A20/AN1 zinc-finger domain-containing proteins in plants and animals represent common elements in stress response
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- Vij, S. & Tyagi, A.K. Funct Integr Genomics (2008) 8: 301. doi:10.1007/s10142-008-0078-7
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A20/AN1 zinc-finger domain-containing proteins are well characterized in animals, and their role in regulating the immune response is established. Recently, such A20/AN1 zinc-finger proteins have been reported from plants. These plant proteins are involved in stress response, but their exact molecular mechanism of action is yet to be deciphered. Sequence information available in public databases has been used to conduct a survey of A20/AN1 zinc-finger proteins across diverse organisms with a special emphasis on plants. Domain analysis provides some interesting insights into their biological function, the most important being that A20/AN1 zinc-finger proteins could represent common elements of stress response in plants and animals.
KeywordsA20 zinc-fingerAN1 zinc-fingerStress response
The A20/AN1 zinc-finger domain-containing family of proteins is well characterized in animals and is known to play a central role in regulating the immune response (Huang et al. 2004; Heyninck and Beyaert 2005; Hishiya et al. 2006). The most well characterized plant A20/AN1 zinc-finger domain protein is OSISAP1 (Mukhopadhyay et al. 2004; Vij and Tyagi 2006). The gene was found to be responsive to different types of stresses including cold, desiccation, salt, submergence, heavy metals, wounding, and the stress hormone, ABA. Transgenic tobacco overexpressing OSISAP1 were found to be tolerant to cold, salt, and dehydration stress (Mukhopadhyay et al. 2004). A genome-wide analysis to identify similar genes from the complete sequence of rice and Arabidopsis (The Arabidopsis Genome Initiative 2000; International Rice Genome Sequencing Project 2005) led to the identification of 18 and 14 members, respectively, pointing to the existence of a family of such proteins in plants (Vij and Tyagi 2006). More recently, the survey was extended to Populus trichocarpa and Zea mays where 19 and 11 members containing at least the AN1 zinc-finger domain were identified, respectively (Jin et al. 2007). Expression analysis of the rice A20/AN1 zinc-finger encoding genes indicated that all the members were responsive to abiotic stress conditions (Vij and Tyagi 2006). Furthermore, the expression profile of maize and Arabidopsis genes analyzed under abiotic stress conditions using northern blot and digital northern analysis, respectively, showed the involvement of a large number of these genes in abiotic stress response (Jin et al. 2007). Thus, the plant A20/AN1 zinc-finger domain-encoding genes are associated with the plant’s response to abiotic stress.
Largely due to its polygenic nature, abiotic stress has been a complex trait to decipher (Vij and Tyagi 2007). Our knowledge of the genes involved in abiotic stress has come through conventional approaches and more lately through genome-wide expression profiling. The genes involved in abiotic stress can be broadly classified as those involved in regulation including genes involved in stress perception and signal transduction which serve to amplify the signal and notify parallel pathways to regulate the expression of the second class of proteins, the effectors which actually help the plant to mitigate stress (Vij and Tyagi 2007). The A20/AN1 zinc-finger domain proteins most probably belong to the regulatory class of proteins in the stress signaling cascade, as they lack any typical nuclear localization signal, and such proteins in animals have already been shown to function in the cytosol. The aim of the present analysis was to investigate the diversity and distribution of A20/AN1 zinc-finger domain-containing proteins across various taxa. Towards this end, A20/AN1 domain-containing proteins were searched across 22 organisms chosen in such a way so as to represent diverse taxonomic groups. Domain analysis of this class of proteins revealed diversity in architecture and in the type of domains with which the A20/AN1 zinc-finger domains were associated across protists, fungi, plants, and animals.
Materials and methods
A20/AN1 zinc-finger domain-containing protein sequences were retrieved from NCBI protein database using ‘A20 zinc-finger’ and ‘AN1 zinc-finger’ as keywords. As an alternate route, an A20/AN1 zinc-finger domain-containing protein was used for BLAST search in the NCBI protein (nr) database. The sequences retrieved in this way were BLAT searched (at 100% identity) followed by a BLAST search at >99% coverage and >99% identity to identify and remove potentially redundant sequences. SMART database (http://smart.embl-heidelberg.de/) was used for identification and retrieval of A20/AN1 zinc-finger domain-specific sequence. These sequences were aligned using ClustalX multiple alignment program (Thompson et al. 1997) and used for building an HMM model (Eddy 1998; http://hmmer.wustl.edu) for A20 and AN1 zinc-finger domains.
