From microbiology to cancer biology: the Rid protein family prevents cellular damage caused by endogenously generated reactive nitrogen species

The Rid family of proteins is highly conserved and broadly distributed throughout the domains of life. Genetic and biochemical studies, primarily in Salmonella enterica, have defined a role for RidA in responding to endogenously generated reactive metabolites. The data show that 2‐aminoacrylate (2AA), a reactive enamine intermediate generated by some pyridoxal 5′‐phosphate‐dependent enzymes, accumulates in the absence of RidA. The accumulation of 2AA leads to covalent modification and inactivation of several enzymes involved in essential metabolic processes. This review describes the 2AA hydrolyzing activity of RidA and the effect of this biochemical activity on the metabolic network, which impacts organism fitness. The reported activity of RidA and the consequences encountered in vivo when RidA is absent have challenged fundamental assumptions in enzymology, biochemistry and cell metabolism regarding the fate of transiently generated reactive enamine intermediates. The current understanding of RidA in Salmonella and the broad distribution of Rid family proteins provide exciting opportunities for future studies to define metabolic roles of Rid family members from microbes to man.     

A computational study of elongation factor G (EFG) duplicated genes: diverged nature underlying the innovation on the same structural template

Background

Elongation factor G (EFG) is a core translational protein that catalyzes the elongation and recycling phases of translation. A more complex picture of EFG''s evolution and function than previously accepted is emerging from analyzes of heterogeneous EFG family members. Whereas the gene duplication is postulated to be a prominent factor creating functional novelty, the striking divergence between EFG paralogs can be interpreted in terms of innovation in gene function.

Methodology/Principal Findings

We present a computational study of the EFG protein family to cover the role of gene duplication in the evolution of protein function. Using phylogenetic methods, genome context conservation and insertion/deletion (indel) analysis we demonstrate that the EFG gene copies form four subfamilies: EFG I, spdEFG1, spdEFG2, and EFG II. These ancient gene families differ by their indispensability, degree of divergence and number of indels. We show the distribution of EFG subfamilies and describe evidences for lateral gene transfer and recent duplications. Extended studies of the EFG II subfamily concern its diverged nature. Remarkably, EFG II appears to be a widely distributed and a much-diversified subfamily whose subdivisions correlate with phylum or class borders. The EFG II subfamily specific characteristics are low conservation of the GTPase domain, domains II and III; absence of the trGTPase specific G2 consensus motif “RGITI”; and twelve conserved positions common to the whole subfamily. The EFG II specific functional changes could be related to changes in the properties of nucleotide binding and hydrolysis and strengthened ionic interactions between EFG II and the ribosome, particularly between parts of the decoding site and loop I of domain IV.

Conclusions/Significance

Our work, for the first time, comprehensively identifies and describes EFG subfamilies and improves our understanding of the function and evolution of EFG duplicated genes.     

Structural relationships in the lysozyme superfamily: significant evidence for glycoside hydrolase signature motifs

Background

Chitin is a polysaccharide that forms the hard, outer shell of arthropods and the cell walls of fungi and some algae. Peptidoglycan is a polymer of sugars and amino acids constituting the cell walls of most bacteria. Enzymes that are able to hydrolyze these cell membrane polymers generally play important roles for protecting plants and animals against infection with insects and pathogens. A particular group of such glycoside hydrolase enzymes share some common features in their three-dimensional structure and in their molecular mechanism, forming the lysozyme superfamily.

Results

Besides having a similar fold, all known catalytic domains of glycoside hydrolase proteins of lysozyme superfamily (families and subfamilies GH19, GH22, GH23, GH24 and GH46) share in common two structural elements: the central helix of the all-α domain, which invariably contains the catalytic glutamate residue acting as general-acid catalyst, and a β-hairpin pointed towards the substrate binding cleft. The invariant β-hairpin structure is interestingly found to display the highest amino acid conservation in aligned sequences of a given family, thereby allowing to define signature motifs for each GH family. Most of such signature motifs are found to have promising performances for searching sequence databases. Our structural analysis further indicates that the GH motifs participate in enzymatic catalysis essentially by containing the catalytic water positioning residue of inverting mechanism.

