Who Is at Risk of Developing Cannabis Dependence? Findings From an Extensive Online Study on Cannabis Users.

OBJECTIVES: Although frequency of cannabis use is considered to be the strongest risk factor for developing cannabis dependence, only up to half of daily users become dependent. In this study, we explored an array of risk factors and moderators of cannabis dependence symptoms from the International Classification of Diseases, Tenth Edition endorsed by participants. METHODS: A sample of 1,635 cannabis users completed an Internet survey consisting of measures of cannabis and other drug use. Multiple linear regression with a backward elimination method was employed to identify predictors of cannabis dependence symptoms. After that, a series of hierarchical multiple regression analyses were performed to test the predictive validity of the interactions between frequency of cannabis use and other predictors. RESULTS: Frequency of cannabis use appeared to be the strongest predictor of developing cannabis dependence symptoms; other significant predictors of cannabis dependence symptoms were substance-dependency-related treatment seeking, mental health problems in the family and pattern of substance use. Duration of cannabis use, relationship status, and drug use history in the family were identified as significant moderators of the relationship between frequency of cannabis use and the number of cannabis dependence symptoms. CONCLUSIONS: This study confirms that the frequency of cannabis use is the strongest predictor of cannabis dependence symptoms but this relationship is significantly moderated by three abovementioned factors.

Recombinant immunotoxin targeting GPC3 is cytotoxic to H446 small cell lung cancer cells.

Glypican-3 (GPC3) is a cell membrane glycoprotein that regulates cell growth and proliferation. Aberrant expression or distribution of GPC3 underlies developmental abnormalities and the development of solid tumours. The strongest evidence for the participation of GPC3 in carcinogenesis stems from studies on hepatocellular carcinoma and lung squamous cell carcinoma. To the best of our knowledge, the role of the GPC3 protein and its potential therapeutic application have never been studied in small cell lung carcinoma (SCLC), despite the known involvement of associated pathways and the high mortality caused by this disease. Therefore, the aim of the present study was to examine GPC3 targeting for SCLC immunotherapy. An immunotoxin carrying an anti-GPC3 antibody (hGC33) and Pseudomonas aeruginosa exotoxin A 38 (PE38) was generated. This hGC33-PE38 protein was overexpressed in E. coli and purified. ADP-ribosylation activity was tested in vitro against eukaryotic translation elongation factor 2. Cell internalisation ability was confirmed by confocal microscopy. Cytotoxicity was analysed by treating liver cancer (HepG2, SNU-398 and SNU-449) and lung cancer (NCI-H510A, NCI-H446, A549 and SK-MES1) cell lines with hGC33-PE38 and estimating viable cells number. A BrdU assay was employed to verify anti-proliferative activity of hGC33-PE38 on treated cells. Fluorescence-activated cell sorting was used for the detection of cell membrane-bound GPC3. The hGC33-PE38 immunotoxin displayed enzymatic activity comparable to native PE38. The protein was efficiently internalised by GPC3-positive cells. Moreover, hGC33-PE38 was cytotoxic to HepG2 cells but had no effect on known GPC3-negative cell lines. The H446 cells were sensitive to hGC33-PE38 (IC50, 70.6±4.6 ng/ml), whereas H510A cells were resistant. Cell surface-bound GPC3 was abundant on the membranes of H446 cells, but absent on H510A. Altogether, the present findings suggested that GPC3 could be considered as a potential therapeutic target for SCLC immunotherapy.

High-density Peptide Arrays Help to Identify Linear Immunogenic B-cell Epitopes in Individuals Naturally Exposed to Malaria Infection.

