Structural and Evolutionary Analysis of PARPs in D. discoideum

Problem statement: Dictyostelium discoideum, a unicellular eukaryote, exhibits multicellularity upon nutrient starvation, making i t a better model for developmental studies and for the study of various signal transduction pathways. The most felicitous point of interest is that many of its genes show high degree of similarity to verteb rate genes. Poly (ADP-ribose) polymerase (PARP), a ubiquitous and abundant nuclear protein, has a num ber of distinct biochemical activities well suited for both structural and regulatory roles throughou t its life cycle. We have analysed structural and evolutionary significance of PARP. Approach: D. discoideum lacks caspases and hence it exhibits caspase independent cell death which is of unique i nterest. PARP is a key protein involved in cell death in D. discoideum. An in silico approach to study the domain organization of PARP’ s in D. discoideum would help us to understand evolution of the struc tural pattern from prokaryotes to eukaryotes. Results: Our previous studies showed involvement of PARP in D. discoideum cell death and development. We have attempted to probe the sig nificance of PARPs in D. discoideum using bioinformatics approach. In this organism PARPs are encoded by 8 members whereas in H. sapiens there are 17 such members encoding PARP family. Conclusion: Our analysis suggests out of 8 genes, adprt1a and adprt1b seem to be involved in DNA damage response. Our app o ch with different bioinformatics tools suggests that these proteins a lso show homology with the H. sapiens counterparts. This article summarizes the domain organization of PARPs to throw light on the biological role of these proteins which will be helpful for further ex p rimental studies in our model organism.


INTRODUCTION
DNA damaging agents like ROS, MNNG and UV irradiation are known to activate PARP, a nuclear enzyme that has various physiological functions (Rajawat et al., 2007;Burkle, 2001;De et al., 1994;Lautier et al., 1993;Shall and Murcia, 2000;Vodenicharov et al., 2005). Activated PARP cleaves its substrate NAD + and transfers ADP-ribose units to several target proteins including itself (Burkle, 2001;Shall and Murcia, 2000;Smulson et al., 2000. Poly ADP-ribosylation is a unique post-translational modification playing crucial role in various cellular processes such as DNA damage signaling, repair, transcription regulation, chromatin modification, intracellular trafficking, mitotic apparatus formation and cell death. In response to DNA damage, PARP-1 uses NAD + as a substrate and attaches polymers of ADP-ribose on different acceptor proteins (heteromodification) or on PARP-1 itself (auto-modification), resulting into a branched polymer known as PAR (Poly ADP-ribose) which can be covalently linked mainly to glutamic acid residues (Hakme et al., 2008) of acceptor proteins i.e., the polymerization starts at a glutamic acid residue (Skalitzky et al., 2003). PAR moieties thus formed are degraded by Poly (ADP-ribose) Glycohydrolase (PARG) and lyase (Shunya et al., 2006;Hayaishi and Ueda, 1982;Okayama et al., 1978). Recently ADP-Ribosyl Hydrolase-3 (ARH3) in human has been identified to have PARG like activity (Mueller-Dieckmann et al., 2003). The role of PARGlike activity of ARH3 seems to be not vital for cell death processes. Also, this enzyme does not significantly contribute to the cell survival process or PAR hydrolysis during cell stress conditions (Koh et al., 2004).
Dictyostelium discoideum is a soil living amoeba that grows as separate, independent cells but interacts to form multicellular structures when challenged by adverse conditions such as starvation. Its genome consists of 34 Mb of DNA which is compacted into six chromosomes ranging from 4-7 Mb each (Eichinger et al., 2005). It comprises of nearly 8,000-10,000 genes and the most interesting point is that many of the genes show high degree of similarity with those of higher organisms. Structural studies with different bioinformatics tools revealed high homology of D. discoideum PARPs with those of H. sapiens. This article summarizes the domain organization of PARPs to throw light on the biological role of these proteins which will be helpful for further studies in our model organism.

