GRP78 recognizes EV-F 3D protein and activates NF-κB to repress virus replication by interacting with CHUK/IKBKB.

Enteroviruses are the causative agents associated with several human and animal diseases, posing a significant threat to human and animal health. As one of the host immune defense strategies, innate immunity plays a crucial role in defending against invading pathogens, where the host utilizes a variety of mechanisms to inhibit or eliminate the pathogen. Here, we report a new strategy for the host to repress enterovirus replication by the 78 kDa glucose-regulated protein (GRP78), also known as heat shock protein family A member 5 (HSPA5). The GRP78 recognizes the EV-encoded RNA-dependent RNA polymerases (RdRPs) 3D protein and interacts with the nuclear factor kappa B kinase complex (CHUK) and subunit beta gene (IKBKB) to facilitate the phosphorylation and nuclear translocation of NF-κB, which induces the production of inflammatory factors and leads to a broad inhibition of enterovirus replication. These findings demonstrate a new role of GRP78 in regulating host innate immunity in response to viral infection and provide new insights into the mechanism underlying enterovirus replication and NF-κB activation.IMPORTANCEGRP78 is known as a molecular chaperone for protein folding and plays a critical role in maintaining protein folding and participating in cell proliferation, cell survival, apoptosis, and metabolism. However, the functions of GRP78 to participate in enterovirus genome replication and innate immune responses are rarely documented. In this study, we explored the functions of the EV-3D-interacting protein GRP78 and found that GRP78 inhibits enterovirus replication by activating NF-κB through binding to EV-F 3D and interacting with the NF-κB signaling molecules CHUK/IKBKB. This is the first report that GRP78 interacts with CHUK/IKBKB to activate the NF-κB signaling pathway, which leads to the expression of the proinflammatory cytokines and inhibition of enterovirus replication. These results demonstrate a unique mechanism of virus replication regulation by GRP78 and provide insights into the prevention and treatment of viral infections.

Genomic profile of eGFP-tagged senecavirus A subjected to serial plaque-to-plaque transfers.

Senecavirus A (SVA) belongs to the genus Senecavirus in the family Picornaviridae. This virus possesses a positive-sense, single-stranded RNA genome, approximately 7200 nt in length, composed of a single 5' untranslated region, encoding region and 3' untranslated region. In this study, a recombinant SVA tagged with enhanced green fluorescent protein (eGFP) sequence, rSVA-eGFP, was rescued from its cDNA clone using reverse genetics. The passage-5 (P5) rSVA-eGFP was totally subjected to 55 rounds of consecutive fluorescent plaque-to-fluorescent plaque (FP-FP) transfers, and one extra common passaging in vitro. The P61 viral stock was analyzed by next-generation sequencing. The result showed ten single-nucleotide mutations (SNMs) in the rSVA-eGFP genome, including nine transitions and only one transversion. The P61 progeny still showed a complete eGFP sequence, indicating no occurrence of copy-choice recombination within the eGFP region during serial FP-FP transfers. In other words, this progeny was genetically deficient in the recombination of eGFP sequence (RES), namely, an RES-deficient strain. Out of ten SNMs, three were missense mutations, leading to single-amino acid mutations (SAAMs): F15V in L protein, A74T in VP2, and E53R in 3D protein. The E53R was predicted to be spatially adjacent to the RNA channel of 3D protein, perhaps involved in the emergence of RES-deficient strain. In conclusion, this study uncovered a global landscape of rSVA-eGFP genome after serial FP-FP transfers, and moreover shed light on a putative SAAM possibly related to the RES-deficient mechanism.

EV71 5'UTR interacts with 3D protein affecting replication through the AKT-mTOR pathway.

BACKGROUND: EV71 is one of the important pathogens of Hand-foot-and-mouth disease (HFMD), which causes serious neurological symptoms. Several studies have speculated that there will be interaction between 5'UTR and 3D protein. However, whether 5'UTR interacts with the 3D protein in regulating virus replication has not been clarified. METHODS: Four 5'UTR mutation sites (nt88C/T, nt90-102-3C, nt157G/A and nt574T/A) and two 3D protein mutation sites (S37N and R142K) were mutated or co-mutated using virulent strains as templates. The replication of these mutant viruses and their effect on autophagy were determined. RESULTS: 5'UTR single-point mutant strains, except for EGFP-EV71(nt90-102-3C), triggered replication attenuation. The replication ability of them was weaker than that of the parent strain the virulent strain SDLY107 which is the fatal strain that can cause severe neurological complications. While the replication level of the co-mutant strains showed different characteristics. 5 co-mutant strains with interaction were screened: EGFP-EV71(S37N-nt88C/T), EGFP-EV71(S37N-nt574T/A), EGFP-EV71(R142K-nt574T/A), EGFP-EV71(R142K-nt88C/T), and EGFP-EV71(R142K-nt157G/A). The results showed that the high replicative strains significantly promoted the accumulation of autophagosomes in host cells and hindered the degradation of autolysosomes. The low replicative strains had a low ability to regulate the autophagy of host cells. In addition, the high replicative strains also significantly inhibited the phosphorylation of AKT and mTOR. CONCLUSIONS: EV71 5'UTR interacted with the 3D protein during virus replication. The co-mutation of S37N and nt88C/T, S37N and nt574T/ A, R142K and nt574T/A induced incomplete autophagy of host cells and promoted virus replication by inhibiting the autophagy pathway AKT-mTOR. The co-mutation of R142K and nt88C/T, and R142K and nt157G/A significantly reduced the inhibitory effect of EV71 on the AKT-mTOR pathway and reduced the replication ability of the virus.

