Family Of Ubiquitous Proteins Research Articles

Noninflammatory 97-amino acid High Mobility Group Box 1 derived polypeptide disrupts and prevents diverse biofilms

Bacterial biofilm communities are embedded in a protective extracellular matrix comprised of various components, with its' integrity largely owed to a 3-dimensional lattice of extracellular DNA (eDNA) interconnected by Holliday Junction (HJ)-like structures and stabilised by the ubiquitous eubacterial DNABII family of DNA-binding architectural proteins. We recently showed that the host innate immune effector High Mobility Group Box 1 (HMGB1) protein possesses extracellular anti-biofilm activity by destabilising these HJ-like structures, resulting in release of biofilm-resident bacteria into a vulnerable state. Herein, we showed that HMGB1's anti-biofilm activity was completely contained within a contiguous 97 amino acid region that retained DNA-binding activity, called 'mB Box-97'. We engineered a synthetic version of this 97-mer and introduced a single amino acid change which lacked any post-translational modifications, and tested its activity independently and in combination with a humanised monoclonal antibody that disrupts biofilms by the distinct mechanism of DNABII protein sequestration. mB Box-97 disrupted and prevented biofilms, including those formed by the ESKAPEE pathogens, and importantly reduced measurable proinflammatory activity normally associated with HMGB1 in a murine lung infection model. Herein, we discuss the value of targeting the ubiquitous eDNA-dependent matrix of biofilms via mB Box-97 used singly or in a dual host-augmenting/pathogen-targeted cocktail to resolve bacterial biofilm infections. This work was supported by NIH/NIDCD R01DC011818 to L.O.B. and S.D.G. and NIH/NIAID R01AI155501 to S.D.G.

Copine7 deficiency leads to hepatic fat accumulation via mitochondrial dysfunction

ObjectiveMitochondrial dysfunction affects hepatic lipid homeostasis and promotes ROS generation. Copine7 (CPNE7) belongs to the ubiquitous copine family of calcium-dependent phospholipid binding proteins. CPNE7 has a high calcium ion binding affinity and the capacity to scavenge reactive oxygen species (ROS). A recent study reported that abnormalities in fatty acid and lipid metabolism were linked to the gene variant of CPNE7. Therefore, the purpose of this study is to examine the role of Cpne7 in hepatic lipid metabolism based on mitochondrial function. MethodsLipid metabolism, mitochondrial function, and ROS production were investigated in high-fat diet (HFD)-fed Cpne7−/− mice and H2O2-damaged HepG2 hepatocytes following CPNE7 silencing or overexpression. ResultsCpne7 deficiency promoted severe hepatic steatosis in the HFD-induced NAFLD model. More importantly, mitochondrial dysfunction was observed along with an imbalance of mitochondrial dynamics in the livers of HFD-fed Cpne7−/−mice, resulting in high ROS levels. Similarly, CPNE7-silenced HepG2 hepatocytes showed high ROS levels, mitochondrial dysfunction, and increased lipid contents. On the contrary, CPNE7-overexpressed HepG2 cells showed low ROS levels, enhanced mitochondrial function and decreased lipid contents under H2O2-induced oxidative stress. ConclusionsIn the liver, Cpne7 deficiency causes excessive ROS formation and mitochondrial dysfunction, which aggravates lipid metabolism abnormalities. These findings provide evidence that Cpne7 deficiency contributes to the pathogenesis of NAFLD, suggesting Cpne7 as a novel therapeutic target for NAFLD.

Overexpression of peanut (Arachis hypogaea L.) AhGRFi gene enhanced root growth inhibition under exogenous NAA treatment in Arabidopsis thaliana.