High-density peptide arrays are an excellent means to profile anti-plasmodial antibody responses. Different protein intrinsic epitopes can be distinguished, and additional insights are gained, when compared with assays involving the full-length protein. Distinct reactivities to specific epitopes within one protein may explain differences in published results, regarding immunity or susceptibility to malaria. We pursued three approaches to find specific epitopes within important plasmodial proteins, (1) twelve leading vaccine candidates were mapped as overlapping 15-mer peptides, (2) a bioinformatical approach served to predict immunogenic malaria epitopes which were subsequently validated in the assay, and (3) randomly selected peptides from the malaria proteome were screened as a control. Several peptide array replicas were prepared, employing particle-based laser printing, and were used to screen 27 serum samples from a malaria-endemic area in Burkina Faso, West Africa. The immunological status of the individuals was classified as "protected" or "unprotected" based on clinical symptoms, parasite density, and age. The vaccine candidate screening approach resulted in significant hits in all twelve proteins and allowed us (1) to verify many known immunogenic structures, (2) to map B-cell epitopes across the entire sequence of each antigen and (3) to uncover novel immunogenic epitopes. Predicting immunogenic regions in the proteome of the human malaria parasite Plasmodium falciparum, via the bioinformatics approach and subsequent array screening, confirmed known immunogenic sequences, such as in the leading malaria vaccine candidate CSP and discovered immunogenic epitopes derived from hypothetical or unknown proteins.

Natural Compounds as Inhibitors of Tyrosyl-tRNA Synthetase.

Tyrosyl-tRNA synthetases (TyrRSs) as essential enzymes for all living organisms are good candidates for therapeutic target in the prevention and therapy of microbial infection. We examined the effect of various polyphenols, alkaloids, and terpenes-secondary metabolites produced by higher plants showing many beneficial properties for the human organism, on bacterial aminoacylation reaction. The most potent inhibitors of Escherichia coli TyrRS are epigallocatechin gallate, acacetin, kaempferide, and chrysin, whereas the enzymes from Staphylococcus aureus and Pseudomonas aeruginosa are inhibited mainly by acacetin and chrysin. Most of them act as competitive inhibitors. Structure-activity relationship showed that the most potent flavonoid inhibitors contain hydroxyl group at position 5 and 7 of A ring and OCH3 group at position 4' of B ring.

Towards Engineering Novel PE-Based Immunotoxins by Targeting Them to the Nucleus.

Exotoxin A (PE) from Pseudomonas aeruginosa is a bacterial ADP-ribosyltransferase, which can permanently inhibit translation in the attacked cells. Consequently, this toxin is frequently used in immunotoxins for targeted cancer therapies. In this study, we propose a novel modification to PE by incorporating the NLS sequence at its C-terminus, to make it a selective agent against fast-proliferating cancer cells, as a nucleus-accumulated toxin should be separated from its natural substrate (eEF2) in slowly dividing cells. Here, we report the cytotoxic activity and selected biochemical properties of newly designed PE mutein using two cellular models: A549 and HepG2. We also present a newly developed protocol for efficient purification of recombinant PE and its muteins with very high purity and activity. We found that furin cleavage is not critical for the activity of PE in the analyzed cell lines. Surprisingly, we observed increased toxicity of the toxin accumulated in the nucleus. This might be explained by unexpected nuclease activity of PE and its potential ability to cleave chromosomal DNA, which seems to be a putative alternative intoxication mechanism. Further experimental investigations should address this newly detected activity to identify catalytic residues and elucidate the molecular mechanism responsible for this action.

FAM46 proteins are novel eukaryotic non-canonical poly(A) polymerases.

FAM46 proteins, encoded in all known animal genomes, belong to the nucleotidyltransferase (NTase) fold superfamily. All four human FAM46 paralogs (FAM46A, FAM46B, FAM46C, FAM46D) are thought to be involved in several diseases, with FAM46C reported as a causal driver of multiple myeloma; however, their exact functions remain unknown. By using a combination of various bioinformatics analyses (e.g. domain architecture, cellular localization) and exhaustive literature and database searches (e.g. expression profiles, protein interactors), we classified FAM46 proteins as active non-canonical poly(A) polymerases, which modify cytosolic and/or nuclear RNA 3' ends. These proteins may thus regulate gene expression and probably play a critical role during cell differentiation. A detailed analysis of sequence and structure diversity of known NTases possessing PAP/OAS1 SBD domain, combined with state-of-the-art comparative modelling, allowed us to identify potential active site residues responsible for catalysis and substrate binding. We also explored the role of single point mutations found in human cancers and propose that FAM46 genes may be involved in the development of other major malignancies including lung, colorectal, hepatocellular, head and neck, urothelial, endometrial and renal papillary carcinomas and melanoma. Identification of these novel enzymes taking part in RNA metabolism in eukaryotes may guide their further functional studies.

