( 18,19 ) , the development of analog - sensitive adp - ribosyltransferases ( 4749 ) as well as the improvement in performing genetic knockdown by rna interference and knockout using crispr in mammalian cell culture . therefore , future development of these parp - specific inhibitors for clinical use should couple with our basic scientific knowledge in how the endogenous adp - ribosylated proteome is affected ( 9,50 ) . as a result , adpribodb starts to collect information under the field drug sensitivity to report whether certain substrates / sites are sensitive to particular parp inhibitors ( 37 ) . with adpribodb , we aim to provide a one - stop portal for the community to search adp - ribosylation substrates and site information , where basic scientists and clinicians can use this cumulative information to understand basic biology of adp - ribosylation , generate hypotheses in examining adp - ribosylation functions and explain clinical benefits and side effects observed in patients at the molecular level . allegheny health network  johns hopkins cancer research fund ; johns hopkins catalyst award ; american cancer society research scholar award [ 129539-rsg-16 - 062 - 01-rmc ] ; national institute of health [ r01-gm104135 ] . the trainee was funded by an nci training grant [ 5t32ca009110 to c.a.v ] ; khorana program [ to c.a ] . funding for open access charge : allegheny health network  johns hopkins cancer research fund .
adp - ribosylation refers to the addition of one or more adp - ribose units onto proteins post - translationally 
 . this protein modification is often added by adp - ribosyltransferases , commonly known as parps , but it can also be added by other enzymes , including sirtuins or bacterial toxins . while past literature has utilized a variety of methods to identify adp - ribosylated proteins , recent proteomics studies bring the power of mass spectrometry to determine sites of the modification . 
 to appreciate the diverse roles of adp - ribosylation across the proteome , we have created adpribodb  a database of adp - ribosylated proteins ( http://adpribodb.leunglab.org ) . 
 each entry of adpribodb is annotated manually by at least two independent curators from the literature between january 1975 and july 2015 . 
 the current database includes over 12 400 protein entries from 459 publications , identifying 2389 unique proteins . here 
 , we describe the structure and the current state of adpribodb as well as the criteria for entry inclusion . using this aggregate data 
 , we identified a statistically significant enrichment of adp - ribosylated proteins in non - membranous rna granules . 
 to our knowledge , adpribodb is the first publicly available database encapsulating adp - ribosylated proteins identified from the past 40 years , with a hope to facilitate the research of both basic scientists and clinicians to better understand adp - ribosylation at the molecular level .