Note for: PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes (2017)

Note for: PARPs and ADP-ribosylation: recent advances linking molecular functions to biological outcomes (2017)

doi: 10.1101/gad.291518.116

- Discovery of Poly(ADP-ribose) – more than 50 years

- Only PARP1 has been extensively studied but not other PARPs

- PARP1 has been studied in DNA damage detection and repair

- Actually, PARPs have diverse range of biological roles (one of the PTMs)

- Substrate for PARPs -- > protein or nucleicacid + NAD with ADP-ribose

ADP-ribosylation

- Reversible process which adding ADP-ribose from beta-NAD+ to protein – mono- or poly-

- Catalyze the reaction called ADP-ribosylation reaction

- This review focuses on

o Mono(ADP-ribosylation) -- MARylation

o Poly(ADP-ribosylation) -- PARylation

o At amino acid site – glutamate, aspartate, and lysine residues

PARPs

- Writers of ADP-ribose

Accessory proteins

- Readers – contain ADP-ribose-binding domains (ARBD)

Erasers

- ADP-ribose and PAR hydrolases

Feeders

- Providing NAD+ (NAD+ synthases)

Consumer

- Eradicating NAD+ (NAD+ hydrolases)

 

Figure: Dynamics of ADP-ribosylation

PARPSs

- 17 members in human

- Distinct structural domains

- Distinct activities

- Distinct subcellular localization

- Distinct function

- NAD+-dependent enzyme

Classification of PARPs 

- based on structural domains and functions

o DNA-dependent PARPs (PARP1-3) -  3 PARPs

o Tankyrases (PARP-5a and PARP5b) – 2 PARPs

o Cys-Cys-Cys-His zinc finger (CCCH)-containing and WWE PAR-binding domain-containing PARPs (PARP-7, PARP-12, PARP-13.1, and PARP-13.2) – 4 PARPs

o PAR-binding macrodomain-containing ‘macro’ PARPs (PARP-9, PARP-14, and PARP-15) – 3 PARPs

- Based on catalytic activities – mono, poly or inactive

o Mono – PARP-3, PARP-4, PARP-6, PARP-10, PARP-14, PARP-15, and PARP-16

o Poly – PARP-1, PARP-2, PARP-5a, PARP-5b

o No enzymatic activity – PARP-9, PARP-13

- Catalytic domain of many PARPs – H-Y-E (His-Tyr-Glu) motif (not all PARPs)

o His and Tyr --- proper orientation of NAD+

o Glu – catalytic activity

ARBDs: ADP-ribose readers

- Well characterized

o PAR-binding motifs (PBMs)

o Macrodomains

o PAR-binding zinc finger (PBZ) modules

o WWE domains

- Less well characterized

o Phosphopeptide-binding Fork-head-associated (FHA)

o BRCA1 C-terminal (BRCT) domain

o RNA recognition modules

o Arginine and Glycine-rich motifs

PAR-binding domain

 PBM (PAR binding motif)

- ~20 AA

- Loosely defined consensus -- ([HKR]1-X2-X3-[AIQVY]4-[KR]5-[KR]6-[AILV]7-[FILPV]8,) – not certainty

- Gap remains

Macrodomains

- Larger ~130- 190 AA

- Globular domain

- Recognize either ADP-ribose monomer or terminal ADP-ribose moiety 

PBZ

- ~30 AA

- Recognize consensus sequence [K/R]-X-X-C-X-[F/Y]-G-X-X-C-X-[K/R]-[K/R]-X-X-X-X-H-X-X-X-[F/Y]-X-H

- Bind ADP-ribose-ADP-ribose junctions of PAR chain

- Bind ADP-ribose monomer

WWE

- Has conserved W and E 

- Bind to iso-ADP-ribose moiety in PAR chains

- Bind exclusively to ADP-ribose oligomers or polymers

Eraser

- Two types

o Cleavage of ribose-ribose bonds but leaves a terminal ADP-residue moiety attached to acceptor AA – leaving a scar

o Hydrolyze the ester bond between ribose and acceptor AA – complete remove

- Turnover of PAR is fast

- Dynamic turnover of ADP-ribose is critical for multiple cellular processes

- Has different subcellular localizations

- Thus, suggesting different turnover rate in different compartment

Feeder

- Nicotinamide mononucleotide adenylyl transferases (nicotinamide mononucleotide + ATP = NAD+)

- NAD+-dependent enzyme

- Three enzymes involved and each locates in different subcellular compartment

o NMNAT-1 – nucleus

o NMNAT-2 – cytoplasm and golgi

o NMNAT-3 – mitochondria

- Thus, the amount of NAD+ affects the activity of PARPs

NAD+ consumer

- Besides PARPs, there are other enzymes using NAD+

o NADase

o CD38 (enzyme)

o Sirtuins

 

Roles of PARPs in cellular processes

 

