Littérature scientifique sur le sujet « DUF3494 »

Ice-binding proteins (IBPs) affect ice crystal growth by attaching to crystal faces. We present the effects on the growth of an ice single crystal caused by an ice-binding protein from the sea ice microalga Fragilariopsis cylindrus (fcIBP) that is characterized by the widespread domain of unknown function 3494 (DUF3494) and known to cause a moderate freezing point depression (below 1 °C). By the application of interferometry, bright-field microscopy, and fluorescence microscopy, we observed that the fcIBP attaches to the basal faces of ice crystals, thereby inhibiting their growth in the c direction and resulting in an increase in the effective supercooling with increasing fcIBP concentration. In addition, we observed that the fcIBP attaches to prism faces and inhibits their growth. In the event that the effective supercooling is small and crystals are faceted, this process causes an emergence of prism faces and suppresses crystal growth in the a direction. When the effective supercooling is large and ice crystals have developed into a dendritic shape, the suppression of prism face growth results in thinner dendrite branches, and growth in the a direction is accelerated due to enhanced latent heat dissipation. Our observations clearly indicate that the fcIBP occupies a separate position in the classification of IBPs due to the fact that it suppresses the growth of basal faces, despite its moderate freezing point depression.

Abstract Modification dependent restriction endonucleases (MDREs) often have separate catalytic and modification dependent domains. We systematically looked for previously uncharacterized fusion proteins featuring a PUA or DUF3427 domain and HNH or PD-(D/E)XK catalytic domain. The enzymes were clustered by similarity of their putative modification sensing domains into several groups. The TspA15I (VcaM4I, CmeDI), ScoA3IV (MsiJI, VcaCI) and YenY4I groups, all featuring a PUA superfamily domain, preferentially cleaved DNA containing 5-methylcytosine or 5-hydroxymethylcytosine. ScoA3V, also featuring a PUA superfamily domain, but of a different clade, exhibited 6-methyladenine stimulated nicking activity. With few exceptions, ORFs for PUA-superfamily domain containing endonucleases were not close to DNA methyltransferase ORFs, strongly supporting modification dependent activity of the endonucleases. DUF3427 domain containing fusion proteins had very little or no endonuclease activity, despite the presence of a putative PD-(D/E)XK catalytic domain. However, their expression potently restricted phage T4gt in Escherichia coli cells. In contrast to the ORFs for PUA domain containing endonucleases, the ORFs for DUF3427 fusion proteins were frequently found in defense islands, often also featuring DNA methyltransferases.

Members of the DUF34 (domain of unknown function 34) family, also known as the NIF3 protein superfamily, are ubiquitous across superkingdoms. Proteins of this family have been widely annotated as “GTP cyclohydrolase I type 2” through electronic propagation based on one study. Here, the annotation status of this protein family was examined through a comprehensive literature review and integrative bioinformatic analyses that revealed varied pleiotropic associations and phenotypes. This analysis combined with functional complementation studies strongly challenges the current annotation and suggests that DUF34 family members may serve as metal ion insertases, chaperones, or metallocofactor maturases. This general molecular function could explain how DUF34 subgroups participate in highly diversified pathways such as cell differentiation, metal ion homeostasis, pathogen virulence, redox, and universal stress responses.

The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

The Zn(II)2Cys6 zinc cluster gene family is a subclass of zinc-finger proteins, which are transcriptional regulators involved in a wide variety of biological processes in fungi. We performed genome-wide identification and characterization of Zn(II)2Cys6 zinc-cluster gene (C6 zinc gene) family in Tolypocladium guangdongense, Cordyceps militaris and Ophiocordyceps sinensis. Based on the structures of the C6 zinc domains, these proteins were observed to be evolutionarily conserved in ascomycete fungi. We focused on T. guangdongense, a medicinal fungus, and identified 139 C6 zinc genes which could be divided into three groups. Among them, 49.6% belonged to the fungal specific transcriptional factors, and 16% had a DUF3468 domain. Homologous and phylogenetic analysis indicated that 29 C6 zinc genes were possibly involved in the metabolic process, while five C6 zinc genes were supposed to be involved in asexual or sexual development. Gene expression analysis revealed that 54 C6 zinc genes were differentially expressed under light, including two genes that possibly influenced the development, and seven genes that possibly influenced the metabolic processes. This indicated that light may affect the development and metabolic processes, at least partially, through the regulation of C6 zinc genes in T. guangdongense. Our results provide comprehensive data for further analyzing the functions of the C6 zinc genes.

