#parps

[ginger-karkitty]

More Adventure on MSPARP

I was Eridan

[biochemistries]

Strain alleviation

One class of post-translational modification (PTM) applied to proteins in the cell is the addition of ADP-ribose (ADPr). There are three classes of enzyme responsible for handling this PTM in humans, forming a family of some 17 members. The H-Y-E (His-Try-Glu) triad-containing PARPs (poly-ADP ribose polymerases) possess PARylation activity (PARPs 1-5).

Counterintuitively, despite their names PARPs 6-8, 10-12, and 14-16 don't contain this triad, and as such can only carry out mono-ADP-ribosylation (elsewhere they're termed MARTs, and all the PARPs have alternative ART-prefix names, but the HUGO committee has left them with this 'official' misnomer). The third class of PARPs are non-functional, lacking the NAD+ binding residues needed to hold close the enzyme's cofactor.

PARP1 is the most abundant of its family, with 200k to a million copies in every cell (generating >95% of intracellular PAR), yet rapid degradation and tight control over catalytic activity keep its levels low until DNA damage occurs (both single and double-stranded breaks). When they do spring to action, the primary acceptor of the PTM is none other than PARP1 itself. Beyond PARP self-PARylation ("auto-modification"), DNA ligases, histones, and p53 also take on ADPr additions, for rapid recruitment to appropriate sites, or towards modulating their function.

The first moiety is joined via ester bond onto Glu or Asp residues (sometimes Lys, including on histone tails as I've written about previously where it appears Glu isn't used), after which the polymer assembles through a unique 2′,1′′-O-glycosidic ribose-ribose bond to make linear chains of up to 200 ADPr-unit PAR in vitro (the length and branching of such chains in vivo are unknown).

In concert with these polymerisations, 'readers' transduce the physical signal: PAR-binding linear motifs (PBMs), PAR-binding zinc fingers (PBZs), macro domains which are the only type to recognise MAR (see Karras 2005), and WWE domains. If not catabolised upon release into AMP and ribose-5-phosphate, as a reducing sugar ADPr can cause protein glycation and glycoxidation. Thus, following DNA damage, ADP-ribose signalling doesn't just add warning flags to proteins, but becomes directly mixed up in modulating histone-histone cross-linking in the nucleus via Schiff base (imine) adduct formation on their amino groups.

Last summer, a group reported that Cys could also be ADP-ribosylated on PARPs (6, 8, 11, and 12), presumably overlooked through earlier experimental fixation on PARP1.

Why do cells need both types of ​ADPr modifications? MAR and PAR synthesis activities are both evolutionarily conserved, indicating that both have important functions in cellular physiology. PAR functions during stress responses and physiological pathways that require the rapid assembly of multiprotein complexes, acting as a protein binding scaffold. The consequences of MAR modifications on target protein are less understood. Recent work showing that ​ADPr-binding Macro domain-containing proteins can specifically bind MARylated targets suggests that one function could be to regulate specific protein-protein interactions, similar to SH2 domains binding to phosphoproteins (Karras '05, Verheugd '13, Forst '13). MAR modifications are also especially interesting because they could serve as primers for further elongation to PAR, with PARPs functioning cooperatively to synthesize polymer, allowing a cell to tightly regulate each step of PAR generation. This possibility is supported by in vitro data showing heterodimerization and activation of PARPs with distinct activities, by the fact that PARPs with each activity are localized in both the cytoplasm and nucleus and by the presence of multiple physiological protein complexes containing MAR and PAR generating PARPs.

Perhaps most ingeniously, through similarity to RNA, PAR can compete for RNA-recognition domains and thus control protein function. It may prevent RNA splicing for example, by specifically binding the prototypical SR splicing factor ASF/SR2 which as I've described previously promotes splicing through binding exonic splicing enhancer (ESE) sequences in pre-mRNA transcripts. Siphoning off the available SR protein in this way produces an unlikely switch (systems biologists call the phenomenon 'thresholding by molecular titration').