The 14-3-3 protein is a kind of evolutionary ubiquitous protein family highly conserved in eukaryotes. Initially, 14-3-3 proteins were reported in mammalian nervous tissues, but in the last decade, their role in various metabolic pathways in plants established the importance of 14-3-3 proteins. In the present study, a total of 22 14-3-3 genes, also called general regulatory factors (GRF), were identified in the peanut (Arachis hypogaea) genome, out of which 12 belonged to the ε group, whereas 10 of them belonged to the non- ε-group. Tissue-specific expression of identified 14-3-3 genes were studied using transcriptome analysis. The peanut AhGRFi gene was cloned and transformed into Arabidopsis thaliana. The investigation of subcellular localization indicated that AhGRFi is localized in the cytoplasm. Overexpression of the AhGRFi gene in transgenic Arabidopsis showed that under exogenous 1-naphthaleneacetic acid (NAA) treatment, root growth inhibition in transgenic plants was enhanced. Further analysis indicated that the expression of auxin-responsive genes IAA3, IAA7, IAA17, and SAUR-AC1 was upregulated and GH3.2 and GH3.3 were downregulated in transgenic plants, but the expression of GH3.2, GH3.3, and SAUR-AC1 showed opposite trends of change under NAA treatment. These results suggest that AhGRFi may be involved in auxin signaling during seedling root development. An in-depth study of the molecular mechanism of this process remains to be further explored.

Evolutionary Aspects of Selenium Binding Protein (SBP)

Selenium-binding proteins represent a ubiquitous protein family and recently SBP1 was described as a new stress response regulator in plants. SBP1 has been characterized as a methanethiol oxidase, however its exact role remains unclear. Moreover, in mammals, it is involved in the regulation of anti-carcinogenic growth and progression as well as reduction/oxidation modulation and detoxification. In this work, we delineate the functional potential of certain motifs of SBP in the context of evolutionary relationships. The phylogenetic profiling approach revealed the absence of SBP in the fungi phylum as well as in most non eukaryotic organisms. The phylogenetic tree also indicates the differentiation and evolution of characteristic SBP motifs. Main evolutionary events concern the CSSC motif for which Acidobacteria, Fungi and Archaea carry modifications. Moreover, the CC motif is harbored by some bacteria and remains conserved in Plants, while modified to CxxC in Animals. Thus, the characteristic sequence motifs of SBPs mainly appeared in Archaea and Bacteria and retained in Animals and Plants. Our results demonstrate the emergence of SBP from bacteria and most likely as a methanethiol oxidase.

Functional characterization of two WD40 family proteins, Alr0671 and All2352, from Anabaena PCC 7120 and deciphering their role in abiotic stress management

WD40 domain-containing proteins are one of the eukaryotes' most ancient and ubiquitous protein families. Little is known about the presence and function of these proteins in cyanobacteria in general and Anabaena in particular. In silico analysis confirmed the presence of WD40 repeats. Gene expression analysis indicated that the transcript levels of both the target proteins were up-regulated up to 4 fold in Cd and drought and 2-3 fold in heat, salt, and UV-B stress. Using a fluorescent oxidative stress indicator, we showed that the recombinant proteins were scavenging reactive oxygen species (ROS) (4-5 fold) more efficiently than empty vectors. Chromatin immunoprecipitation analysis (ChIP) and electrophoretic mobility shift assay (EMSA) revealed that the target proteins function as transcription factors after binding to the promoter sequences. The presence of kinase activity (2-4 fold) in the selected proteins indicated that these proteins could modulate the functions of other cellular proteins under stress conditions by inducing phosphorylation of specific amino acids. The chosen proteins also demonstrated interaction with Zn, Cd, and Cu (1.4-2.5 fold), which might stabilize the proteins' structure and biophysical functions under multiple abiotic stresses. The functionally characterized Alr0671 and All2352 proteins act as transcription factors and offer tolerance to agriculturally relevant abiotic stresses.