In silico analysis of the possible crosstalk between O-linked ß-GlcNAcylation and phosphorylation sites of Disabled 1 adaptor protein in vertebrates.

Disabled 1 (Dab1) is an adaptor protein with essential functions regulated by reelin signaling and affects many biological processes in the nervous system, including cell motility, adhesion, cortical development, maturation, and synaptic plasticity. Posttranslational modifications directly guide the fates of cytoplasmic proteins to complete their functions correctly. Reciprocal crosstalk between O-GlcNAcylation and phosphorylation is a dynamic modification in cytoplasmic proteins. It modulates the functions of the proteins by regulating their interactions with other molecules in response to the continuously changeable cell microenvironment. Although Dab1 contains conserved recognition sites for phosphorylation in their N-terminal protein interaction domain, the O-ß-GlcNAcylation and phosphorylation sites of human Dab1 sequence, their reciprocal crosstalk, and potential kinases catalyzing the phosphorylation remain unknown. In this study, we determined potential thirty-seven O-ß-GlcNAcylation and sixty-seven phosphorylation sites. Conserved twenty-one residues of these glycosylated sites were also phosphorylated with various kinases, including ATM, CKI, DNAPK, GSK3, PKC, PKG, RSK, cdc2, cdk5, and p38MAPK. In addition, we analyzed these conserved sites at our constructed two- and three-dimensional structures of human Dab1 protein. Dab1 protein models were frequently composed of coil structures as well as α-helix and ß-strands. Many of these conserved crosstalk sites between O-ß-GlcNAcylation and phosphorylation were localized at the coil region of the protein model. These findings may guide biochemical, genetic, and glyco-biology based on further experiments about the Dab1 signaling process. Understanding these modifications might change the point of view of the Dab1 signaling process and treatment for pathological conditions in neurodegenerative diseases such as Alzheimer's disease.

Determine genetic variations in heat shock factor gene family (HSFs) and study their effect on the functional and structural characterization of protein in Tali goat.

In this study, the effect of genetic variations of four heat shock transcription factor genes (HSF1, HSF2, HSF4, and HSF5) on the 3 D protein structure and function were studied. We defined the breed-specific genetic variations of pooled DNA of Tali goat that differed from the goat reference sequence (CHI2.0). Disordered regions of HSF proteins were predicted using PONDR. Post-translation changes were studied by several predicted online servers. Then, the structure of the order region of proteins was anticipated by using the Swiss model. Tali goat HSF genes contain a total number of 181, 679, 91, and 301 SNPs for HSF1, 2, 4, and 5, respectively. Also, 5 and 3 variants were identified as nsSNPs in the coding region of HSF4 and HSF5, respectively. (r.145A/S), (r.322P/Y), (r.379T/C) in HSF4 and (r.300Q/P), (r.573E/Q) in HSF5 obtained the tolerant and high confidence (SIFT score) for nsSNPs. More than half of these proteins are predicted to be disordered (56, 50, 52, and 50%, respectively for HSF1, 2, 4, and 5). Phosphorylation, acetylation, glycosylation, and Sumoylation sites of HSFs were compared between Tali goat and reference goat. Three residues S145, S263, and S322 of HSF4 in Tali goat were phosphorylation sites, and in HSF5, the reference goat has a phosphorylation site in S593.

Phylogenetic analysis and characterization of arsenic (As) transforming bacterial marker proteins following isolation of As-tolerant indigenous bacteria.