HarmonyDOCK: the structural analysis of poses in protein-ligand docking.

Molecular docking is a widely used method for lead optimization. However, docking tools often fail to predict how a ligand (the smaller molecule, such as a substrate or drug candidate) binds to a receptor (the accepting part of a protein). We present here the HarmonyDOCK, a novel method for assessing the docking software accuracy, and creating the scoring function which would determine consensus protein-ligand pose among those generated by available docking programs. Conformations for few hundred protein-ligand complexes with known three-dimensional structure were predicted on a benchmark set by set of different docking programs. On the basis of the derived ranking, the point of reference and the lower score limit were determined for subsequent investigations. The focus of the methodology is on the top-ranked poses, with the assumption being that the conformation of the docked molecules is the most accurate. We found out that some docking programs perform considerably better than the others, yet in all cases the proper selection of decoys, namely HarmonyDOCK, is needed for successful docking procedure.

Alphaherpesvirinae and Gammaherpesvirinae glycoprotein L and CMV UL130 originate from chemokines.

Herpesviridae is a large family of DNA viruses divided into three subfamilies: Alpha-, Beta- and Gammaherpesvirinae. The process of herpesvirus transmission is mediated by a range of proteins, one of which is glycoprotein L (gL). Based on our analysis of the solved structures of HSV2 and EBV gH/gL complexes, we propose that Alphaherpesvirinae and Gammaherpesvirinae glycoprotein L and Betaherpesvirinae UL130 originate from chemokines. Herpes simplex virus type 2 gL and human cytomegalovirus homolog (UL130) adopt a novel C chemokine-like fold, while Epstein-Barr virus gL mimics a CC chemokine structure. Hence, it is possible that gL interface with specific chemokine receptors during the transmission of Herpesviridae. We conclude that the further understanding of the function of viral chemokine-like proteins in Herpesviridae infection may lead to development of novel prophylactic and therapeutic treatment.

Structural bioinformatics of the general transcription factor TFIID.

The Transcription Factor IID is a large macromolecular complex composed of the TATA-box binding protein (TBP) and a group of 13-14 conserved TBP-associated factors (TAFs). TAFs are known to regulate transcription at various levels - mediating transcription via interaction with activators, histone modifications; recognition and binding to promoters; acting as a platform for other Transcription Factors and RNA polymerase II. Despite numerous previous studies of the TFIID complex, the knowledge concerning the structure of its components, and thus the exact mechanism of its function, remains undetermined. To carry out an in-depth analysis of TFIID we performed the structural bioinformatic analysis of the TFIID complex. The sequence identity and similarity of 13.74% and 37.56%, respectively (calculated with PAM250 matrix) between M1 aminopeptidase protein and TAF2 and the high similarity of their putative secondary structures allowed us to model a large part of the TAF2 structure. The sequence analysis enabled the mapping of previously not fully characterized structural domains in well-studied TAF proteins (including the full histone domains of TAF4 and 12 or TAF3 and 8). In this study we provided detailed structural models for all the elements of human analyzed in the context of TFIID activity, along with indications of structural alterations within TFIID in various animal model species.

Sequence, structure and functional diversity of PD-(D/E)XK phosphodiesterase superfamily.