The eukaryotic genome projects listed in NCBI (http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi) were searched to identify suitable genomes based on two main criteria: taxonomic position and sequencing status. Sequence data for Entamoeba histolytica, Tetrahymena thermophila, Trypanosoma brucei, Trichomonas vaginalis was obtained from TIGR (http://www.tigr.org); Plasmodium falciparum, Caenorhabditis elegans from Welcome Trust Sanger Institute (http://www.sanger.ac.uk); Thalassiosira pseudonana, Monosiga brevicollis, Naegleria gruberi, Nematostella vectensis, Phytophthora sojae, Chlamydomonas reinhardtii, Physcomitrella patens and Populus trichocarpa from DOE Joint Genome Institute (http://www.jgi.doe.gov); Saccharomyces cerevisiae from Stanford Genome Technology Centre (http://www-sequence.stanford.edu); Giardia lamblia from GiardiaDB (http://gmod.mbl.edu/perl/site/giardia14); Drosophila melanogaster and Homo sapiens from EMBL-EBI (http://www.ebi.ac.uk); Sorghum bicolor from DOE-JGI Community Sequencing Program (CSP; http://www.phytozome.net/sorghum); Vitis vinifera from Genoscope (http://www.genoscope.cns.fr/externe/English/Projets/Projet_ML/index.html). Arabidopsis thaliana and Oryza sativa A20/AN1 zinc-finger domain-containing proteins had been identified in a previous analysis (Vij and Tyagi 2006).
These datasets were searched for A20/AN1 zinc-finger domain-containing proteins using the HMM models built for A20 and the AN1 zinc-finger domain. The presence of A20/AN1 domains in the retrieved sequences was verified using SMART (http://smart.embl-heidelberg.de) and Interpro scan (http://www.ebi.ac.uk/interpro).
Identification of A20/AN1 zinc-finger domain-containing proteins
A20/AN1 zinc-finger domain containing proteins in various organisms
The number of A20/AN1 proteins identified was 1 in Entamoeba histolytica, Trypanosoma brucei, and Trichomonas vaginalis, 2 in Plasmodium falciparum, Thalassiosira pseudonana, and Saccharomyces cerevisiae, 3 in Monosiga brevicollis, Tetrahymena thermophila, Phytophthora sojae, Chlamydomonas reinhardtii, 4 in Caenorhabditis elegans, 7 in Drosophila melanogaster, 8 in Nematostella vectensis, 10 in Naegleria gruberi, Physcomitrella patens, and Vitis vinifera, 15 in Homo sapiens, 18 in Sorghum bicolor, and 19 in Populus trichocarpa as against 14 and 18 identified previously in Arabidopsis and rice, respectively (Vij and Tyagi 2006). No A20/AN1 zinc-finger domain-containing protein was identified in G. lamblia.
Domain organization reveals a much higher diversity in animals in comparison to plants
SMART database (http://smart.embl-heidelberg.de) and Interpro scan (http://www.ebi.ac.uk/interpro) were used to study the domain organization of the A20/AN1 zinc-finger domain-containing proteins. Enatmoba (Entamoebidae), Plasmodium (Apicomplexa), Saccharomyces (Fungi), Tetrahymena (Intramacronucleata), Trypanosoma (Kinetoplastida), and Trichomonas (Trichomonada) were characterized by the presence of only the AN1 zinc-finger domain either present singly or doubly, and there was a total absence of the A20 zinc-finger domain. On the other hand, no AN1 domain was present in Thalassiosira (Bacillariophyta). Of the remaining organisms, in Naegleria (Heterolobosea), both the A20 and AN1 zinc-finger domains were present but not associated with each other. The most abundant type of domain organization (A20 zinc-finger domain at the N-terminus and AN1 zinc-finger domain at the C-terminus) is seen in all the remaining organisms used for analysis. In addition to being associated with the AN1 zinc-finger domain, the A20 zinc-finger domain was seen associated with VPS9, HdAC, WD40, and OTU domains. On the other hand, the AN1 zinc-finger domain was associated with UIM, UBQ, C2H2, AAA, and R3H domains in addition to the A20 zinc-finger.