Conclusions

The seven families and subfamilies of the lysozyme superfamily all have in common a β-hairpin structure which displays a family-specific sequence motif. These GH β-hairpin motifs contain potentially important residues for the catalytic activity, thereby suggesting the participation of the GH motif to catalysis and also revealing a common catalytic scheme utilized by enzymes of the lysozyme superfamily.     

Molecular evolution and functional divergence of the cytochrome P450 3 (CYP3) Family in Actinopterygii (ray-finned fish)

Background

The cytochrome P450 (CYP) superfamily is a multifunctional hemethiolate enzyme that is widely distributed from Bacteria to Eukarya. The CYP3 family contains mainly the four subfamilies CYP3A, CYP3B, CYP3C and CYP3D in vertebrates; however, only the Actinopterygii (ray-finned fish) have all four subfamilies and detailed understanding of the evolutionary relationship of Actinopterygii CYP3 family members would be valuable.

Methods and Findings

Phylogenetic relationships were constructed to trace the evolutionary history of the Actinopterygii CYP3 family genes. Selection analysis, relative rate tests and functional divergence analysis were combined to interpret the relationship of the site-specific evolution and functional divergence in the Actinopterygii CYP3 family. The results showed that the four CYP3 subfamilies in Actinopterygii might be formed by gene duplication. The first gene duplication event was responsible for divergence of the CYP3B/C clusters from ancient CYP3 before the origin of the Actinopterygii, which corresponded to the fish-specific whole genome duplication (WGD). Tandem repeat duplication in each of the homologue clusters produced stable CYP3B, CYP3C, CYP3A and CYP3D subfamilies. Acceleration of asymmetric evolutionary rates and purifying selection together were the main force for the production of new subfamilies and functional divergence in the new subset after gene duplication, whereas positive selection was detected only in the retained CYP3A subfamily. Furthermore, nearly half of the functional divergence sites appear to be related to substrate recognition, which suggests that site-specific evolution is closely related with functional divergence in the Actinopterygii CYP3 family.

Conclusions

The split of fish-specific CYP3 subfamilies was related to the fish-specific WGD, and site-specific acceleration of asymmetric evolutionary rates and purifying selection was the main force for the origin of the new subfamilies and functional divergence in the new subset after gene duplication. Site-specific evolution in substrate recognition was related to functional divergence in the Actinopterygii CYP3 family.     

Evolution of the karyopherin-β family of nucleocytoplasmic transport factors; ancient origins and continued specialization

Background

Macromolecular transport across the nuclear envelope (NE) is achieved through nuclear pore complexes (NPCs) and requires karyopherin-βs (KAP-βs), a family of soluble receptors, for recognition of embedded transport signals within cargo. We recently demonstrated, through proteomic analysis of trypanosomes, that NPC architecture is likely highly conserved across the Eukaryota, which in turn suggests conservation of the transport mechanisms. To determine if KAP-β diversity was similarly established early in eukaryotic evolution or if it was subsequently layered onto a conserved NPC, we chose to identify KAP-β sequences in a diverse range of eukaryotes and to investigate their evolutionary history.

Results

Thirty six predicted proteomes were scanned for candidate KAP-β family members. These resulting sequences were resolved into fifteen KAP-β subfamilies which, due to broad supergroup representation, were most likely represented in the last eukaryotic common ancestor (LECA). Candidate members of each KAP-β subfamily were found in all eukaryotic supergroups, except XPO6, which is absent from Archaeplastida. Phylogenetic reconstruction revealed the likely evolutionary relationships between these different subfamilies. Many species contain more than one representative of each KAP-β subfamily; many duplications are apparently taxon-specific but others result from duplications occurring earlier in eukaryotic history.

Conclusions

At least fifteen KAP-β subfamilies were established early in eukaryote evolution and likely before the LECA. In addition we identified expansions at multiple stages within eukaryote evolution, including a multicellular plant-specific KAP-β, together with frequent secondary losses. Taken with evidence for early establishment of NPC architecture, these data demonstrate that multiple pathways for nucleocytoplasmic transport were established prior to the radiation of modern eukaryotes but that selective pressure continues to sculpt the KAP-β family.     

A Cytohesin Homolog in Dictyostelium Amoebae

Background

Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration.

Principal Findings

Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG⁻ cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced.

Significance

The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.     

