Andreja Mikoč

Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

Highlights • PARPs are present in representatives from all six major eukaryotic supergroups.• Reversible PAR metabolism was established early in eukaryotic evolution.• The last common ancestor of all eukaryotes possessed five types of PARP proteins.• PARPs are associated to a large variety of different pathways.

Molecular evidence for the presence of a developmental gene in the lowest animals: identification of a homeobox-like gene in the marine sponge Geodia cydonium

During the development of higher animals, morphogenetic programs are switched on which are frequently controlled by homeotic genes. Until now these genes have not been identified in the lowest animals, the marine sponges. Since sponges show (i) an antero-posterior and/or dorso-ventral axis during embryogenesis and (ii) a complex differentiation pattern during spicula formation, we hypothesized that in sponges homeotic genes--if present--are also involved in the control of these processes. Therefore, we searched for homeobox or homeobox-like sequences in the marine sponge Geodia cydonium. Here we describe a homeobox-like sequence from these animals; it was isolated from a cDNA library of an adult specimen. The deduced amino acid sequence of the complete homeodomain shares over 70% similarity with other homeodomain sequences, including those from hydra, insects and vertebrates. These data indicate that the sponge homeodomain-like sequence is similar with respect to structure to those of other animals and may suggest that the sponge homeodomain-like sequence(s) might function during developmental processes and/or during spiculogenesis in a similar manner to that known for higher animals.

Molecular Insights into Poly(ADP-ribose) Recognition and Processing

Poly(ADP-ribosyl)ation is a post-translational protein modification involved in the regulation of important cellular functions including DNA repair, transcription, mitosis and apoptosis. The amount of poly(ADP-ribosyl)ation (PAR) in cells reflects the balance of synthesis, mediated by the PARP protein family, and degradation, which is catalyzed by a glycohydrolase, PARG. Many of the proteins mediating PAR metabolism possess specialised high affinity PAR-binding modules that allow the efficient sensing or processing of the PAR signal. The identification of four such PAR-binding modules and the characterization of a number of proteins utilising these elements during the last decade has provided important insights into how PAR regulates different cellular activities. The macrodomain represents a unique PAR-binding module which is, in some instances, known to possess enzymatic activity on ADP-ribose derivatives (in addition to PAR-binding). The most recently discovered example for this is the PARG protein, and several available PARG structures have provided an understanding into how the PARG macrodomain evolved into a major enzyme that maintains PAR homeostasis in living cells.

ADP‐ribosylation: new facets of an ancient modification

Rapid response to environmental changes is achieved by uni‐ and multicellular organisms through a series of molecular events, often involving modification of macromolecules, including proteins, nucleic acids and lipids. Amongst these, ADP‐ribosylation is of emerging interest because of its ability to modify different macromolecules in the cells, and its association with many key biological processes, such as DNA‐damage repair, DNA replication, transcription, cell division, signal transduction, stress and infection responses, microbial pathogenicity and aging. In this review, we provide an update on novel pathways and mechanisms regulated by ADP‐ribosylation in organisms coming from all kingdoms of life.

Sponge non-metastatic Group I Nme gene/protein - structure and function is conserved from sponges to humans

BackgroundNucleoside diphosphate kinases NDPK are evolutionarily conserved enzymes present in Bacteria, Archaea and Eukarya, with human Nme1 the most studied representative of the family and the first identified metastasis suppressor. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestors genomic features. Recent studies show that sponges have a wide repertoire of genes many of which are involved in diseases in more complex metazoans. The original function of those genes and the way it has evolved in the animal lineage is largely unknown. Here we report new results on the metastasis suppressor gene/protein homolog from the marine sponge Suberites domuncula, NmeGp1Sd. The purpose of this study was to investigate the properties of the sponge Group I Nme gene and protein, and compare it to its human homolog in order to elucidate the evolution of the structure and function of Nme.ResultsWe found that sponge genes coding for Group I Nme protein are intron-rich. Furthermore, we discovered that the sponge NmeGp1Sd protein has a similar level of kinase activity as its human homolog Nme1, does not cleave negatively supercoiled DNA and shows nonspecific DNA-binding activity. The sponge NmeGp1Sd forms a hexamer, like human Nme1, and all other eukaryotic Nme proteins. NmeGp1Sd interacts with human Nme1 in human cells and exhibits the same subcellular localization. Stable clones expressing sponge NmeGp1Sd inhibited the migratory potential of CAL 27 cells, as already reported for human Nme1, which suggests that Nmes function in migratory processes was engaged long before the composition of true tissues.ConclusionsThis study suggests that the ancestor of all animals possessed a NmeGp1 protein with properties and functions similar to evolutionarily recent versions of the protein, even before the appearance of true tissues and the origin of tumors and metastasis.

Ras-like small GTPases form a large family of proteins in the marine sponge Suberites domuncula.