Domain organization of A20/AN1 zinc-finger proteins
Plfa1, Mobr1, Drme1, Hosa2, Hosa3, Hosa7, Neve1, Neve3, Cael2, Phso1, Chre2, Phpa1, Phpa2, Phpa3, Phpa4, Phpa5, Phpa6, Potr1, Potr2, Potr3, Potr4, Potr5, Potr6, Potr7, Potr8, Potr9, Potr10, Potr11, Potr12, Potr13, Potr14, Potr15, Sobi1, Sobi2, Sobi3, Sobi4, Sobi5, Sobi6, Sobi7, Sobi8, Sobi9, Sobi10, Sobi11, Vivi1, Vivi2, Vivi3, Vivi4, Vivi5, Vivi6, Vivi7, OsSAP1, OsSAP2, OsSAP3, OsSAP4, OsSAP5, OsSAP6, OsSAP7, OsSAP8, OsSAP9, OsSAP10, OsSAP11, AtSAP1, AtSAP2, AtSAP3, AtSAP4, AtSAP5, AtSAP6, AtSAP7, AtSAP8, AtSAP9, AtSAP10
OsSAP12, Sobi12, Sobi13, Sobi14
Thps1, Thps2, Nagr1, Nagr2, Nagr4, Nagr5, Hosa4, Chre1, OsSAP18
Drme2, Hosa5, Hosa6, Neve2, Cael1
Sace1, Sace2, Nagr10, Teth1, Teth3, Trbr1, Drme4, Drme5, Drme6, Drme7, Hosa14, Neve6, Neve7, Trva1, Phpa8, Phpa9, Phpa10, Potr17, Potr18, Potr19, Sobi15, Vivi8, OsSAP13, OsSAP14, OsSAP15, AtSAP14
Enhi1, Plfa2, Mobr3, Nagr6, Nagr7, Nagr8, Nagr9, Teth2, Drme3, Hosa10, Hosa12, Neve4, Neve5, Cael3, Sobi16, Sobi17, Vivi9, OsSAP17, AtSAP12
Phso2, Chre3, Phpa7, Potr16, Sobi18, Vivi10, OsSAP16, AtSAP11
The human A20 protein is the most well-characterized A20 zinc-finger domain-containing protein. It is a cytoplasmic protein whose domain structure comprises of an ovarian tumor domain (OTU) at the N-terminus and seven A20 zinc-finger domains present in the C-terminus (Opipari et al. 1990; Heyninck and Beyaert 2005). A20 down-regulates the activation of NF-κB in response to TNFα, a pro-inflammatory cytokine. Its effect is mediated by its dual ubiquitin-editing function, with the OTU domain involved in de-ubiquitination and the A20 zinc-fingers involved in the ubiquitination of the TNF receptor-interacting protein (RIP) thus targeting it for proteasomal degradation (Wertz et al. 2004; Heyninck and Beyaert 2005). In addition, another class of well-studied A20/AN1 zinc-finger proteins in mammals is the one which is characterized by the presence of an A20 zinc-finger domain at the N-terminus and an AN1 zinc-finger domain at the C-terminus (Scott et al. 1998; Duan et al. 2000; Huang et al. 2004; Hishiya et al. 2006). One such well-characterized protein from humans is ZNF216. Detailed domain mapping experiments with ZNF216 have shown that its A20 zinc-finger domain interacts with RIP and IKKγ, whereas the AN1 domain interacts with TRAF6 to mediate its function (Huang et al. 2004). Furthermore, ZNF216 performs a function similar to the human A20 protein, as it is capable of inhibition of NF-κB activation (Huang et al. 2004). A similar protein has been characterized in mouse and found to function in protein degradation via the ubiquitin–proteasome system (UPS). The most well-characterized plant protein in this class is rice A20/AN1 zinc-finger domain stress associated protein 1 (OSISAP1). It has the same domain organization as the class with A20 zinc-finger at the N-terminus and AN1 zinc-finger at the C-terminus. OSISAP1 is responsive to multiple environmental stresses and confers abiotic stress tolerance when overexpressed in tobacco (Mukhopadhyay et al. 2004). In a later study, it was found that there are 18 and 14 such A20/AN1 zinc-finger domain-encoding genes in the rice and Arabidopsis genomes, respectively (Vij and Tyagi 2006). Furthermore, 11 and 19 proteins containing at least the AN1 zinc-finger domain have been identified in Zea mays and Populus trichocarpa, respectively (Jin et al. 2007). Expression profiling of the rice genes under cold, salt, and dehydration stress conditions revealed that each member of the family was up-regulated in response to one or the other abiotic stress, albeit at variable levels. Interestingly, expression analysis of the maize and Arabidopsis genes under abiotic stress conditions also showed that most of the genes showed stress-responsive expression (Vij and Tyagi 2006; Jin et al. 2007). In addition, a Capsicum A20/AN1 zinc-finger encoding gene has been deposited in the NCBI database as a cold-induced gene (GenBank Accession Number AAR83854), and it is identified as an AtSAP5 ortholog. PvPR3 from soybean has been previously reported as a gene coding for a pathogenesis-related protein not only due to sequence homology but also, as it was seen to be inducible by elicitor treatment (Sharma et al. 1992). A Chrysanthemum A20/AN1 zinc-finger class protein has also been deposited as a stress tolerance zinc-finger protein in the NCBI database (GenBank Accession Number ABI23728). Its AN1 zinc-finger domain is 100% identical to OsiSAP1. With the aim of studying the distribution and diversity of the A20/AN1 zinc-finger