Comparative Mitogenomic Analysis of Damsel Bugs Representing Three Tribes in the Family Nabidae (Insecta: Hemiptera)

Background

Nabidae, a family of predatory heteropterans, includes two subfamilies and five tribes. We previously reported the complete mitogenome of Alloeorhynchus bakeri, a representative of the tribe Prostemmatini in the subfamily Prostemmatinae. To gain a better understanding of architecture and evolution of mitogenome in Nabidae, mitogenomes of five species representing two tribes (Gorpini and Nabini) in the subfamily Nabinae were sequenced, and a comparative mitogenomic analysis of three nabid tribes in two subfamilies was carried out.

Methodology/Principal Findings

Nabid mitogenomes share a similar nucleotide composition and base bias, except for the control region, where differences are observed at the subfamily level. In addition, the pattern of codon usage is influenced by the GC content and consistent with the standard invertebrate mitochondrial genetic code and the preference for A+T-rich codons. The comparison among orthologous protein-coding genes shows that different genes have been subject to different rates of molecular evolution correlated with the GC content. The stems and anticodon loops of tRNAs are extremely conserved, and the nucleotide substitutions are largely restricted to TψC and DHU loops and extra arms, with insertion-deletion polymorphisms. Comparative analysis shows similar rates of substitution between the two rRNAs. Long non-coding regions are observed in most Gorpini and Nabini mtDNAs in-between trnI-trnQ and/or trnS2-nad1. The lone exception, Nabis apicalis, however, has lost three tRNAs. Overall, phylogenetic analysis using mitogenomic data is consistent with phylogenies constructed mainly form morphological traits.

Conclusions/Significance

This comparative mitogenomic analysis sheds light on the architecture and evolution of mitogenomes in the family Nabidae. Nucleotide diversity and mitogenomic traits are phylogenetically informative at subfamily level. Furthermore, inclusion of a broader range of samples representing various taxonomic levels is critical for the understanding of mitogenomic evolution in damsel bugs.     

Classification of fungal and bacterial lytic polysaccharide monooxygenases

Background

Lytic polysaccharide monooxygenases are important enzymes for the decomposition of recalcitrant biological macromolecules such as plant cell wall and chitin polymers. These enzymes were originally designated glycoside hydrolase family 61 and carbohydrate-binding module family 33 but are now classified as auxiliary activities 9, 10 and 11 in the CAZy database. To obtain a systematic analysis of the divergent families of lytic polysaccharide monooxygenases we used Peptide Pattern Recognition to divide 5396 protein sequences resembling enzymes from families AA9 (1828 proteins), AA10 (2799 proteins) and AA11 (769 proteins) into subfamilies.

Results

The results showed that the lytic polysaccharide monooxygenases have two conserved regions identified by conserved peptides specific for each AA family. The peptides were used for in silico PCR discovery of the lytic polysaccharide monooxygenases in 79 fungal and 95 bacterial genomes. The bacterial genomes encoded 0 – 7 AA10s (average 0.6). No AA9 or AA11 were found in the bacteria. The fungal genomes encoded 0 – 40 AA9s (average 7) and 0 – 15 AA11s (average 2) and two of the fungi possessed a gene encoding a putative AA10. The AA9s were mainly found in plant cell wall-degrading asco- and basidiomycetes in agreement with the described role of AA9 enzymes. In contrast, the AA11 proteins were found in 36 of the 39 ascomycetes and in only two of the 32 basidiomycetes and their abundance did not correlate to the degradation of cellulose and hemicellulose.

Conclusions

These results provides an overview of the sequence characteristics and occurrence of the divergent AA9, AA10 and AA11 families and pave the way for systematic investigations of the of lytic polysaccharide monooxygenases and for structure-function studies of these enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1601-6) contains supplementary material, which is available to authorized users.     

Family caregivers' experiences of involuntary psychiatric hospital admissions of their relatives--a qualitative study

Background

Family caregivers of people with mental disorders are frequently involved in involuntary hospital admissions of their relatives.

Objective

To explore family caregivers'' experience of involuntary admission of their relative.

Method

30 in-depth interviews were conducted with family caregivers of 29 patients who had been involuntarily admitted to 12 hospitals across England. Interviews were analysed using thematic analysis.

