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)
- 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) -- MARylationo Poly(ADP-ribosylation) -- PARylationo 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 PARPso Tankyrases (PARP-5a and PARP5b) – 2 PARPso 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 PARPso 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-16o Poly – PARP-1, PARP-2, PARP-5a, PARP-5bo 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 Macrodomainso PAR-binding zinc finger (PBZ) moduleso WWE domains
- Less well characterized
o Phosphopeptide-binding Fork-head-associated (FHA)o BRCA1 C-terminal (BRCT) domaino RNA recognition moduleso 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 – nucleuso NMNAT-2 – cytoplasm and golgio NMNAT-3 – mitochondria
- Thus, the amount of NAD+ affects the activity of PARPs
NAD+ consumer
- Besides PARPs, there are other enzymes using NAD+
o NADaseo 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 enzymeo PARP1 recognizes nick as well as double strand breakso 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 repairo Thus, different level of PARylation contributes to different cellular processes – different protein recognizes different degree of PARylationo PARP3 (mono ADP-ribose) – involves in DNA repairo PARP3 – critical for DSB repairo PARP3 – add ADP-ribose to KU80 – directing DNA repair to NHEJStructural 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 moietyo Cleave ADP-ribosylation moiety to produce identifiable mass shift
- Chemical cleavage
o Hydroxylamine chemistry – using with aspartic acid and glutamic acid onlyo Other linkage, etc,. lysine or arginine – cannot use this approach
- Enzymatic cleavage
o Snake venom phosphodiesterase, ARH3, PARG or NUDIX hydrolaseso 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
- 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
Comments
Post a Comment