Time line of discoveries for PARPs and ADP-ribosylation

- DNA damage repair, cell survival and cell death

o PARP1 is the most studied enzyme

o PARP1 recognizes nick as well as double strand breaks

o DNA damage – production of long PAR chains on PARP1 itself and the other repair-related protein – recruit other PAR-binding proteins

 XRCC1 – scaffolding protein (BER player)

 CHD4

 APLF and CHFR

 Macrodomain containing proteins

 MRE11 and ATM also recognized PAR on PARP1

o Excessive PARylation by PARP-1 can direct cell away from DNA repair pathways toward the activation of cell death pathways (parthanatos – caspase independent)

o Stress stimulant increase – higher activity of PAPR1 – higher PARylation – induce cell-death rather than repair

o Thus, different level of PARylation contributes to different cellular processes – different protein recognizes different degree of PARylation

o PARP3 (mono ADP-ribose) – involves in DNA repair 

o PARP3 – critical for DSB repair

o PARP3 – add ADP-ribose to KU80 – directing DNA repair to NHEJ

Structural component of PARP1-3

PARP1

- Zn1 and Zn2 domains – contact DNAasdf44829

- Zn3 domain DNA-dependent PARP-1 catalytic activity and chromatin compaction

- Zn1, Zn3 and WGR domains – activation of PARP-1 at DNA damage site

- Upon DNA damage binding – induce conformational change – enable for catalytic activity by which opening up helical domain for NAD+ entering

PARP2

- No Zn finger domain, short N-terminal region with WGR and catalytic domain

- PARP-2 requires 5’ phosphorylation at the DNA breaks for activation

Interplay between ADP-ribosylation and ubiquitylation

- There is PAR-dependent ubiquitylation – leading to proteasome degradation

- The E3 CHFR can bind autoPARylated PARP-1 through its PBZ domain – ubiquitylation and degradation

- This is thought to be a general mechanism controlling the stability and degradation of many PARP substrate proteins

PARPs localization

- Cytosol

o Can be found in specific sub organelle

- Nucleus

- Shuttle between cytosol and nucleus and related to cell cycle

  

Many roles of PARPs 

Method to study the modification

- Reagent to detect MAR, oligo-(ADP-ribose) and PAR in cells and biochemical assays

- Mass spectrometry to identify ADP-ribosylated proteins and the specific sites of ADP-ribosylation

- Chemical biology to assign ADP-ribosylation events to determine the sites of ADP-ribosylation

- Genomic assays to determine the site of ADP-ribosylation across the genome

- Assay the levels of NAD+ in various cellular compartments

Methodology overview to detect the ADP-ribosylation

ADP-ribose detection reagents

- Anti-PAR monoclonal antibody 10H – bind PAR chains > 10 ADP-ribose units

- Using ARBD GFP fusion 

- Using ARBD fuses with Fc region of Ig

- Using chemical biology approach labeling ADP-ribose (through substrate corporated into ADP-ribose) – using click chemistry to visualize the labelling

Proteomics and MS

- Detecting ADP-ribosylation is very difficult

- Labile

- Heterogeneous

- Polymers are bulky and charged

- Presence of multiple active PARP making it hard to study on specific PARP on ADP-ribosylation proteome

- Recent advance in proteomics

o Degrade PAR chains down to a single moiety 

o Cleave ADP-ribosylation moiety to produce identifiable mass shift

- Chemical cleavage

o Hydroxylamine chemistry – using with aspartic acid and glutamic acid only

o Other linkage, etc,. lysine or arginine – cannot use this approach

 

- Enzymatic cleavage

o Snake venom phosphodiesterase, ARH3, PARG or NUDIX hydrolases

o They are seeking for the alternate enzymes which can be used to elucidate the ADP-ribosylation sites in many different proteins

- Chemical biology approach

o Using NAD+ analog as the substrate and using special mutant PARP to detect the targeted protein that has been ADP-ribosylation

 

Database for ADP-ribosylated proteins

- Leung group provides database for the list of ADP-ribosylated proteins derived from >400 publication

- http://adpribodb.leunglab.org/

- Contain 2389 unique protein targets

Genomic analyses of ADP-ribosylation

- ADP-ribose chromatin affinity precipitation (ADPr-ChAP)

- Click-ChIP (click chemistry-based chromatin isolation and precipitation)

Cellular NAD+ sensor

- Dynamic NAD+ synthesis and its subcellular localization are important factors for PARP function

- Using Preredox sensor – determine NADH/NAD+ level in cells

- Using the protein domain, which bind to either NADH/NAD+, tags with FP

PARP inhibitors

- Veliparib and niraparib have the highes selectivity for PARP1 and PARP2

- Other inhibitors, olaparib, has high potency but lower selectivity for PARP 1 and 2

Gap

- Factor drives selectivity and specificity for different substrates by different PARPs

- Sits of ADP-ribosylation in theirs target proteins

- Function of ADP-ribosylation  of target proteins

- Therapeutic potential of PARPs in diseases