Conserved Amino Acid Motifs Research Articles

Recent Molecular Epidemiology of Echovirus 11 Throughout North and West Africa Resulted in the First Identification of a Recombinant Strain from an Acute Flaccid Paralysis Case in West Africa

Echovirus 11 has emerged as a major public health concern, causing sepsis in neonates in many European countries in recent years. In Africa, especially West Africa, where resources and diagnostic capacities are limited, only sporadic cases have been reported. To better understand the recent molecular epidemiology of E11 in West Africa, we characterized twenty-three echovirus 11 strains isolated through the acute flaccid paralysis and environmental surveillance systems for polio from 2013 to 2023, using high-throughput sequencing. Our data are noteworthy due to identifying for the first time a recombinant strain from an acute flaccid paralysis case and represent the first focus to date on molecular characterization of echovirus 11 in West Africa. Moreover, our data show that echovirus 11 diverged from 1970 (95% HPD range, 1961–1979) and evolved into four distinct clades, with the virus spread from West Africa to Europe, exhibiting two introductions in France around 2017, from Senegal and Guinea. Furthermore, the in silico analysis reveals four non-conservative amino acid substitutions in the VP1 sequences of the European strains associated with neonatal sepsis in newborns and a conserved amino acid motif in the VP1 protein toward enterovirus genotypes. Our data provide new insights into the epidemiology of echovirus 11 and point to the crucial need to implement specific surveillance programs targeting non-polio enteroviruses for the rapid identification of emerging or re-emerging enterovirus species, particularly in Africa.

The Role of the CX3CR1-CX3CL1 Axis in Respiratory Syncytial Virus Infection and the Triggered Immune Response.

Respiratory syncytial virus (RSV) is a common respiratory pathogen that causes respiratory illnesses, ranging from mild symptoms to severe lower respiratory tract infections in infants and older adults. This virus is responsible for one-third of pneumonia deaths in the pediatric population; however, there are currently only a few effective vaccines. A better understanding of the RSV-host relationship at the molecular level may lead to a more effective management of RSV-related symptoms. The fractalkine (CX3CL1) receptor (CX3CR1) is a co-receptor for RSV expressed by airway epithelial cells and diverse immune cells. RSV G protein binds to the CX3CR1 receptor via a highly conserved amino acid motif (CX3C motif), which is also present in CX3CL1. The CX3CL1-CX3CR1 axis is involved in the activation and infiltration of immune cells into the infected lung. The presence of the RSV G protein alters the natural functions of the CX3CR1-CX3CL1 axis and modifies the host's immune response, an aspects that need to be considered in the development of an efficient vaccine and specific pharmacological treatment.

Genome-wide analysis and identification of the TBL gene family in Eucalyptus grandis.

The TRICHOME BIREFRINGENCE-LIKE (TBL) gene encodes a class of proteins related to xylan acetylation, which has been shown to play an important role in plant response to environmental stresses. This gene family has been meticulously investigated in Arabidopsis thaliana, whereas there have been no related reports in Eucalyptus grandis. In this study, we identified 49 TBL genes in E. grandis. A conserved amino acid motif was identified, which plays an important role in the execution of the function of TBL gene family members. The expression of TBL genes was generally upregulated in jasmonic acid-treated experiments, whereas it has been found that jasmonic acid activates the expression of genes involved in the defense functions of the plant body, suggesting that TBL genes play an important function in the response of the plant to stress. The principle of the action of TBL genes is supported by the finding that the xylan acetylation process increases the rigidity of the cell wall of the plant body and thus improves the plant's resistance to stress. The results of this study provide new information about the TBL gene family in E. grandis and will help in the study of the evolution, inheritance, and function of TBL genes in E. grandis, while confirming their functions.

Allosensitisation in NHP results in cross-reactive anti-SLA antibodies not detected by a lymphocyte-based flow cytometry crossmatch.