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by an unusually complex GPI-N-acetylglucosaminyltransferase (GPI-GnT) consisting of at least six proteins. Here, we report that human GPI-GnT requires another component, termed PIG-Y, a 71 amino acid protein with two transmembrane domains. The Burkitt lymphoma cell line Daudi, severely defective in the surface expression of GPI-anchored proteins, was a null mutant of PIG-Y. A complex of six components was formed without PIG-Y. PIG-Y appeared to be directly associated with PIG-A, implying that PIG-Y is the key molecule that regulates GPI-GnT activity by binding directly to the catalytic subunit PIG-A. PIG-Y is probably homologous to yeast Eri1p, a component of GPI-GnT. We did not obtain evidence for a functional linkage between GPI-GnT and ras GTPases in mammalian cells as has been reported for yeast cells. A single transcript encoded PIG-Y and, to its 5′ side, another protein PreY that has homologues in a wide range of organisms and is characterized by a conserved domain termed DUF343. These two proteins are translated from one mRNA by leaky scanning of the PreY initiation site.

In this study, we conducted whole-genome sequencing with six species of Pectobacterium composed of seven strains, JR1.1, BP201601.1, JK2.1, HNP201719, MYP201603, PZ1, and HC, for the analysis of pathogenic factors associated with the genome of Pectobacterium. The genome sizes ranged from 4,724,337 bp to 5,208,618 bp, with the GC content ranging from 50.4% to 52.3%. The average nucleotide identity was 98% among the two Pectobacterium species and ranged from 88% to 96% among the remaining six species. A similar distribution was observed in the carbohydrate-active enzymes (CAZymes) class and extracellular plant cell wall degrading enzymes (PCWDEs). HC showed the highest number of enzymes in CAZymes and the lowest number in the extracellular PCWDEs. Six strains showed four subsets, and HC demonstrated three subsets, except hasDEF, in type I secretion system, while the type II secretion system of the seven strains was conserved. Components of human pathogens, such as Salmonella pathogenicity island 1 type type III secretion system (T3SS) and effectors, were identified in PZ1; T3SSa was not identified in HC. Two putative effectors, including hrpK, were identified in seven strains along with dspEF. We also identified 13 structural genes, six regulator genes, and five accessory genes in the type VI secretion system (T6SS) gene cluster of six Pectobacterium species, along with the loss of T6SS in PZ1. HC had two subsets, and JK2.1 had three subsets of T6SS. With the GxSxG motif, the phospholipase A gene did locate among tssID and duf4123 genes in the T6SSa cluster of all strains. Important domains were identified in the vgrG/paar islands, including duf4123, duf2235, vrr-nuc, and duf3396.

Dormancy is a common strategy adopted by bacterial cells as a means of surviving adverse environmental conditions. ForStreptomycesbacteria, this involves developing chains of dormant exospores that extend away from the colony surface. Both spore formation and subsequent spore germination are tightly controlled processes, and while significant progress has been made in understanding the underlying regulatory and enzymatic bases for these, there are still significant gaps in our understanding. One class of proteins with a potential role in spore-associated processes are the so-called resuscitation-promoting factors, or Rpfs, which in other actinobacteria are needed to restore active growth to dormant cell populations. The model speciesStreptomyces coelicolorencodes five Rpf proteins (RpfA to RfpE), and here we show that these proteins have overlapping functions during growth. Collectively, theS. coelicolorRpfs promote spore germination and are critical for growth under nutrient-limiting conditions. Previous studies have revealed structural similarities between the Rpf domain and lysozyme, and ourin vitrobiochemical assays revealed various levels of peptidoglycan cleavage capabilities for each of these fiveStreptomycesenzymes. Peptidoglycan remodeling by enzymes such as these must be stringently governed so as to retain the structural integrity of the cell wall. Our results suggest that one of the Rpfs, RpfB, is subject to a unique mode of enzymatic autoregulation, mediated by a domain of previously unknown function (DUF348) located within the N terminus of the protein; removal of this domain led to significantly enhanced peptidoglycan cleavage.