DNA topoisomerase I is an enzyme with two mutually exclusive functions: in-keeping with its name, it controls DNA supercoiling, relaxing the molecule of torsional stress that builds up during DNA repair (see prev.) but less well known is that it specifically phosphorylates these SR proteins. When PAR is introduced and SR proteins thus rendered inaccessible, topo. I's workload shifts from phosphorylating these proteins to cleaving the double helix (the only way to relieve torsional stress in such a long polymer).

topo I bears three PAR-binding sites localized in domains that are critical for the catalytic activity of the enzyme on DNA and for its regulation (Malanga 2004). In fact, PAR has a dual effect on topo I: it inhibits DNA cleavage (thus preventing initiation of new catalytic cycles) while it stimulates the re-ligation activity of the enzyme blocked in a ternary complex with nicked DNA and [anticancer drug camptothecin] CPT (thus counteracting the poisoning effect of the drug)

More direct topological effects are seen in the mechanism of ADP-ribosylation by Ia, the enzyme component of Clostridium perfringens iota toxin. As shown above (from a 2013 commentary on the experiments that chanced upon this mechanism), the Ia ADP-ribosyltransferase component binds the AMP moiety of NAD+, releasing nicotinamide and forming an oxocarbenium cation intermediate.

Thereafter, a rotation around the axes of the phosphodiester bond forms a second oxocarbenium cation, which results in strain relief and brings the C1 (NC1) of the ribosyl moiety near to arginine177 (R177) of actin to complete the transfer of ADP ribose.

From the original report by Tsurumura et al. :

The mechanism by which an SN1 reaction [nucleophilic substitution, unimolecular rds] leads to ADP ribosylation of Arg177 was suggested by studies of the Ia structure and its site-directed mutagenesis. The positively charged Arg295 and Arg352 (Ia) interact electrostatically with NAD+ phosphate and contribute to the highly folded and strained conformation of the NMN [nicotinamide mononucleotide] ring-like conformation. This specific structure of NAD+ is conserved among all ART [ADP-ribosyltransferase] family members. The specific conformation appears to induce an equilibrium shift toward formation of an oxocarbenium cation. After cleavage of the nicotinamide, the oxocarbenium cation may be stabilized via Tyr251 through a cation-pi interaction. In the present NAD+-Ia-actin structure, the distance between the nucleophile (Arg177) and the electrophile (NC1 of N-ribose) is 8.2 Å, making it necessary to reduce the distance between Arg177 and NC1 of N-ribose. In an earlier paper, we proposed a strain-alleviation model in which a second oxocarbenium ion acts as an intermediary. In that model, "strain" referred to the highly folded and strained conformation of NMN, whereas "alleviation" referred to the rotation of the N-ribose after scission of the nicotinamide. That is, SN1 cleavage produced the first oxocarbenium cation, after which rotation occurred via NP-NO5 of ADP ribose to produce a second cationic intermediate, which enabled NC1 of N-ribose to approach the guanidyl nitrogen of Arg177. In the present pre- and postreaction state structural study, the strain-alleviation model was confirmed experimentally and was improved.

The key features of the improved model are as follows (Fig. 7A, cf. Ueda 1985). The ADP moiety is gripped by Ia, and then the ADP ribosylation reaction occurs. The grip is necessary for the subsequent rotation (Gill 1969). To reduce the distance between the nucleophile (Arg177) and the electrophile (NC1 of N-ribose), after the first oxocarbenium ion intermediate is produced (SN1 reaction), the rotation of N-ribose occurs mainly via rotation of both O3-NP and NP-NO5 (Moss 1977). The present structure confirms that the N-ribose 3′OH is in close proximity to Asp179 (3.5 Å) within Ia-ADPR-actin. We envision that actin Asp179 plays a stabilizing role by making contact with the N-ribose (Gill 1978). Arg177 tilts slightly to react with N-ribose.