Proteins belonging to PD-(D/E)XK phosphodiesterases constitute a functionally diverse superfamily with representatives involved in replication, restriction, DNA repair and tRNA-intron splicing. Their malfunction in humans triggers severe diseases, such as Fanconi anemia and Xeroderma pigmentosum. To date there have been several attempts to identify and classify new PD-(D/E)KK phosphodiesterases using remote homology detection methods. Such efforts are complicated, because the superfamily exhibits extreme sequence and structural divergence. Using advanced homology detection methods supported with superfamily-wide domain architecture and horizontal gene transfer analyses, we provide a comprehensive reclassification of proteins containing a PD-(D/E)XK domain. The PD-(D/E)XK phosphodiesterases span over 21,900 proteins, which can be classified into 121 groups of various families. Eleven of them, including DUF4420, DUF3883, DUF4263, COG5482, COG1395, Tsp45I, HaeII, Eco47II, ScaI, HpaII and Replic_Relax, are newly assigned to the PD-(D/E)XK superfamily. Some groups of PD-(D/E)XK proteins are present in all domains of life, whereas others occur within small numbers of organisms. We observed multiple horizontal gene transfers even between human pathogenic bacteria or from Prokaryota to Eukaryota. Uncommon domain arrangements greatly elaborate the PD-(D/E)XK world. These include domain architectures suggesting regulatory roles in Eukaryotes, like stress sensing and cell-cycle regulation. Our results may inspire further experimental studies aimed at identification of exact biological functions, specific substrates and molecular mechanisms of reactions performed by these highly diverse proteins.

Squalene monooxygenase - a target for hypercholesterolemic therapy.

Squalene monooxygenase catalyzes the epoxidation of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compounds of these organisms and squalene epoxidation is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively, which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clinical use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clinical practice. After many years of use of statins there is some clinical evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, especially in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the future.

Comprehensive structural and substrate specificity classification of the Saccharomyces cerevisiae methyltransferome.

Methylation is one of the most common chemical modifications of biologically active molecules and it occurs in all life forms. Its functional role is very diverse and involves many essential cellular processes, such as signal transduction, transcriptional control, biosynthesis, and metabolism. Here, we provide further insight into the enzymatic methylation in S. cerevisiae by conducting a comprehensive structural and functional survey of all the methyltransferases encoded in its genome. Using distant homology detection and fold recognition, we found that the S. cerevisiae methyltransferome comprises 86 MTases (53 well-known and 33 putative with unknown substrate specificity). Structural classification of their catalytic domains shows that these enzymes may adopt nine different folds, the most common being the Rossmann-like. We also analyzed the domain architecture of these proteins and identified several new domain contexts. Interestingly, we found that the majority of MTase genes are periodically expressed during yeast metabolic cycle. This finding, together with calculated isoelectric point, fold assignment and cellular localization, was used to develop a novel approach for predicting substrate specificity. Using this approach, we predicted the general substrates for 24 of 33 putative MTases and confirmed these predictions experimentally in both cases tested. Finally, we show that, in S. cerevisiae, methylation is carried out by 34 RNA MTases, 32 protein MTases, eight small molecule MTases, three lipid MTases, and nine MTases with still unknown substrate specificity.

Mapping the substrate binding site of phenylacetone monooxygenase from Thermobifida fusca by mutational analysis.

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope.

Detailed mechanism of squalene epoxidase inhibition by terbinafine.