PheGRF4e initiated auxin signaling during moso bamboo shoot development

As a ubiquitous acid-regulating protein family in eukaryotes, general regulatory factors (GRFs) are active in various life activities of plants. However, detailed investigations of the GRFs gene family in moso bamboo are scarce. Genome-wide characteristics of the GRF gene family in moso bamboo were analyzed using the moso bamboo genome. GRF phylogeny, gene structure, conserved domains, cis-element promoters, and gene expression were systematically analyzed. A total of 20 GRF gene family members were identified in the moso bamboo genome. These genes were divided into ε and non-ε groups. qRT-PCR (real-time quantitative reverse transcription polymerase chain reaction) showed that PheGRF genes responded to auxin and gibberellin treatment. To further study PheGRF gene functions, a yeast two-hybrid experiment was performed and verified by a bimolecular fluorescence complementation experiment. The results showed that PheGRF4e could interact with PheIAA30 (auxin/indole-3-acetic acid, an Aux/IAA family gene), and both were found to act mainly on the root tip meristem and vascular bundle cells of developing shoots by in situ hybridization assay. This study revealed that PheGRF genes were involved in hormone response during moso bamboo shoot development, and the possible regulatory functions of PheGRF genes were enriched by the fact that PheGRF4e initiated auxin signaling by binding to PheIAA30.

Selenium-binding Protein 1 (SBD1): A stress response regulator in Chlamydomonas reinhardtii.

Selenium-binding proteins (SBPs) represent a ubiquitous protein family implicated in various environmental stress responses, although the exact molecular and physiological role of the SBP family remains elusive. In this work, we report the identification and characterization of CrSBD1, an SBP homolog from the model microalgae Chlamydomonas reinhardtii. Growth analysis of the C. reinhardtii sbd1 mutant strain revealed that the absence of a functional CrSBD1 resulted in increased growth under mild oxidative stress conditions, although cell viability rapidly declined at higher hydrogen peroxide (H2O2) concentrations. Furthermore, a combined global transcriptomic and metabolomic analysis indicated that the sbd1 mutant exhibited a dramatic quenching of the molecular and biochemical responses upon H2O2-induced oxidative stress when compared to the wild-type. Our results indicate that CrSBD1 represents a cell regulator, which is involved in the modulation of C. reinhardtii early responses to oxidative stress. We assert that CrSBD1 acts as a member of an extensive and conserved protein-protein interaction network including Fructose-bisphosphate aldolase 3, Cysteine endopeptidase 2, and Glutaredoxin 6 proteins, as indicated by yeast two-hybrid assays.

Functional analysis of cyantraniliprole tolerance ability mediated by ATP-binding cassette transporters in Aphis gossypii glover

Cyantraniliprole, a second-generation anthranilic diamide insecticide, is widely used to control chewing and sucking pests. ATP-binding cassette transporters (ABCs) are a ubiquitous family of membrane proteins that play important roles in insect detoxification mechanisms. However, the potential effects of ABCs on cyantraniliprole-resistance remain unclear. In the present study, synergism bioassays revealed that verapamil, an ABC inhibitor, increased the toxicity of cyantraniliprole by 2.00- and 12.25-fold in the susceptible and cyantraniliprole-resistant strains of Aphis gossypii. Based on transcriptome data, the expression levels of ABCB4, ABCB5, ABCD1, ABCG4, ABCG7, ABCG13, ABCG16, ABCG17, ABCG26 and MRP12 were upregulated 1.56-, 1.32-, 1.51-, 2.03-, 1.65-, 1.50-, 4.18-, 6.07-, 4.68- and 4.69-fold, respectively, in the cyantraniliprole-resistant strain (CyR) compared to the susceptible strain (SS), as determined using RT-qPCR. Drosophila melanogaster ectopically overexpressing ABCB5, ABCG4, ABCG7, ABCG16, ABCG17, ABCG26 and MRP12 exhibited significantly increased tolerance to cyantraniliprole by 11.71-, 2.39-, 4.85-, 2.06-, 3.75-, 4.20- and 3.50-fold, respectively, with ABCB5 and ABCG family members being the most effective. Furthermore, the suppression of ABCB5, ABCG4, ABCG7, ABCG16, ABCG17, ABCG26 and MRP12 significantly increased the sensitivity of the CyR strain to cyantraniliprole. These results indicate that ABCs may play crucial roles in cyantraniliprole resistance and may provide information for shaping resistance management strategies.

Integration of family-wide information to probe the conformational landscape of sugar transporters

Sugar transporters belong to a ubiquitous family of membrane proteins that transport sugarmolecules across the membrane cycling cycles through 4 conformational states following a rocker-switch mechanism. To transport sugar molecules, the large conformational changes required are induced by substrate binding. The current view of the mechanism has evolved with the deposition of new structures in the PDB database, currently numbering 17. However, the present view is based on comparing structures of different transporters, between which factors such as membrane environment, substrate specificity, and kinetics vary significantly.