Xenotransplantation is a potential option for individuals for whom an acceptable human allograft is unavailable. Individuals with broadly reactive HLA antibodies due to prior exposure to foreign HLA are potential candidates for a clinical xenotransplant trial. It remains controversial if allosensitisation results in the development of cross-reactive antibodies against SLA. This may require increased histocompatibility scrutiny for highly sensitised individuals prior to enrollment in a clinical trial. Serum samples were obtained from non-human primates sensitised via serial skin transplantation from maximally MHC-mismatched donor, as reported. Sera from pre- and post-allosensitisation timepoints were assessed in a flow crossmatch (FXM) for IgM and IgG binding to pig splenocytes with or without red blood cell adsorption. Xenoreactive antibodies were eluted from pig splenocytes and screened on a single antigen HLA bead assay. A MHC Matchmaker algorithm was developed to predict potential conserved amino acid motifs among the pig, NHP, and human. Our sensitised NHP model was used to demonstrate that allosensitisation does not result in an appreciable difference in xenoreactive antibody binding in a cell-based FXM. However, antibody elution and screening on single antigen HLA beads suggest the existence of potential cross-reactive antibodies against SLA. The cross-reactive IgG after allosensitisation were predicted by comparing the recipient Mamu alleles against its previous allograft donor Mamu alleles and the donor pig SLA alleles. Our study suggests that allosensitisation could elevate cross-reactive antibodies, but a more sensitive assay than a cell-based FXM is required to detect them. The MHC Matchmaker algorithm was developed as a potential tool to help determine amino acid motif conservation and reactivity pattern.

Tubb4b is required for multi-ciliogenesis in the mouse.

Cilia are microtubule (MT)-based organelles present on the surface of nearly all vertebrate cells. MTs are polymers of α- and β-tubulins that are each encoded by multiple, individual isotype genes. Tubulin isotype composition is thought to influence MT behaviors. Ciliary MTs differ from other MTs in the cell in terms of organization, stability and post-translational modifications. However, little is known about the tubulin isotypes that build ciliary MTs and the functional requirements for tubulin isotypes in cilia have not been examined in vertebrates. Here, we have tested the role of the β-tubulin isotype genes in the mouse that harbor a conserved amino acid motif associated with ciliated organisms. We found that Tubb4b localizes to cilia in multi-ciliated cells (MCCs) specifically. In respiratory and oviduct MCCs, Tubb4b is asymmetrically localized within multi-cilia, indicating that the tubulin isotype composition changes along the length of the ciliary axonemal MTs. Deletion of Tubb4b resulted in striking structural defects within the axonemes of multi-cilia, without affecting primary cilia. These studies show that Tubb4b is essential for the formation of a specific MT-based subcellular organelle and sheds light on the requirements of tubulin isotypes in cilia.

Phylogenetic and Protein Structure Analyses Provide Insight into the Evolution and Diversification of the CD36 Domain "Apex" among Scavenger Receptor Class B Proteins across Eukarya.

The cluster of differentiation 36 (CD36) domain defines the characteristic ectodomain associated with class B scavenger receptor (SR-B) proteins. In bilaterians, SR-Bs play critical roles in diverse biological processes including innate immunity functions such as pathogen recognition and apoptotic cell clearance, as well as metabolic sensing associated with fatty acid uptake and cholesterol transport. Although previous studies suggest this protein family is ancient, SR-B diversity across Eukarya has not been robustly characterized. We analyzed SR-B homologs identified from the genomes and transcriptomes of 165 diverse eukaryotic species. The presence of highly conserved amino acid motifs across major eukaryotic supergroups supports the presence of a SR-B homolog in the last eukaryotic common ancestor. Our comparative analyses of SR-B protein structure identify the retention of a canonical asymmetric beta barrel tertiary structure within the CD36 ectodomain across Eukarya. We also identify multiple instances of independent lineage-specific sequence expansions in the apex region of the CD36 ectodomain-a region functionally associated with ligand-sensing. We hypothesize that a combination of both sequence expansion and structural variation in the CD36 apex region may reflect the evolution of SR-B ligand-sensing specificity between diverse eukaryotic clades.

Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena thermophila

Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila.

Presence of Multiple Herpesvirus Variants in Australian Flying Foxes (Pteropus spp.).