ADP-ribosylated actin is then said to be 'capped': the cytoskeletal polymer will no longer elongate (while the levels of PARP1 are highly dynamic, the PTM itself is stable). The finding was incidental:

By chance, the authors obtained the complex NAD+-Ia-actin as a prereaction state by using the cryoprotectant ethylene glycol, which blocked the ADP ribosylation reaction. All arginine-modifying ADP-ribosylating toxins are characterized by an EXE motif (378E-X-E380 in Ia). This motif is part of ADP ribosylation turn-turn loop (7) and plays a pivotal role in catalysis and protein substrate recognition. Although the second Glu is essential for ADP-ribosylation and NADase activity, the first Glu (E378) is needed for the ADP-ribosyltransferase reaction but not for NAD+ hydrolysis. Therefore, by exchanging E380 to serine NAD+ hydrolysis was blocked and a NAD+-Ia-actin complex could be crystallized.

In this prereaction form, the ADP of NAD+ is held in the grip of Gln300, Asn335, and Arg352 of Ia (shown above). The nicotinamide mononucleotide (NMN) phosphate is coordinated by Arg295, while the NMN ribose interacts with the EXE motif glutamines of the Ia toxin. The Arg296 residue of Ia stabilises with the carboxyl amide group of the nicotinic acid moiety, producing the distorted and strained form of NAD's NMN moiety, typical for all known ADP-ribosyltransferases.

The acidic Glu380 of the EXE motif provides stability for the NMN ribose conjoining glycosidic bond to break, and these glutamine residues also help to keep the oxocarbenium ion that develops from reacting with "unfavourable water". Tsurumura and colleagues wrote that bond cleavage here (and the subsequent protein conformational trigger) is a delicate interplay of physical properties and the chemical factor.

There should be an equilibrium between NAD+ and oxocarbenium cation/nicotinamide. If there is still strain in the first oxocarbenium cation intermediate, it creates the second conformation of the oxocarbenium cation intermediate by alleviation and finally interacts with arginine. It seems that the strain strongly supports the forward reaction not to go backward.

As mentioned above, ADP-ribosylation impinges on chromatin packaging of the genome, but also with p53, and as such is of interest to anticancer drug development. PARP1, PARP6 and PARP10 are themselves tumour suppressors for example, though a recent review was hesitant to speculate on roles of PARPs in cancer more generally.

Leung (2011) PAR regulates stress responses and miRNA activity in the cytoplasm

In addition to DNA lesions, they also follow up on the unfolded protein response (the PERK and IRE1-, but not ATF6-mediated routes), miRNA gene regulation (connected to PARPs in stress granules), cancer-related signal transduction (NF-kB, JNK, and STAT pathways), and cell migration.

PARP10, a MAR-generating PARP with RNA- and ubiquitin-binding domains, has functions in multiple signalling pathways. It was initially identified as a MYC-interacting protein that inhibits the transformation of rat embryo fibroblasts by MYC and HRAS, but its effect was independent of its ADP-ribosylase activity. This was the first indication of a potential tumour-suppressive role for PARP10 because MYC — a transcription factor that regulates many cellular processes, including cell proliferation — is frequently deregulated in cancer cells and is associated with tumour progression.

PARP10 was also recently shown to regulate NF-κB signalling in a manner that is dependent on both PARP10 interacting with polyubiquitin chains on proteins that regulate NF-κB activity and its ADP-ribosylase activity. Exogenous PARP10 expression in both HeLa and U2OS cells resulted in the inhibition of downstream NF-κB target gene expression in response to interleukin-1β (IL-1β) and tumour necrosis factor (TNF) by altering the polyubiquitylation state of several NF-κB signalling intermediates and preventing the translocation of the NF-κB transcription factor subunit p65 (also known as RELA) to the nucleus. Additionally, PARP10 overexpression in HeLa cells inhibited cell proliferation through the induction of apoptosis, although the contribution of altered NF-κB and/or MYC signalling to apoptosis induction was not investigated. PARP10 function in the regulation of NF-κB signalling is physiologically relevant, as short hairpin RNA (shRNA)-mediated and siRNA-mediated knockdown of endogenous PARP10 increased the expression of NF-κB targets in HeLa and U2OS [cervical and bone cancer] cell lines57.