Structures of multidrug resistance protein MRP4 reveal basis of substrate specificity

ATP-binding cassette (ABC) transporters are a ubiquitous family of transmembrane proteins which hydrolyze ATP to facilitate the movement of substrates across cell membranes. Despite common architectural features, individual eukaryotic ABC transporters have evolved to transport a broad spectrum of substrates. The multidrug resistance protein MRP4, a member of the MRP sub-family of transporters, has been linked to the efflux of diverse endogenous and exogenous substrates, including prostaglandins, cyclic nucleotides, conjugated steroids, and other chemical scaffolds.

Metallothionein1A Regulates Rhizobial Infection and Nodulation in Phaseolus vulgaris.

Metallothioneins (MTs) constitute a heterogeneous family of ubiquitous metal ion-binding proteins. In plants, MTs participate in the regulation of cell growth and proliferation, protection against heavy metal stress, oxidative stress responses, and responses to pathogen attack. Despite their wide variety of functions, the role of MTs in symbiotic associations, specifically nodule-fabacean symbiosis, is poorly understood. Here, we analyzed the role of the PvMT1A gene in Phaseolus vulgaris-Rhizobium tropici symbiosis using bioinformatics and reverse genetics approaches. Using in silico analysis, we identified six genes encoding MTs in P. vulgaris, which were clustered into three of the four classes described in plants. PvMT1A transcript levels were significantly higher in roots inoculated with R. tropici at 7 and 30 days post inoculation (dpi) than in non-inoculated roots. Functional analysis showed that downregulating PvMT1A by RNA interference (RNAi) reduced the number of infection events at 7 and 10 dpi and the number of nodules at 14 and 21 dpi. In addition, nodule development was negatively affected in PvMT1A:RNAi transgenic roots, and these nodules displayed a reduced nitrogen fixation rate at 21 dpi. These results strongly suggest that PvMT1A plays an important role in the infection process and nodule development in P. vulgaris during rhizobial symbiosis.

An Integrative Sialomic Analysis Reveals Molecules From Triatoma sordida (Hemiptera: Reduviidae).

Triatomines have evolved salivary glands that produce versatile molecules with various biological functions, including those leading their interactions with vertebrate hosts’ hemostatic and immunological systems. Here, using high-throughput transcriptomics and proteomics, we report the first sialome study on the synanthropic triatomine Triatoma sordida. As a result, 57,645,372 reads were assembled into 26,670 coding sequences (CDS). From these, a total of 16,683 were successfully annotated. The sialotranscriptomic profile shows Lipocalin as the most abundant protein family within putative secreted transcripts. Trialysins and Kazal-type protease inhibitors have high transcript levels followed by ubiquitous protein families and enzyme classes. Interestingly, abundant trialysin and Kazal-type members are highlighted in this triatomine sialotranscriptome. Furthermore, we identified 132 proteins in T. sordida salivary gland soluble extract through LC-MS/MS spectrometry. Lipocalins, Hemiptera specific families, CRISP/Antigen-5 and Kazal-type protein inhibitors proteins were identified. Our study provides a comprehensive description of the transcript and protein compositions of the salivary glands of T. sordida. It significantly enhances the information in the Triatominae sialome databanks reported so far, improving the understanding of the vector’s biology, the hematophagous behaviour, and the Triatominae subfamily’s evolution.

Structure of recombinantly expressed cockroach Lili-Mip protein in glycosylated and deglycosylated forms