Marker proteins play a significant role in bacterial arsenic (As) transformation. Phylogenetic analysis and three-dimensional (3D) characteristics of As transforming bacterial marker proteins guide the evolutionary origin and As transforming potential of the species. Indeed, As-tolerant bacteria also show a significant level of As transformation. Hence, characterization of As transforming bacterial marker proteins, isolation of As transforming bacteria, and proper integration of the findings may guide to elucidate how bacteria transform As. Therefore, phylogenetic analysis and 3D characterization of As transforming bacterial marker protein following isolation of potential indigenous As-tolerant indigenous bacteria were done to explore the mechanism of bacterial As transformation. Phylogenetic analysis of ten As transforming marker proteins (arsA, arsB, arsC, arsD, arsR, aioA, arrA, aioB, acr1, and acr3) in 20 potential bacterial genomes (except 19 for the acr3) were studied. Some bacterial genomes featured up to five marker proteins, and therefore, 3D characteristics of the marker proteins were analyzed in those genomes having three-to-five marker proteins. In phylogeny, species in close clades represent their phylogenetic resemblances and may have similar functions. P. aeruginosa, E. coli, and K. pneumonia were found to be more effective due to having the highest number (five) of marker proteins. In 3D protein modeling, most of the marker proteins were found to be active. Among 19 indigenous bacterial isolates, multiple isolates showed tolerance up to 50 mM As(III) and 250 mM As(V), which may potentially transform a significant quantities of As. Hence, integration of the results of phylogenetic analysis, 3D protein characteristics, and As tolerance in the bacterial isolates could guide to explore the mechanism of how bacteria transform As at cellular and molecular levels.

In Silico Characterisation of the Late Embryogenesis Abundant (LEA) Protein Families and Their Role in Desiccation Tolerance in Ramonda serbica Panc.

Ramonda serbica Panc. is an ancient resurrection plant able to survive a long desiccation period and recover metabolic functions upon watering. The accumulation of protective late embryogenesis abundant proteins (LEAPs) is a desiccation tolerance hallmark. To propose their role in R. serbica desiccation tolerance, we structurally characterised LEAPs and evaluated LEA gene expression levels in hydrated and desiccated leaves. By integrating de novo transcriptomics and homologues LEAP domains, 318 R. serbica LEAPs were identified and classified according to their conserved motifs and phylogeny. The in silico analysis revealed that hydrophilic LEA4 proteins exhibited an exceptionally high tendency to form amphipathic α-helices. The most abundant, atypical LEA2 group contained more hydrophobic proteins predicted to fold into the defined globular domains. Within the desiccation-upregulated LEA genes, the majority encoded highly disordered DEH1, LEA1, LEA4.2, and LEA4.3 proteins, while the greatest portion of downregulated genes encoded LEA2.3 and LEA2.5 proteins. While dehydrins might chelate metals and bind DNA under water deficit, other intrinsically disordered LEAPs might participate in forming intracellular proteinaceous condensates or adopt amphipathic α-helical conformation, enabling them to stabilise desiccation-sensitive proteins and membranes. This comprehensive LEAPs structural characterisation is essential to understanding their function and regulation during desiccation aiming at crop drought tolerance improvement.

Structural modeling of two plant UDP-dependent sugar-sugar glycosyltransferases reveals a conserved glutamic acid residue that is a hallmark for sugar acceptor recognition.

Glycosylation is one of the common modifications of plant metabolites, playing a major role in the chemical/biological diversity of a wide range of compounds. Plant metabolite glycosylation is catalyzed almost exclusively by glycosyltransferases, mainly by Uridine-diphosphate dependent Glycosyltransferases (UGTs). Several X-ray structures have been determined for primary glycosyltransferases, however, little is known regarding structure-function aspects of sugar-sugar/branch-forming O-linked UGTs (SBGTs) that catalyze the transfer of a sugar from the UDP-sugar donor to an acceptor sugar moiety of a previously glycosylated metabolite substrate. In this study we developed novel insights into the structural basis for SBGT catalytic activity by modelling the 3d-structures of two enzymes; a rhamnosyl-transferase Cs1,6RhaT - that catalyzes rhamnosylation of flavonoid-3-glucosides and flavonoid-7-glucosides and a UGT94D1 - that catalyzes glucosylation of (+)-Sesaminol 2-O-ß-d-glucoside at the C6 of the primary sugar moiety. Based on these structural models and docking studies a glutamate (E290 or E268 in Cs1,6RhaT or UGT94D1, respectively) and a tryptophan (W28 or W15 in Cs1,6RhaT or UGT94D1, respectively) appear to interact with the sugar acceptor and are suggested to be important for the recognition of the sugar-moiety of the acceptor-substrate. Functional analysis of substitution mutants for the glutamate and tryptophan residues in Cs1,6RhaT further support their role in determining sugar-sugar/branch-forming GT specificity. Phylogenetic analysis of the UGT family in plants demonstrates that the glutamic-acid residue is a hallmark of SBGTs that is entirely absent from the corresponding position in primary UGTs.