Herpesviruses have been detected in bat species from several countries, with a limited number of studies examining herpesviruses in Pteropus spp. (flying foxes) and no investigation of herpesviruses in Australian flying foxes. We examined the presence and prevalence of herpesviruses in the four mainland Australian flying fox species. A nested PCR targeting highly conserved amino acid motifs in the DNA polymerase (DPOL) gene of herpesviruses was used to analyze 564 samples collected from 514 individual Pteropus scapulatus, Pteropus poliocephalus, Pteropus alecto, and Pteropus conspicillatus. The prevalence of herpesvirus DNA in blood, urine, oral, and fecal swabs from the four species was 17% in P. scapulatus, 11% in P. poliocephalus, 10% in P. alecto, and 9% in P. conspicillatus (31% in P. conspicillatus spleen tissue). Five putative novel herpesviruses were detected. Following PCR amplicon sequence analysis, four of the herpesviruses grouped phylogenetically with the gammaherpesviruses, with nucleotide identities between 79% and 90% to gammaherpesviruses from Asian megabats. A betaherpesvirus was detected in P. scapulatus with 99% nucleotide identity to the partial DPOL gene sequence of an Indonesian fruit bat betaherpesvirus. This study lays the foundation for future epidemiology research of herpesviruses in Australian Pteropus spp. and adds to the discussion of hypotheses surrounding the evolutionary epidemiology of bat-borne viruses on a global scale.

De novo transcriptome of a bast fibre crop Crotalaria juncea reveals T2 ribonuclease genes to investigate late-acting self-rejection of pollens

The biology of gametophytic late-acting self-rejection of pollen in pre-zygotic ovules is relatively unknown in the Fabaceae family of plants. Our understanding of the genetic basis of late-acting self-incompatibility (LSI) is limited due to a lack of sequence data and candidate genes. Crotalaria juncea, a Fabaceae family member that produces commercially important phloem fiber, possesses LSI. Due to a lack of selfed seeds, it is difficult to maintain genetic purity in germplasm and develop breeding lines for improved fiber quality and yield. To investigate candidate genes for LSI in C. juncea, a high-quality de novo transcriptome was generated. It facilitated the identification of genes from the self-incompatibility-related ribonuclease (RNase) family, specifically a Class III T2/RNase gene with sequence properties similar to S-RNase proteins. Based on conserved amino acid motifs, histidine residues, and a high isoelectric point (pI ≥9.0), Cjun_RNS3.1 was identified as an S-RNase homologue (non-S-RNase) or relic S-RNase gene. Unlike typical S-RNases found in known SI plant systems, evolutionary analysis of the Cjun_RNS3.1 protein revealed its ancestral origin. The expression of Cjun_RNS3.1 and other T2/RNase genes revealed differential expression patterns in the pistils of LSI and self-compatible Crotalaria species. The upregulation of the Cjun_RNS3.1 gene during the different stages of pollen tube development indicates that it may be involved in the LSI. In summary, the sunn hemp transcriptome is the first genomic resource reported from a Fabaceae family plant with the LSI trait. A non-S-RNase gene with an ancestral evolutionary origin and a diverse function was found, which could be related to gametophytic LSI. This could be used as a model system to investigate the molecular basis of LSI in the Fabaceae family and help develop C. juncea breeding lines for fibre improvement.

Symport and antiport mechanisms of human glutamate transporters

Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.

Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus.

Nucleoprotein (NP) functions crucially in the replicative cycle of influenza A virus (IAV) via forming the ribonucleoprotein complex together with PB2, PB1, and PA proteins. As its high conservation, NP ranks one of the hot targets for design of universal diagnostic reagents and antiviral drugs for IAV. Here, we report an anti-NP murine monoclonal antibody (mAb) 5F10 prepared from traditional lymphocyte hybridoma technique with the immunogen of a clade 2.3.4.4 H5N1 subtype avian influenza virus. The specificity of mAb 5F10 to NP protein was confirmed by immunofluorescence assay and western blotting, and the mAb 5F10 could be used in immunoprecipitation and immunohistochemistry assays. Importantly, mAb 5F10 possessed broad-spectrum reactivity against H1~H11 subtypes of avian influenza viruses, including various HA clades of H5Nx subtype. In addition, mAb 5F10 also showed good affinity with H1N1 and H3N2 subtype influenza viruses of swine and human origin. Furthermore, the recognized antigenic epitope of mAb 5F10 was identified to consist of the conserved amino acid motif 81EHPSA85 in the second flexible loop region of NP protein through screening the phage display peptide library. Collectively, the mAb 5F10 which recognizes the novel universal NP linear B-cell epitope of IAV with diverse origins and subtypes will be a powerful tool for NP protein-based structural, functional, and mechanistic studies, as well as the development of detection methods and universal vaccines for IAV. KEY POINTS: • A broad-spectrum mAb against various subtypes and sources of IAV was developed • The mAb possessed good reactivity in IFA, western blot, IP, and IHC assays • The mAb targeted a novel conserved linear B-cell epitope involving 81EHPSA85 on NP protein.