Vyas & Chang (2014) New PARP targets for cancer therapy

The enigmatic vault particle, whose arresting structure I wrote of recently, contains a vault PARP (VPARP or PARP4), yet knockout showed no change of phenotype in mice (reviewed in 2013).

In January this year, the first PARP inhibitor was approved by the European Commission: olaparib, from a Cambridge researcher's drug development company that was acquired by AstraZeneca — see Reuters press release and preceding Cancer Research UK blog post, whose scientists played a role in bringing it to market "for women with advanced serous ovarian cancer, fallopian tube and peritoneal cancer who carry a faulty BRCA1 or BRCA2 gene". It inhibits PARP1, PARP2, and PARP3 in these individuals' BRCA-mutated cells, meaning tumour DNA repair at lesions is scaled back, the chromosomal instability is not rescued, tumours stop growing as the cell cycle arrests, and die off through subsequent apoptosis.

As mentioned above, DNA repair is not the only site of PARP involvement. Similarly, inhibiting the unfolded protein response with these drugs activates the apopotic programme. This was mentioned in passing within a review last year of New PARP targets for cancer therapy:

PARP10 is enriched in cytoplasmic polyubiquitin-containing foci that can interact with autophagosomes marked by p62 (also known as SQSTM1 [sequestome 1]), which is a ubiquitin-binding autophagy adaptor protein. Although the role of PARP10 in autophagy has not been investigated, this association is particularly interesting because autophagy is activated in cancer cells and might represent another mechanism to cope with cellular stress (Mathew '07, Yang '11, Sui '13). Inhibition of autophagy can sensitize cancer cells to chemotherapy or inhibit tumour growth. The autophagy pathway exerts cytoprotective effects on cancer cells by inhibiting apoptosis and necrosis in response to metabolic stress. As NF-κB signalling can regulate autophagy, PARP10 might represent an important link between these two pathways. Further study of the function of PARP10 in NF-κB signalling will be important to understand its role in normal physiology and disease, and to determine if it does indeed have a function in autophagy regulation.

Langelier (2013, Current Opinion in Structural Biology) noted that "recent structural studies have illustrated how PARP-1 uses specialized zinc fingers to detect DNA breaks through sequence-independent interaction with exposed nucleotide bases, a common feature of damaged and abnormal DNA structures", but notes there's little knowledge of how substrate selection occurs. While we have clues to human PARP mechanism through homology to bacterial toxins, as of yet it is currently too complex for detailed chemical studies.

As mentioned above, the PAR modification is removed by PAR glycohydrolases (PARGs), and the subsequent balance serves as a signal transduction in the cell. While PARPs constitute a large family, we currently only know of one PARG. A distinction is made between 'endo' and 'exo' glycohydrolase activity: debranching enzymes vs. those that make their cut at the end. Biochemical and modelling results seem to show the human PARG is predominantly exo, thanks to a particular residue which in humans gives only low-affinity binding in endo-mode (specifically Phe908 breaks 3 or 4 important H bonds, giving >100-fold difference in Kd).

It's been suggested that this balance is baked in to allow for death signalling when the PAR:PARG ratio becomes too high. One of the lesser known modes of cell death, 'parthanatos', takes place when the "otherwise beneficial" mitochondrial-associated apoptosis inducing factor flavoprotein is released from mitochondria (by PAR upon PARP overactivation), and moves to the nucleus where it causes large-scale DNA fragmentation and spells the bitter end of that cell.

See also: a 2013 FEBS minireview from Barkauskaite et al. covering PARPs, PARGs and how Macro domains effect the complete reversibility of the process, The recognition and removal of cellular poly(ADP-ribose) signals — which notes a 'growing vibe' :-)

The vibe in the ADP-ribosylation field is growing, with promising new developments in our understanding of how PAR is both recognized and removed. The appreciation that ADP-ribosylation signalling is involved in diverse disease phenotypes is interesting and provides added impetus to further dissect this elusive post-translational modification.