PARPs in D. discoideum:
Though the domain architecture analysis by PROSITE (Hulo et al., 2006;De et al., 2006), an ExPASy database of protein domains, suggests differences among H. sapiens and D. discoideum PARPs but their functions remain the same. It remains a matter of fascination that how PARPs interact with diverse set of proteins though functions of major domains have been dissected out. Also functions of a few domains like macro domain, WWE domain and WGR domains are still to be studied adequately. The only major difference observed on the evolutionary ladder is absence of Zn Finger (ZnF) in lower organisms (prokaryotes) while the transition from lower to higher forms of life the number of ZnF increases.
D. discoideum genes possess high degree of similarity with those of higher eukaryotes, here we demonstrate the domain homology of D. discoideum PARPs with that of H. sapiens. As shown in Table 1 in D. discoideum PARPs are encoded by 8 members whereas in H. sapiens there are 17 such members encoding PARP family (Otto et al., 2005). BLAST (Altschul et al., 1990) analysis shows that ADPRT1A and ADPRT1B of D. discoideum show ~50% similarity to human PARP-1 ( Fig. 1 and 2     These two proteins possess similar domains as human PARP-1 excepting being less by one ZnF domain. ADPRT2 of D. discoideum carries an extra BRCT (breast cancer susceptibility protein C terminus motif) domain and shares similarity to human PARP-2 by 57% ( Fig. 3) whereas ADPRT4 which is similar to ADPRT2 in domain composition aligns better with human PARP-4, however ADPRT4 lacks VIT and VWFA domains (Fig. 4). ADPRT3, pARTg and pARTf also show different degree of homology with different human PARPs ( Fig. 5-8). Table 2 summarizes the similarity between D. discoideum and human PARPs. Representation of all the PARPs/pARTs present in D. discoideum with gene ID and protein ID sequence and it is fetched through the Dictybase (Kreppel et al., 2004;Fey et al., 2009) and EMBLmm (Guenter et al., 1999), (Fig 1-8). Schematic representation of 8 different members of PARP superfamily of D. discoideum compared with that of H. sapiens PARPs. It is well established that all the members of D. discoideum show high degree of similarity with that of higher eukaryotes. This study (B and C) has been done by using T-coffee online server (Notredame et al., 2000) Our BLAST search shows an ADPRH (D. discoideum) protein ( Fig. 9) which shows ~53% similarity to (H. sapien) ARH3 protein which is shown to have PARG-like activity (Mueller-Dieckmann et al., 2003). Nonetheless the biological significance of PARG like activity of ARH3 is still poorly understood (Shunya et al., 2006). Fig. 10A form characteristic of PARP. Presence of Zn Finger (ZnF) domain is essential for sensing DNA damage and further recruiting DNA repair machinery. ZnF-like motifs of the form CX2C-X28/30-WHX2C are present in 1-3 copies at the Nterminus of different DNA repair enzymes (Caldecott et al., 1996;Mackey et al., 1999;D'Amours et al., 1999) hence PARP ZnF is associated with the function of strand-break sensing (Caldecott et al., 1996;Mackey et al., 1999;D'Amours et al., 1999;Gradwohl et al., 1990;Ikejima et al., 1990). This motif is also present in the sequence of ADPRT1A ZnF domain of D. discoideum (CX2C-X28/30-WHX2…… ..YEIEYAKSDRSTCSTCQRGINKEAVRIGYKTKSK HFDGMDVSWHHLKCKCPQVPSFTDLIHWEYLR WE…) (highlighted in red) hence this protein could also be involved in DNA damage sensing in D. discoideum. This BRCT domain is present in ADPRT1A, PARP-2 and PARP-4 in D. discoideum. This structure is modeled with template (2COK) Solution structure of BRCT domain of PARP-1 of Homo sapiens. The BRCT domain of PARP consists of AMD i.e., automodification domain. The automodification site comprises of~9-15 glutamates residues which are thought to be important for automodification (Ikejima et al., 1990).

Structural modeling of different domains of PARP in D. discoideum: As mentioned earlier certain domains shown in
BRCT domain is also essential for protein-protein interaction which in turn is important for recruitment of XRCC1 to DNA damage sites in addition to the recruitment of PARG (necessary for PAR turnover) (D'Amours et al., 1999). WGR domain of PARP has been named after the most conserved central motif of the domain W-G-R as shown in the figure 10B and C. This domain is present in many of polymerases and other proteins with unknown function and ranges between 70-89 residues. The function of this domain is still unclear however it is proposed to be important in nucleic acid binding. The regulatory domain of the protein is in association with the C-terminal catalytic domain and consists of ~130 amino acids with duplication of 2 helix-loop-helix structural repeats. It is thought to relay the activation signal issued on binding to damaged DNA (Pion et al., 2005;Ruf et al., 1996;Oliver et al., 2004). Macro domain is ADP-ribose binding module (Karras et al., 2005). The 3D structure of the macro domain has a mixed α/β fold of a mixed β sheet sandwiched between four helices and consisting of ~180 amino acids. It has been suggested to play a regulatory role in ADP-ribosylation (Oliver et al., 2004). Catalytic domain possesses NAD + binding site (Oliver et al., 2004). T coffee alignment results (Fig. 1) suggest that the catalytic domains of ADPRT1A, ADPRT1B, PARP-2, PARP-3, PARP-4, PARTf and PARTg have throughout conserved glutamate residue which is very essential for its activity. It has been reported that role of GLU 988 in human PARP catalytic domain is very important for its enzymatic activity (Gerald et al., 1995) hence the presence of GLU residue in catalytic domain of D. discoideum PARP reflects its function similar to that of human PARPs. Although proteins of the PARP family are related through their PARP catalytic domains, they may not resemble each other outside of that region.