Squalene epoxidase (SE) is a key flavin adenine dinucleotide (FAD)-dependent enzyme of ergosterol and cholesterol biosynthetic pathways and an attractive potential target for drugs used to inhibit the growth of pathogenic fungi or to lower cholesterol level. Although many studies on allylamine drugs activity have been published during the last 30 years, up until now no detailed mechanism of the squalene epoxidase inhibition has been presented. Our study brings such a model at atomic resolution in the case of yeast Saccharomyces cerevisiae . Presented data resulting from modeling studies are in excellent agreement with experimental findings. A fully atomic three-dimensional (3D) model of squalene epoxidase (EC 1.14.99.7) from S. cerevisiae was built with the help of 3D-Jury approach and further screened based on data known from mutation experiments leading to terbinafine resistance. Docking studies followed by molecular dynamics simulations and quantum interaction energy calculations [MP2/6-31G(d)] resulted in the identification of the terbinafine-squalene epoxidase mode of interaction. In the energetically most likely orientation of terbinafine its interaction energy with the protein is ca. 120 kJ/mol. In the favorable position the terbinafine lipophilic moiety is located vertically inside the squalene epoxidase binding pocket with the tert-butyl group oriented toward its center. Such a position results in the SE conformational changes and prevents the natural substrate from being able to bind to the enzyme's active site. That would explain the noncompetitive manner of SE inhibition. We found that the strongest interaction between terbinafine and SE stems from hydrogen bonding between hydrogen-bond donors, hydroxyl group of Tyr90 and amine nitrogen atom of terbinafine. Moreover, strong attractive interactions were recorded for amino acids whose mutations resulted in terbinafine resistance. Our results, elucidating at a molecular level the mode of terbinafine inhibitory activity, can be utilized in designing more potent or selective antifungal drugs or even medicines lowering cholesterol in humans.

VoteDock: consensus docking method for prediction of protein-ligand interactions.

Molecular recognition plays a fundamental role in all biological processes, and that is why great efforts have been made to understand and predict protein-ligand interactions. Finding a molecule that can potentially bind to a target protein is particularly essential in drug discovery and still remains an expensive and time-consuming task. In silico, tools are frequently used to screen molecular libraries to identify new lead compounds, and if protein structure is known, various protein-ligand docking programs can be used. The aim of docking procedure is to predict correct poses of ligand in the binding site of the protein as well as to score them according to the strength of interaction in a reasonable time frame. The purpose of our studies was to present the novel consensus approach to predict both protein-ligand complex structure and its corresponding binding affinity. Our method used as the input the results from seven docking programs (Surflex, LigandFit, Glide, GOLD, FlexX, eHiTS, and AutoDock) that are widely used for docking of ligands. We evaluated it on the extensive benchmark dataset of 1300 protein-ligands pairs from refined PDBbind database for which the structural and affinity data was available. We compared independently its ability of proper scoring and posing to the previously proposed methods. In most cases, our method is able to dock properly approximately 20% of pairs more than docking methods on average, and over 10% of pairs more than the best single program. The RMSD value of the predicted complex conformation versus its native one is reduced by a factor of 0.5 Å. Finally, we were able to increase the Pearson correlation of the predicted binding affinity in comparison with the experimental value up to 0.5.

Distant homologs of anti-apoptotic factor HAX1 encode parvalbumin-like calcium binding proteins.

BACKGROUND: Apoptosis is a highly ordered and orchestrated multiphase process controlled by the numerous cellular and extra-cellular signals, which executes the programmed cell death via release of cytochrome c alterations in calcium signaling, caspase-dependent limited proteolysis and DNA fragmentation. Besides the general modifiers of apoptosis, several tissue-specific regulators of this process were identified including HAX1 (HS-1 associated protein X-1) - an anti-apoptotic factor active in myeloid cells. Although HAX1 was the subject of various experimental studies, the mechanisms of its action and a functional link connected with the regulation of apoptosis still remains highly speculative. FINDINGS: Here we provide the data which suggests that HAX1 may act as a regulator or as a sensor of calcium. On the basis of iterative similarity searches, we identified a set of distant homologs of HAX1 in insects. The applied fold recognition protocol gives us strong evidence that the distant insects' homologs of HAX1 are novel parvalbumin-like calcium binding proteins. Although the whole three EF-hands fold is not preserved in vertebrate our analysis suggests that there is an existence of a potential single EF-hand calcium binding site in HAX1. The molecular mechanism of its action remains to be identified, but the risen hypothesis easily translates into previously reported lines of various data on the HAX1 biology as well as, provides us a direct link to the regulation of apoptosis. Moreover, we also report that other family of myeloid specific apoptosis regulators - myeloid leukemia factors (MLF1, MLF2) share the homologous C-terminal domain and taxonomic distribution with HAX1. CONCLUSIONS: Performed structural and active sites analyses gave new insights into mechanisms of HAX1 and MLF families in apoptosis process and suggested possible role of HAX1 in calcium-binding, still the analyses require further experimental verification.