Una proteina in grado di legare i cristalli di ghiaccio è definita proteina legante il ghiaccio o IBP acronimo dall’inglese ice-binding protein. Le IBP grazie alla loro capacità di abbassare il punto di congelamento dell’acqua, aumentando il gap di isteresi termica (TH). Questo intervallo è definito come la differenza tra il punto di fusione e di congelamento dell’acqua. La seconda attività delle IBP è l’inibizione della ricristallizzazione del ghiaccio (ice recrystallization inhibition, IRI). Infatti, queste proteine stabilizzano i piccoli cristalli di ghiaccio impedendo la formazione di cristalli di ghiaccio di grosse dimensioni che sono dannosi per le cellule. Le IBP sono state identificate in numerosi organismi tra cui pesci, insetti, batteri, alghe e lieviti. Queste proteine rappresentano un esempio di evoluzione convergente, infatti tutte le IBP condividono lo stesso meccanismo di legame con il ghiaccio nonostante una sorprendente diversità strutturale e funzionale. Questo lavoro di tesi è focalizzato sulla caratterizzazione funzionale e strutturale di EfcIBP, una IBP batterica identificata da analisi di metagenomica effettuate sul ciliato Antartico Euplotes focardii e sul consorzio batterico ad esso associato. La struttura 3D di EfcIBP è stata risolta mediante cristallografia ai raggi X e consiste in un β-solenoide con un α-elica parallela all’asse principale della proteina. L’analisi strutturale ha permesso di identificare tre diverse facce del solenoide denominate A, B e C. Simulazioni di docking suggeriscono che EfcIBP è in grado di legare i cristalli di ghiaccio tramite le facce B e C del solenoide. Questa ipotesi è stata verificata attraverso la progettazione razionale di 6 varianti che sono state prodotte e saggiate per la loro attività. In generale, questi risultati indicano che EfcIBP è in grado di legare i cristalli di ghiaccio attraverso le facce B e C del solenoide. Questa peculiarità strutturale si riflette in un’insolita combinazione di attività di IRI e TH. Infatti, EfcIBP presenta una notevole attività di IRI in un intervallo di concentrazione nanomolare e una attività di isteresi termica di 0.53°C alla concentrazione di 50 μM che la rende una IBP moderata. All’interno del gap di TH, i cristalli di ghiaccio presentano una forma esagonale, mentre a temperature al di sotto della temperatura di congelamento presentano una forma a “Saturno". La proteina chimerica formata dalla “green fluorescent protein” e da EfcIBP è stata utilizzata per determinare a quali piani del cristallo di ghiaccio la proteina è in grado di legarsi e con quale cinetica. I dati sperimentali suggeriscono che le peculiarità funzionali di EfcIBP sono dovute alla sua capacità di legare velocemente i piani basali e piramidali del cristallo di ghiaccio. Questi dati, insieme alla presenza di una sequenza segnale per la secrezione, suggeriscono che EfcIBP è secreta e svolge la funzione di mantenere liquido l’ambiente circostante aumentando lo spazio vitale. In conclusione, EfcIBP è un nuovo tipo di IBP con proprietà insolite di legame al ghiaccio e di attività di IRI. Questo studio ha contribuito ad identificare una nuova classe di IBP moderate che potrebbero essere sfruttate come crioprotettori in diversi settori come la criobiologia e quello alimentare.
Ice-binding proteins (IBPs) are characterized by the ability to control the growth of ice crystals. IBPs are active in increasing thermal hysteresis (TH) gap as they decrease the freezing point of water. On the other hand, IBPs can inhibit ice recrystallization (IRI) and stabilize small ice crystals at the expense of the harmful, large ones. IBPs have been identified in several organisms including higher Eukaryotes and microorganisms such as bacteria, yeasts and algae. Although IBPs share the ability to bind ice crystals, proteins from different sources present different 3D structures, from α-helix to β-solenoid proteins. This thesis is focused on the structural and functional characterization of EfcIBP, a bacterial IBP identified by metagenomic analysis of the Antarctic ciliate Euplotes focardii and the associated consortium of non-cultivable bacteria. The 3D structure of EfcIBP, solved by X-ray crystallography, consists in a β-solenoid with an α-helix aligned along the axis of the β-helix. It is possible to distinguish three different faces: A, B and C. Docking simulations suggest that B and C faces are involved in ice binding. This hypothesis was tested by the rational design of six variants that were produced and assayed for their activity. Overall, these experiments indicate that both solenoid faces contribute to the activity of EfcIBP. EfcIBP displays remarkable IRI activity at nanomolar concentration and a TH activity of 0.53°C at the concentration of 50 μM. The atypical combination between these two activities could stem from the ability of this protein to bind ice crystals through two faces of the solenoid. In the presence of EfcIBP, ice crystals show a hexagonal trapezohedron shape within the TH gap, and a unique “Saturn-shape” below the freezing point. A chimeric protein consisting of the fusion between EfcIBP and the green fluorescent protein was used to deeper investigate on this aspects by analyses of fluorescence ice plane affinity and binding kinetics. Overall, experimental data suggest that the EfcIBP unique pattern of ice growth and burst are due to its high rate of binding at the basal and the pyramidal near-basal planes of ice crystals. These data, together with the signal sequence for the secretion, suggest that EfcIBP is secreted in local environment where it becomes active in increasing the habitable space. In conclusion, EfcIBP is a new type of IBP with unusual properties of ice shaping and IRI activity. This study opens new scenarios in the field of IBPs by contributing to identify a new class of moderate IBPs potentially exploitable as cryoprotectants in several fields, such as cryobiology and food science.