BackgroundThe Pacific Beetle Cockroach is the only known viviparous cockroach. The pregnant females provide nutrition to the embryos by secreting milk proteins (Lili-Mips), which crystallize in vivo. The crystals that grow in the embryo are heterogeneous in their protein sequence. It is not apparent from the structure determined what role heterogeneity and glycosylation played in crystallization. Lili-Mips are very nutritious. MethodsHere, we report the cloning of synthesized Lili-Mip genes, their expression in Saccharomyces cerevisiae as secreted proteins, purification, crystallization, and the determination of a three-dimensional structure of one glycosylated and one deglycosylated form. ResultsA 2.35 Å structure of the glycosylated form is bound to palmitoleic acid and has several Zn atom mediated interactions. A 1.45 Å structure of the deglycosylated protein reveals a binding pocket that has both oleic and palmitoleic acid bound. Mass-spectrometry shows that oleic acid and palmitoleic acid are bound to the protein. Docking studies suggest that aliphatic chains of lengths 15, 16, and 18 carbons bind well in the pocket. ConclusionsThe recombinantly expressed and secreted protein is glycosylated, has a bound fatty acid, is homogenous in its protein sequences, and readily forms crystals. The deglycosylated protein also crystallizes readily, suggesting that the high crystallizability of this protein is independent of glycosylation. General significanceLili-Mips belong to the ubiquitous lipocalin family of proteins that bind to a large variety of ligands. While the residues lining the barrel are essential for the affinity of the ligand, our results show the role of side-chain orientations to ligand selectivity.

Progress in the detection and quantification of collagens: a review

Collagens are an important and ubiquitous family of proteins. They have many functions in the human body and similarly have found numerous, potent applications in various industries including the manufacture of biomaterials. The ever-increasing demand for collagen has made necessary the exploration of alternative sources such as bacterial collagen-like proteins which have a triple-helical domain of Gly-X-Y amino acid repeats. Detection and quantification of native collagens have been well-established. However, collagen-like proteins differ in their composition and do not have the unique abundance of hydroxyproline and hydroxylysine found in vertebrate collagens. Thus, this poses a problem in the detection and quantification of collagen-like proteins. This paper evaluates reports on the detection and quantification of collagens and collagen-like proteins. A systematic search of the PubMed database was conducted in May 2021, to which five additional papers were added. The 310 unique search results were then subjected to a screening and elimination process, at the end of which 22 papers were included in the study. The findings were summarized and presented in a table that highlights progress in this field. While novel methods have been developed for the detection and quantitation of collagens in general, mainly using enzyme digestion, hybridization, and fluorescence, there is a need for a rapid, one-step method that selectively and sensitively detects and quantitates collagen and collagen-like protein samples with ease.

Aquaporins Are Essential to Maintain Motility and Membrane Lipid Architecture During Mammalian Sperm Capacitation.

Aquaporins are a family of ubiquitous transmembrane proteins that allow the transport of water and small molecules across the cell plasma membrane. The different members of this family present a characteristic distribution across different cell types, which is species-specific. In mammalian sperm, different AQPs, including AQP3, AQP7, and AQP11, have been identified; their main roles are related to osmoadaptation and sperm motility activation after ejaculation. Capacitation, which is a post-ejaculatory process that sperm must undergo to achieve fertilizing ability, is triggered by pH changes and different extracellular ions that are present in the female reproductive tract. Considering the function of AQPs and their influence on pH through the regulation of water flow, this study aimed to elucidate the potential role of different AQPs during in vitro sperm capacitation using three different transition metal compounds as AQP inhibitors. Cooper sulfate, a specific inhibitor of AQP3, caused a drastic increase in peroxide intracellular levels compared to the control. Mercury chloride, an unspecific inhibitor of all AQPs except AQP7 produced an increase in membrane lipid disorder and led to a decrease in sperm motility and kinetics parameters. Finally, the addition of silver sulfadiazine, an unspecific inhibitor of all AQPs, generated the same effects than mercury chloride, decreased the intracellular pH and altered tyrosine phosphorylation levels after the induction of the acrosome reaction. In the light of the aforementioned, (a) the permeability of AQP3 to peroxides does not seem to be crucial for sperm capacitation and acrosome reaction; (b) AQPs have a key role in preserving sperm motility during that process; and (c) AQPs as a whole seem to contribute to the maintenance of lipid membrane architecture during capacitation and may be related to the intracellular signaling pathways involved in the acrosome reaction. Hence, further research aimed to elucidate the mechanisms underlying the involvement of AQPs in mammalian sperm capacitation and acrosome reaction is warranted.