Overlap of D. discoideum PARP with human PARP:
We used Superpose Web Server (Maiti et al., 2004) to obtain Root Mean Square Deviation (RMSD) in order to measure average distance and divergence between the backbones of superimposed domains of D. discoideum and H. sapiens PARP. RMSD reflects conformation of the protein backbone as well as the rotameric states of the side chains. Lower is the RMSD value better is the alignment of the superposed proteins.
Results suggest that the RMSD value of ADPRT1A for both the domains are between 0-3, whereas ADPRT1B shows higher RMSD for catalytic domain therefore ADPRT1A is more similar to that of human PARP-1 than ADPRT1B (Fig. 11).

Evolution of PARP:
Using Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) (Jensen et al., 2009) we generated phylogenetic profile of PARP and related proteins i.e. organisms containing PARP and functionally related gene (Fig. 12). Functionally associated proteins often have similar phylogenetic profiles and conserved amino acids (Sanchez-Aguilar et al., 2007). It has been experimentally proved that this organism does not have caspases (Olie et al., 1998) which is also seen in the phylogenetic profile. Wet lab results in our lab have shown the involvement of PARP in D. discoideum cell death and development (Rajawat et al., 2007; which is substantiated by the appearance of PARP in D. discoideum in the phylogenetic profile. We have shown the involvement of PARP during D. discoideum normal development by our PARP down-regulation studies (Rajawat et al., 2011).
Constitutive PARP down-regulation resulted in blocked development while no effect was observed on D. discoideum growth (Rajawat et al., 2011). Interestingly, stage specific down-regulation arrested development at the slug stage (Rajawat et al., 2011). Also results in our lab have shown the role of PARP in oxidative stress and UV-C stress induced delayed development of D. discoideum (Rajawat et al., 2007;. These results emphasize that PARP is essential for complex differentiation and its function may be linked to multicellularity adding a feather to its multitasking characteristic. On the other hand PARP inhibition during oxidative stress in lower organisms like E. coli and B. thuringiensis shows contrasting results. E. coli did not show any effect of PARP inhibition when subjected to oxidative stress unlike B. thuringiensis wherein inhibition of PARP rescued oxidative stress induced cell death. These results (data not shown) are in accordance of the phylogenetic profile (Fig. 12) as done using STRING server which shows E. coli lacks PARP whereas genome of Bacillus spp bears it.
With evolution, proteins gain and lose certain functions, this reflects in terms of gain and lose of domains corresponding to the functions. PARP protein also displays such evolution. The Fig. 13 depicts that PARP like protein of bacteria B. thuringiensis lacks ZnF as well as BRCT domain while BRCT domain is found in PARP protein in A.nidulans nevertheless, the ZnF domain remains absent here also. The appearance of ZnF domain in D. discoideum further signifies the transitional state of this organism between prokaryotes and eukaryotes. PARP shows an increase in the number of domains as well as number of encoding genes in the evolutionary lineage from lower organisms to higher eukaryotes. In higher organisms there is an addition of ZnF domain; H. sapiens contain three ZnF further signifying that there exists an evolutionary transition occurring in the PARP protein. The point of interest remains in the fact that it has been reported that the ZnFs are essential for DNA binding during DNA damage. However, absence of these ZnF in lower organisms is intriguing. It would be interesting to investigate the DNA damage sensing role of PARP in these organisms. Further in the evolutionary tree it has been observed that even though new domains are added, 70% homology in catalytic domain has been observed throughout the lineages.
All these results suggest that the evolution of this protein is directed such that the organisms become more efficient in linking DNA associated processes sensed by ZnF to other systems via protein-protein interactions through BRCT domain within a cell. In addition to the catabolising activity of PARG the presence of AMD in BRCT domain functions to refine the regulations of PARP.

CONCLUSION
Poly (ADP-ribose) polymerase in higher eukaryotes is known to be involved in DNA damage response. We have attempted to generate structure of the various domains as well as the protein folding of D. discoideum PARPs. D. discoideum PARPs show differential homology and domain structure and function. BLAST results show that ADPRT1A and ADPRT1B show maximum homology to H. sapiens PARP-1. Also overlapping studies of the catalytic domains of H. sapiens PARP and D. discoideum ADPRT1A and ADPRT1B depict remarkable resemblances. This study highlights the possibility of both these ZnF bearing proteins to be involved in DNA damage response like their mammalian counterparts. The phylogenetic profile and domain analysis highlight the fact that higher organisms possess more number of genes for PARP protein. This is further substantiated by differential results obtained by PARP inhibition in E. coli, B. thuringiensis and D. discoideum multicellular development. Overall this study points out that PARP protein has evolved to cope up the multitasking function along with the DNA damage response from unicellular prokaryotes to multicellular eukaryotes.