Species used for drug testing reveal different inhibition susceptibility for 17beta-hydroxysteroid dehydrogenase type 1.

Steroid-related cancers can be treated by inhibitors of steroid metabolism. In searching for new inhibitors of human 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) for the treatment of breast cancer or endometriosis, novel substances based on 15-substituted estrone were validated. We checked the specificity for different 17beta-HSD types and species. Compounds were tested for specificity in vitro not only towards recombinant human 17beta-HSD types 1, 2, 4, 5 and 7 but also against 17beta-HSD 1 of several other species including marmoset, pig, mouse, and rat. The latter are used in the processes of pharmacophore screening. We present the quantification of inhibitor preferences between human and animal models. Profound differences in the susceptibility to inhibition of steroid conversion among all 17beta-HSDs analyzed were observed. Especially, the rodent 17beta-HSDs 1 were significantly less sensitive to inhibition compared to the human ortholog, while the most similar inhibition pattern to the human 17beta-HSD 1 was obtained with the marmoset enzyme. Molecular docking experiments predicted estrone as the most potent inhibitor. The best performing compound in enzymatic assays was also highly ranked by docking scoring for the human enzyme. However, species-specific prediction of inhibitor performance by molecular docking was not possible. We show that experiments with good candidate compounds would out-select them in the rodent model during preclinical optimization steps. Potentially active human-relevant drugs, therefore, would no longer be further developed. Activity and efficacy screens in heterologous species systems must be evaluated with caution.

ELM: the status of the 2010 eukaryotic linear motif resource.

Linear motifs are short segments of multidomain proteins that provide regulatory functions independently of protein tertiary structure. Much of intracellular signalling passes through protein modifications at linear motifs. Many thousands of linear motif instances, most notably phosphorylation sites, have now been reported. Although clearly very abundant, linear motifs are difficult to predict de novo in protein sequences due to the difficulty of obtaining robust statistical assessments. The ELM resource at http://elm.eu.org/ provides an expanding knowledge base, currently covering 146 known motifs, with annotation that includes >1300 experimentally reported instances. ELM is also an exploratory tool for suggesting new candidates of known linear motifs in proteins of interest. Information about protein domains, protein structure and native disorder, cellular and taxonomic contexts is used to reduce or deprecate false positive matches. Results are graphically displayed in a 'Bar Code' format, which also displays known instances from homologous proteins through a novel 'Instance Mapper' protocol based on PHI-BLAST. ELM server output provides links to the ELM annotation as well as to a number of remote resources. Using the links, researchers can explore the motifs, proteins, complex structures and associated literature to evaluate whether candidate motifs might be worth experimental investigation.

Comprehensive classification of nucleotidyltransferase fold proteins: identification of novel families and their representatives in human.

This article presents a comprehensive review of large and highly diverse superfamily of nucleotidyltransferase fold proteins by providing a global picture about their evolutionary history, sequence-structure diversity and fulfilled functional roles. Using top-of-the-line homology detection method combined with transitive searches and fold recognition, we revised the realm of these superfamily in numerous databases of catalogued protein families and structures, and identified 10 new families of nucleotidyltransferase fold. These families include hundreds of previously uncharacterized and various poorly annotated proteins such as Fukutin/LICD, NFAT, FAM46, Mab-21 and NRAP. Some of these proteins seem to play novel important roles, not observed before for this superfamily, such as regulation of gene expression or choline incorporation into cell membrane. Importantly, within newly detected families we identified 25 novel superfamily members in human genome. Among these newly assigned members are proteins known to be involved in congenital muscular dystrophy, neurological diseases and retinal pigmentosa what sheds some new light on the molecular background of these genetic disorders. Twelve of new human nucleotidyltransferase fold proteins belong to Mab-21 family known to be involved in organogenesis and development. The determination of specific biological functions of these newly detected proteins remains a challenging task.