Impact of cancer-associated mutations in CC chemokine receptor 2 on receptor function and antagonism

CC chemokine receptor 2 (CCR2), a G protein-coupled receptor, plays a role in many cancer-related processes such as metastasis formation and immunosuppression. Since ∼ 20 % of human cancers contain mutations in G protein-coupled receptors, ten cancer-associated CCR2 mutants obtained from the Genome Data Commons were investigated for their effect on receptor functionality and antagonist binding. Mutations were selected based on either their vicinity to CCR2′s orthosteric or allosteric binding sites or their presence in conserved amino acid motifs. One of the mutant receptors, namely S101P2.63 with a mutation near the orthosteric binding site, did not express on the cell surface. All other studied mutants showed a decrease in or a lack of G protein activation in response to the main endogenous CCR2 ligand CCL2, but no change in potency was observed. Furthermore, INCB3344 and LUF7482 were chosen as representative orthosteric and allosteric antagonists, respectively. No change in potency was observed in a functional assay, but mutations located at F1163.28 impacted orthosteric antagonist binding significantly, while allosteric antagonist binding was abolished for L134Q3.46 and D137N3.49 mutants. As CC chemokine receptor 2 is an attractive drug target in cancer, the negative effect of these mutations on receptor functionality and drugability should be considered in the drug discovery process.

Evolutionary analyses and expression patterns of TCP genes in Ranunculales.

TCP transcription factors play a role in a large number of developmental processes and are at the crossroads of numerous hormonal biosynthetic and signaling pathways. The complete repertoire of TCP genes has already been characterized in several plant species, but not in any species of early diverging eudicots. We focused on the order Ranunculales because of its phylogenetic position as sister group to all other eudicots and its important morphological diversity. Results show that all the TCP genes expressed in the floral transcriptome of Nigella damascena (Ranunculaceae) are the orthologs of the TCP genes previously identified from the fully sequenced genome ofAquilegia coerulea. Phylogenetic analyses combined with the identification of conserved amino acid motifs suggest that six paralogous genes of class I TCP transcription factors were present in the common ancestor of angiosperms. We highlight independent duplications in core eudicots and Ranunculales within the class I and class II subfamilies, resulting in different numbers of paralogs within the main subclasses of TCP genes. This has most probably major consequences on the functional diversification of these genes in different plant clades. The expression patterns of TCP genes in Nigella damascena were consistent with the general suggestion that CIN and class I TCP genes may have redundant roles or take part in same pathways, while CYC/TB1 genes have more specific actions. Our findings open the way for future studies at the tissue level, and for investigating redundancy and subfunctionalisation in TCP genes and their role in the evolution of morphological novelties.

Screening of West Nile Virus, Herpesvirus, and Parvovirus in Rattus spp. in Klang Valley, Malaysia

Free-roaming and scavenging lifestyles of Rattus spp. in densely populated urban areas expose them to multiple pathogens that facilitate the transmission of infection to the human population more rapidly, raising public health concerns. There is limited information on the status of rat susceptibility to virus infection, particularly West Nile virus (WNV), herpesvirus, and parvovirus, to prepare for emerging zoonosis. A total of 23 (n = 23) blood samples collected from Rattus spp. in the wet market areas of Klang Valley, Malaysia, were subjected to molecular assay using a one-step reverse transcription-polymerase chain reaction (RT-PCR) to detect the highly conserved region of the WNV capsid and pre-membrane protein via nested polymerase chain reaction (PCR) assay targeting highly conserved amino acid motifs within the herpesviral DNA-directed DNA polymerase gene (DPOL) and polymerase chain reaction (PCR) assay targeting the parvovirus non-structural (NS) protein. As a result, 4 out of 23 (17.39%) rats were positive for herpesvirus DNA, but none were positive for WNV RNA and parvovirus DNA. The positive PCR amplicons of herpesvirus DNA were subjected to partial DNA sequencing analysis, 100% identical to Acomys herpesvirus SVMS 226,222 from Betaherpesvirinae, which is highly suggestive of rat cytomegalovirus (RCMV). This study has successfully demonstrated the presence of RCMV from Rattus spp. in the Klang Valley. The RCMV potentially crosses species barriers and establishes infection, raising public health concerns. The non-viraemic state of WNV or parvovirus infection, low sample size, and limited niche distribution emphasise the need for the expansion of this study in the future.