Results

Four major themes of experiences were identified: relief and conflicting emotions in response to the relative''s admission; frustration with a delay in getting help; being given the burden of care by services; and difficulties with confidentiality.Relief was a predominant emotion as a response to the relative''s admission and it was accompanied by feelings of guilt and worry. Family caregivers frequently experienced difficulties in obtaining help from services prior to involuntary admission and some thought that services responded to crises rather than prevented them. Family caregivers experienced increased burden when services shifted the responsibility of caring for their mentally unwell relatives to them. Confidentiality was a delicate issue with family caregivers wanting more information and a say in decisions when they were responsible for aftercare, and being concerned about confidentiality of information they provided to services.

Conclusion

Compulsory admission of a close relative can be a complex and stressful experience for family caregivers. In order for caregivers to be effective partners in care, a balance needs to be struck between valuing their involvement in providing care for a patient and not overburdening them.     

Classification of Protein Kinases on the Basis of Both Kinase and Non-Kinase Regions

Background

Protein phosphorylation is a generic way to regulate signal transduction pathways in all kingdoms of life. In many organisms, it is achieved by the large family of Ser/Thr/Tyr protein kinases which are traditionally classified into groups and subfamilies on the basis of the amino acid sequence of their catalytic domains. Many protein kinases are multi-domain in nature but the diversity of the accessory domains and their organization are usually not taken into account while classifying kinases into groups or subfamilies.

Methodology

Here, we present an approach which considers amino acid sequences of complete gene products, in order to suggest refinements in sets of pre-classified sequences. The strategy is based on alignment-free similarity scores and iterative Area Under the Curve (AUC) computation. Similarity scores are computed by detecting common patterns between two sequences and scoring them using a substitution matrix, with a consistent normalization scheme. This allows us to handle full-length sequences, and implicitly takes into account domain diversity and domain shuffling. We quantitatively validate our approach on a subset of 212 human protein kinases. We then employ it on the complete repertoire of human protein kinases and suggest few qualitative refinements in the subfamily assignment stored in the KinG database, which is based on catalytic domains only. Based on our new measure, we delineate 37 cases of potential hybrid kinases: sequences for which classical classification based entirely on catalytic domains is inconsistent with the full-length similarity scores computed here, which implicitly consider multi-domain nature and regions outside the catalytic kinase domain. We also provide some examples of hybrid kinases of the protozoan parasite Entamoeba histolytica.

Conclusions

The implicit consideration of multi-domain architectures is a valuable inclusion to complement other classification schemes. The proposed algorithm may also be employed to classify other families of enzymes with multi-domain architecture.     

Arabidopsis and Maize RidA Proteins Preempt Reactive Enamine/Imine Damage to Branched-Chain Amino Acid Biosynthesis in Plastids

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.     

The Associations between the Family Education and Mortality of Patients on Peritoneal Dialysis

Aims

To investigate whether education level of family members predicts all-cause and cardiovascular death and initial-episode peritonitis in patients on peritoneal dialysis (PD).

Methods

A total of 2264 patients on chronic PD were collected from seven centers affiliated with the Socioeconomic Status on the Outcome of Peritoneal Dialysis (SSOP) Study. All demographic, socioeconomic and laboratory data of patients and the education level of all family members were recorded at baseline. Multivariate Cox regression was used to calculate the hazard ratio (HR) of all-cause and cardiovascular mortality, and initial-episode peritonitis with adjustments for recognized traditional factors.

Results

There were no significant differences in baseline characteristics between patients with (n = 1752) and without (n = 512) complete education information. According to the highest education level of patients'' family, included 1752 patients were divided into four groups, i.e. elementary or lower (15%), middle (27%), high (24%) and more than high school (34%). The family highest education (using elementary school or lower group as reference, hazard ratio and 95% confidence interval of middle school group, high school group and more than high school group was 0.68[0.48–0.96], 0.64[0.45–0.91], 0.66[0.48–0.91], respectively) rather than their average education level or patients'' or spouse''s education was significantly associated with the higher mortality. Neither patients'' nor family education level did correlate to the risk for cardiovascular death or initial-episode peritonitis.

Conclusions

Family members'' education level was found to be a novel predictor of PD outcome. Family, as the main source of health care providers, should be paid more attention in our practice.