class of proteins, A20/AN1 zinc-finger domain-containing proteins were searched across 22 organisms. With the exception of G. lamblia, the A20/AN1 zinc-finger domain-containing proteins were identified in all other organisms used for analysis pointing to the universal existence of this class of proteins. The absence of any such domain-containing protein in G. lamblia is of evolutionary significance but can also be attributed to the fact that its genome sequence available at present is in the assembly (phase II) stage (http://www.ncbi.nlm.nih.gov/genomes/leuks.cgi; http://gmod.mbl.edu/perl/site/giardia14). The AN1/AN1+AN1 class could represent a primitive domain organization with it being the only type seen in Entamoeba histolyticaPlasmodium falciparum, Saccharomyces cerevisiae, Tetrahymena thermophila, Trypanosoma brucei, and Trichomonas vaginalis. In addition, in the majority of protists and fungi, the A20 and AN1 zinc-finger domains usually exist separately or with each other and were rarely seen associated with additional domains. In fact, even though the A20/AN1 zinc-finger domain containing proteins are present universally, a fundamental difference is evident in the domain organization between plants and animals. More specifically, very few types of organizations were shared between plants and animals such as ‘i’, ‘iii’, ‘ix’, and ‘x’ (A20+AN1, A20, AN1 and 2AN1) though the proteins representing these types were most abundant in nature. Very few domain organizations are specific to plants (ii, xii, xiii). In fact, the only additional domain identified with this class of proteins was the C2H2 domain (xii, xiii) in plants. However, in the case of animal-specific domain organizations, a much higher diversity is seen both in the types of domains and their organization. The additional domains identified in animals include VPS9, OTU, AAA, R3H, UBQ, and UIM (iv, vii, viii, xi, xiv, xv, xvii). Of these, at least three domains are related to ubiquitin signaling including OTU domain (ovarian tumor domain involved in de-ubiquitination), UBQ (present in ubiquitin and its homologs) and UIM present in proteasome subunit S5a and other ubiquitin-associated proteins (Hurley et al. 2006; http://smart.embl-heidelberg.de; http://www.ebi.ac.uk/interpro). Notably, the A20-AN1 zinc-finger class of proteins has also been shown to be involved in ubiquitin signaling with direct evidence available for the ubiquitin ligase activity of the A20 zinc-finger domain (Hishiya et al. 2006). Interestingly, though the plants lack in diversity of domains associated with the A20/AN1 zinc-finger domain containing family of proteins, they show expansion of a particular family (A20 zinc-finger domain at the N-terminus and AN1 zinc-finger domain present at the C-terminus) of proteins. In fact, lineage specific expansion (LSE) is evident only after the divergence of chlorophytes and embryophytes. However, it would have occurred before the monocot–dicot divergence ∼200 mya (Wolfe et al. 1989), as the expansion is clearly seen in the moss, Physcomitrella patens.
In conclusion, the A20/AN1 zinc-finger domain-containing proteins are known to be involved in stress response in plants and immune response in animals. In the present study, diversity and domain organization of this class of proteins was studied. Our results indicate that certain domain organizations were taxa-specific with only a few domain organizations shared between plants and animals. In addition, animals exhibit a much higher diversity of domain organization in comparison to plants. Furthermore, in the case of plants, distinct LSE was evident for one particular class of A20/AN1 proteins (A20 zinc-finger at the N-terminus and AN1 zinc-finger at the C-terminus). It has been seen that largely, the same set of domains form the structural and regulatory components of the eukaryotic crown group that includes plants, animals, fungi, and some protists. The major forces shaping the evolution of species have been creation of new domain architectures and expansion/contraction of gene families in a lineage-specific way (Lespinet et al. 2002). The plant A20-AN1 zinc-finger class of proteins is involved in abiotic stress response, and its expansion is in line with the previously observed expansion of gene families involved in stress response across diverse organisms. LSEs of such gene families are thought to contribute to the diversity needed to counter pathogen attack and respond to multiple environmental stresses.
The work is financially supported by the Department of Biotechnology, Government of India.