Sponges (Porifera) are the simplest and the most ancient metazoan animals, which branched off first from the common ancestor of all multicellular animals. We have inspected ∼13,000 partial cDNA sequences (ESTs) from the marine sponge Suberites domuncula and have identified full or partial cDNA sequences coding for ∼50 different Ras-like small GTPases. Forty-four sponge proteins from the Ras family are described here: 6 proteins from the Ras subfamily, 5 from Rho, 6 from Arf, 1 Ran, and 26 Rabs or Rab-like proteins. No isoforms of these proteins were detected; the closest related proteins are two Rho proteins with 74% identity. Small GTPases from sponge display a higher degree of sequence conservation with orthologues from vertebrates (53%–93% identity) than with those from either Caenorhabditis elegans or Drosophila melanogaster. The real number of small GTPases in this sponge is certainly much higher than 50, because the actual S. domuncula database of ∼13,000 ESTs contains at most 3000 nonredundant cDNA sequences. The number of genes for Ras-like small GTPases in yeast, C. elegans, D. melanogaster, and humans is 30, 56, 90, and 174, respectively. Both model invertebrates have only 29 Rabs or Rab-like proteins, compared with 26 already found in sponge, and are missing at least 1 Rab (Rab24) present in S. domuncula and mammals. Our results indicate that duplications and diversifications of genes encoding Ras-like small GTPases, especially the Rab subfamily of small GTPases, happened very early in the evolution of Metazoa.

Characterization of Nme6-like gene/protein from marine sponge Suberites domuncula

Nucleoside diphosphate kinases (NDPKs) are evolutionarily conserved enzymes involved in many biological processes such as metastasis, proliferation, development, differentiation, ciliary functions, vesicle transport and apoptosis in vertebrates. Biochemical mechanisms of these processes are still largely unknown. Sponges (Porifera) are simple metazoans without tissues, closest to the common ancestor of all animals. They changed little during evolution and probably provide the best insight into the metazoan ancestors’ genomic features. The purpose of this study was to address structural and functional properties of group II Nme6 gene/protein ortholog from the marine sponge Suberites domuncula, Nme6, in order to elucidate its evolutionary history. Sponge Nme6 gene and promoter were sequenced and analysed with various bioinformatical tools. Nme6 and Nme6Δ31 proteins were produced in E. coli strain BL21 and NDPK activity was measured using a coupled pyruvate kinase-lactate dehydrogenase assay. Subcellular localization in human tumour cells was examined by confocal scanning microscopy. Our results show that the sponge Nme6 compared to human Nme6 does not possess NDPK activity, does not localize in mitochondria at least in human cells although it has a putative mitochondrial signal sequence, lacks two recent introns that comprise miRNAs and have different transcriptional binding sites in the promoter region. Therefore, we conclude that the structure of Nme6 gene has changed during metazoan evolution possibly in correlation with the function of the protein.

The recA gene from Streptomyces rimosus R6: sequence and expression in Escherichia coli

The recA gene from Streptomyces rimusus encodes a 376-amino acids polypeptide (M(r) 39,702) that is one of the largest bacterial RecA proteins observed. Detailed analyses of the Streptomyces RecA proteins showed that all possess an additional and unique C-terminal, rich in lysines and alanines, which can form an additional terminal alpha helix. Expression of the S. rimosus RecA protein in Escherichia coli FR333 (delta recA306) was demonstrated using antibodies raised against E. coli RecA protein; expression was possible only from the S. rimosus promoter. A Streptomyces-E. coli-like promoter sequence (TTGACA-18bp-TCTTAT) was found in the A+ T-rich region 135-165 base pairs upstream from the initiation codon and was related to Bacillus subtilis DNA damage-inducible promoters.

Specificity of reversible ADP-ribosylation and regulation of cellular processes.

Abstract Proper and timely regulation of cellular processes is fundamental to the overall health and viability of organisms across all kingdoms of life. Thus, organisms have evolved multiple highly dynamic and complex biochemical signaling cascades in order to adapt and survive diverse challenges. One such method of conferring rapid adaptation is the addition or removal of reversible modifications of different chemical groups onto macromolecules which in turn induce the appropriate downstream outcome. ADP-ribosylation, the addition of ADP-ribose (ADPr) groups, represents one of these highly conserved signaling chemicals. Herein we outline the writers, erasers and readers of ADP-ribosylation and dip into the multitude of cellular processes they have been implicated in. We also review what we currently know on how specificity of activity is ensured for this important modification.

Disruption of macrodomain protein SCO6735 increases antibiotic production in Streptomyces coelicolor

ADP-ribosylation is a post-translational modification that can alter the physical and chemical properties of target proteins and that controls many important cellular processes. Macrodomains are evolutionarily conserved structural domains that bind ADP-ribose derivatives and are found in proteins with diverse cellular functions. Some proteins from the macrodomain family can hydrolyze ADP-ribosylated substrates and therefore reverse this post-translational modification. Bacteria and Streptomyces, in particular, are known to utilize protein ADP-ribosylation, yet very little is known about their enzymes that synthesize and remove this modification. We have determined the crystal structure and characterized, both biochemically and functionally, the macrodomain protein SCO6735 from Streptomyces coelicolor. This protein is a member of an uncharacterized subfamily of macrodomain proteins. Its crystal structure revealed a highly conserved macrodomain fold. We showed that SCO6735 possesses the ability to hydrolyze PARP-dependent protein ADP-ribosylation. Furthermore, we showed that expression of this protein is induced upon DNA damage and that deletion of this protein in S. coelicolor increases antibiotic production. Our results provide the first insights into the molecular basis of its action and impact on Streptomyces metabolism.