Diversity of amino acid substitutions of penicillin-binding proteins in penicillin-non-susceptible and non-vaccine type Streptococcus pneumoniae

PurposeIn Japan, the introduction of pneumococcal conjugate vaccine (PCV) in children has decreased vaccine-type (VT) pneumococcal infections caused by penicillin (PEN)-non-susceptible Streptococcus pneumoniae. PEN-non-susceptible strains have gradually emerged among non-vaccine types (NVT). In this study, we aim to investigate the pbp gene mutations and the characteristics of PEN-binding proteins (PBPs) that mediate PEN resistance in NVT strains. Materials and methodsPneumococcal 41 strains of NVT isolated from patients with invasive pneumococcal infection were randomly selected. Nucleotide sequences for pbp genes encoding PBP1A, PBP2X, and PBP2B were analyzed, and amino acid (AA) substitutions that contribute to β-lactam resistance were identified. In addition, the three-dimensional (3D) structure of abnormal PBPs in the resistant strain was compared with that of a reference R6 strain via homology modeling. ResultsIn PEN-non-susceptible NVT strains, Thr to Ala or Ser substitutions in the conserved AA motif (STMK) were important in PBP1A and PBP2X. In PBP2B, substitutions from Thr to Ala, adjacent to the SSN motif, and from Glu to Gly were essential. The 3D structure modeling indicated that AA substitutions are characterized by accumulation around the enzymatic active pocket in PBPs. Many AA substitutions detected throughout the PBP domains were not associated with resistance, except for AA substitutions in or adjacent to AA motifs. Clonal complexes and sequence types showed that almost all NVT cases originated in other countries and spread to Japan via repeat mutations. ConclusionsNVT with diverse AA substitutions increased gradually with pressure from both antimicrobial agents and vaccines.

Atypical cyclic di-AMP signaling is essential for Porphyromonas gingivalis growth and regulation of cell envelope homeostasis and virulence

Microbial pathogens employ signaling systems through cyclic (di-) nucleotide monophosphates serving as second messengers to increase fitness during pathogenesis. However, signaling schemes via second messengers in Porphyromonas gingivalis, a key Gram-negative anaerobic oral pathogen, remain unknown. Here, we report that among various ubiquitous second messengers, P. gingivalis strains predominantly synthesize bis-(3′,5′)-cyclic di-adenosine monophosphate (c-di-AMP), which is essential for their growth and survival. Our findings demonstrate an unusual regulation of c-di-AMP synthesis in P. gingivalis. P. gingivalis c-di-AMP phosphodiesterase (PDE) gene (pdepg) positively regulates c-di-AMP synthesis and impedes a decrease in c-di-AMP concentration despite encoding conserved amino acid motifs for phosphodiesterase activity. Instead, the predicted regulator gene cdaR, unrelated to the c-di-AMP PDE genes, serves as a potent negative regulator of c-di-AMP synthesis in this anaerobe. Further, our findings reveal that pdepg and cdaR are required to regulate the incorporation of ATP into c-di-AMP upon pyruvate utilization, leading to enhanced biofilm formation. We show that shifts in c-di-AMP signaling change the integrity and homeostasis of cell envelope, importantly, the structure and immunoreactivity of the lipopolysaccharide layer. Additionally, microbe–microbe interactions and the virulence potential of P. gingivalis were modulated by c-di-AMP. These studies provide the first glimpse into the scheme of second messenger signaling in P. gingivalis and perhaps other Bacteroidetes. Further, our findings indicate that c-di-AMP signaling promotes the fitness of the residents of the oral cavity and the development of a pathogenic community.

Copper Binding and Oligomerization Studies of the Metal Resistance Determinant CrdA from Helicobacter pylori.

Within this research, the CrdA protein from Helicobacter pylori (HpCrdA), a putative copper-binding protein important for the survival of bacterium, was biophysically characterized in a solution, and its binding affinity toward copper was experimentally determined. Incubation of HpCrdA with Cu(II) ions favors the formation of the monomeric species in the solution. The modeled HpCrdA structure shows a conserved methionine-rich region, a potential binding site for Cu(I), as in the structures of similar copper-binding proteins, CopC and PcoC, from Pseudomonas syringae and from Escherichia coli, respectively. Within the conserved amino acid motif, HpCrdA contains two additional methionines and two glutamic acid residues (MMXEMPGMXXMXEM) in comparison to CopC and PcoC but lacks the canonical Cu(II) binding site (two His) since the sequence has no His residues. The methionine-rich site is in a flexible loop and can adopt different geometries for the two copper oxidation states. It could bind copper in both oxidation states (I and II), but with different binding affinities, micromolar was found for Cu(II), and less than nanomolar is proposed for Cu(I). Considering that CrdA is a periplasmic protein involved in chaperoning copper export and delivery in the H. pylori cell and that the affinity of the interaction corresponds to a middle or strong metal–protein interaction depending on the copper oxidation state, we conclude that the interaction also occurs in vivo and is physiologically relevant for H. pylori.

Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.

Effect of mutations in the 2b protein of tomato aspermy virus on RNA silencing suppressor activity, virulence, and virus-induced gene silencing.

Tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) belong to the genus Cucumovirus in the family Bromoviridae. The cucumovirus 2b protein is one of the first identified viral suppressors of RNA silencing (VSR). The cucumovirus 2b protein contains a conserved amino acid motif consisting of several highly conserved amino acid residues. Here, we demonstrate that the TAV 2b protein N-terminal region, Arg46, Ser40, and Ser42 as well as the CMV 2b protein are essential for VSR activity, virulence, and viral RNA accumulation. Furthermore, we developed the first TAV-induced gene silencing (VIGS) vector. In contrast to other cucumovirus vectors, such as CMV and peanut stunt virus, the TAV vector did not induce a silencing phenotype in Nicotiana benthamiana when 2b protein VSR activity was retained. These findings suggest that the cucumovirus 2b proteins share amino acid residues for VSR activity but may have different roles in VIGS induction.

An Insight into the Proofreading Functions of Multisubunit DNA-Dependent RNA Polymerases and Their Catalytic Mechanism

Aim: To analyze the active sites of the proofreading (PR) functions in the multisubunit DNA-dependent RNA polymerases (MSU RNAPs) from prokaryotes, chloroplasts and eukaryotes, and propose a plausible unified catalytic mechanism for these enzymes.
 Study Design: Data collected on these enzymes from bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallography and cryo-electron microscopy (cryo-EM) were used for the analyses.
 Methodology: The protein sequence data of MSU RNAPs from prokaryotes, prokaryotic-types (plant chloroplasts) and eukaryotes were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available from biochemical and SDM experiments, and X-ray crystallographic and cryo-EM data on these enzymes are also used to confirm the possible amino acids involved in the active site of the PR function in these MSU RNAPs
 Results: All the seven types of MSU RNAPs (I-VII) reported from prokaryotes to eukaryotes were analyzed by the multiple sequence alignment (MSA) software, Clustal Omega, to find out conservations among them. The MSA analysis showed many conserved amino acid motifs including small and large peptide regions from the MSU RNAPs of prokaryotes, eukaryotes and plant chloroplasts. Interestingly, the catalytic amino acid and template-binding pairs are highly conserved in all these polymerases, with a few exceptions. Most of them use a basic amino acid (R/K/H) for initiating catalysis and an -YG/FG- pair for template-binding. Some odd type of catalytic amino acids and template-binding pairs are observed in human pathogens, parasites and organisms which cannot ferment sugars. In all the MSU RNAPs, the proposed polymerase catalytic region also possessed three invariant Cs and an invariant H within it. The invariant Cs is shown to bind a zinc atom and proposed to involve in the PR function by excising any misincorporated nucleotide during the transcription process. In the plant-specific MSU RNAPs IV and V, which involve in transcriptional gene silencing in plants, the catalytic and template-binding pairs do not follow the regular distance conservations as observed with other five of the MSU RNAPs. Their polymerase/PR active site regions are similar to RNAP III rather than to RNAP II, as all three make only low molecular weight RNAs.
 Conclusions: All the known MSU RNAPs possess three invariant Cs and an invariant H embedded within the polymerase active site itself. The three invariant Cs are shown to bind a zinc atom and the invariant H could act as the proton acceptor from a metal-bound water molecule, for initiating excision of the mismatches by a Zn-mediated hydrolysis. Thus, the PR function in MSU RNAPs is integrated within the polymerase active site itself, which is in sharp contrast to the PR functions reported in DNA-dependent DNA polymerases and RNA-dependent RNA polymerases. Therefore, all the seven MSU RNAPs from prokaryotes and eukaryotes are proposed to follow a unified mechanism to excise the mismatches during transcription. The discovery of intrinsic self-correcting RNA transcription mechanism fulfils the missing link in molecular evolution.