The extracellular innate-immune effector HMGB1 limits pathogenic bacterial biofilm proliferation.

Herein, we describe an extracellular function of the vertebrate high-mobility group box 1 protein (HMGB1) in the proliferation of bacterial biofilms. Within host cells, HMGB1 functions as a DNA architectural protein, similar to the ubiquitous DNABII family of bacterial proteins; despite that, these proteins share no amino acid sequence identity. Extracellularly, HMGB1 induces a proinflammatory immune response, whereas the DNABII proteins stabilize the extracellular DNA-dependent matrix that maintains bacterial biofilms. We showed that when both proteins converged on extracellular DNA within bacterial biofilms, HMGB1, unlike the DNABII proteins, disrupted biofilms both in vitro (including the high-priority ESKAPEE pathogens) and in vivo in 2 distinct animal models, albeit with induction of a strong inflammatory response that we attenuated by a single engineered amino acid change. We propose a model where extracellular HMGB1 balances the degree of induced inflammation and biofilm containment without excessive release of biofilm-resident bacteria.

Allergological Importance of Invertebrate Glutathione Transferases in Tropical Environments.

Glutathione-S transferases (GSTs) are part of a ubiquitous family of dimeric proteins that participate in detoxification reactions. It has been demonstrated that various GSTs induce allergic reactions in humans: those originating from house dust mites (HDM), cockroaches, and helminths being the best characterized. Evaluation of their allergenic activity suggests that they have a clinical impact. GST allergens belong to different classes: mu (Blo t 8, Der p 8, Der f 8, and Tyr p 8), sigma (Bla g 5 and Asc s 13), or delta (Per a 5). Also, IgE-binding molecules belonging to the pi-class have been discovered in helminths, but they are not officially recognized as allergens. In this review, we describe some aspects of the biology of GST, analyze their allergenic activity, and explore the structural aspects and clinical impact of their cross-reactivity.

Structural Basis of RICs Iron Donation for Iron-Sulfur Cluster Biogenesis.

Escherichia coli YtfE is a di-iron protein of the widespread Repair of Iron Centers proteins (RIC) family that has the capacity to donate iron, which is a crucial component of the biogenesis of the ubiquitous family of iron-sulfur proteins. In this work we identify in E. coli a previously unrecognized link between the YtfE protein and the major bacterial system for iron-sulfur cluster (ISC) assembly. We show that YtfE establishes protein-protein interactions with the scaffold IscU, where the transient cluster is formed, and the cysteine desulfurase IscS. Moreover, we found that promotion by YtfE of the formation of an Fe-S cluster in IscU requires two glutamates, E125 and E159 in YtfE. Both glutamates form part of the entrance of a protein channel in YtfE that links the di-iron center to the surface. In particular, E125 is crucial for the exit of iron, as a single mutation to leucine closes the channel rendering YtfE inactive for the build-up of Fe-S clusters. Hence, we provide evidence for the key role of RICs as bacterial iron donor proteins involved in the biogenesis of Fe-S clusters.

Binding Partners of 14-3-3 (YWHA) Protein Isoforms among Mammalian Species, Tissues, and Developmental Stages

The 14-3-3 (YWHA or Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation proteins) are a family of abundant, highly conserved, ubiquitous, acidic, and homologous proteins expressed in most eukaryotes ranging from plants to animals, including humans, important in regulating a multitude of cellular processes such as signal transduction, cell cycle, protein trafficking, metabolism, apoptosis, and development. Mammals have been noted contain seven isoforms of these proteins (beta, epsilon, eta, gamma, sigma, tau/theta, and zeta), encoded by separate genes. The 14-3-3 proteins are known to interact with over 200 binding partners in isoform-specific, tissue-specific, and developmental stage-specific ways. The present review article encapsulates previously published research articles that report 14-3-3-interactors, and investigates isoform-specific interactions within a wide array of mammalian species, cells, tissues, organs, and developmental stages. Of the hundreds of binding partners of 14-3-3 discovered till date, this paper focuses on analyzing selected, representative interactors with key functional roles. The study would help a better understanding of isoform-specific interactions of this critical protein family in mammals.

Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsβ1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gβγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.