Identification and characterization of amphibian SLC26A5 using RNA-Seq.

BACKGROUND: Prestin (SLC26A5) is responsible for acute sensitivity and frequency selectivity in the vertebrate auditory system. Limited knowledge of prestin is from experiments using site-directed mutagenesis or domain-swapping techniques after the amino acid residues were identified by comparing the sequence of prestin to those of its paralogs and orthologs. Frog prestin is the only representative in amphibian lineage and the studies of it were quite rare with only one species identified. RESULTS: Here we report a new coding sequence of SLC26A5 for a frog species, Rana catesbeiana (the American bullfrog). In our study, the SLC26A5 gene of Rana has been mapped, sequenced and cloned successively using RNA-Seq. We measured the nonlinear capacitance (NLC) of prestin both in the hair cells of Rana's inner ear and HEK293T cells transfected with this new coding gene. HEK293T cells expressing Rana prestin showed electrophysiological features similar to that of hair cells from its inner ear. Comparative studies of zebrafish, chick, Rana and an ancient frog species showed that chick and zebrafish prestin lacked NLC. Ancient frog's prestin was functionally different from Rana. CONCLUSIONS: We mapped and sequenced the SLC26A5 of the Rana catesbeiana from its inner ear cDNA using RNA-Seq. The Rana SLC26A5 cDNA was 2292 bp long, encoding a polypeptide of 763 amino acid residues, with 40% identity to mammals. This new coding gene could encode a functionally active protein conferring NLC to both frog HCs and the mammalian cell line. While comparing to its orthologs, the amphibian prestin has been evolutionarily changing its function and becomes more advanced than avian and teleost prestin.

On the biological activity of cytokinin free bases and their ribosides.

MAIN CONCLUSION: The free bases of cytokinins are the biologically active forms of the hormone while cytokinin ribosides become active only upon removal of the ribose residue. Cytokinins (CKs) belong to the classical plant hormones. They were discovered more than 65 years ago, but which molecular forms possess genuine CK activity is still matter of debate. Numerous studies support the view that only the free bases are the biologically active molecules. This standpoint has been challenged in a recent review (Nguyen et al. in Planta 254: 45, 2021) proposing that also CK ribosides may have genuine own CK activity. Here we critically discuss the pros and cons of this viewpoint considering the results of biological assays, CK binding studies, 3D structural data of CK-receptor interaction and mutant analyses. It is concluded that all types of study provide clear and convincing evidence only for biological activity of free bases and not ribosides; the latter are rather a transport form of the hormone without their own biological activity.

Monoclonal antibodies against foot-and-mouth disease virus RNA polymerase for detection of virus infection.

Foot-and-mouth disease (FMD) is a major viral disease in farm animals. In the present study, seven monoclonal antibodies (mAbs) were produced against the FMD virus (FMDV)-encoded RNA-dependent RNA polymerase (3D protein) and characterized. Screening of mAb reactivity against three overlapping fragments of the 3D protein expressed in Escherichia coli revealed that the binding sites of all the mAbs were confined to the N-terminal one-third of the 3D protein. A selected mAb was utilized for detecting FMDV in the infected cell culture and tissues obtained from FMDV-infected animals.

Evidence for a Negative Correlation between Human Reactive Enamine-Imine Intermediate Deaminase A (RIDA) Activity and Cell Proliferation Rate: Role of Lysine Succinylation of RIDA.

Reactive intermediate deaminase (Rid) proteins are enzymes conserved in all domains of life. UK114, a mammalian member of RidA subfamily, has been firstly identified as a component of liver perchloric acid-soluble proteins (L-PSP). Although still poorly defined, several functions have been attributed to the mammalian protein UK114/RIDA, including the reactive intermediate deamination activity. The expression of UK114/RIDA has been observed in some tumors, arousing interest in this protein as an evaluable tumor marker. However, other studies reported a negative correlation between UK114/RIDA expression, tumor differentiation degree and cell proliferation. This work addressed the question of UK114/RIDA expression in human non-tumor HEK293 cell lines and in some human tumor cell lines. Here we reported that human RIDA (hRIDA) was expressed in all the analyzed cell line and subjected to lysine (K-)succinylation. In HEK293, hRIDA K-succinylation was negatively correlated to the cell proliferation rate and was under the control of SIRT5. Moreover, K-succinylation clearly altered hRIDA quantification by immunoblotting, explaining, at least in part, some discrepancies about RIDA expression reported in previous studies. We found that hRIDA was able to deaminate reactive enamine-imine intermediates and that K-succinylation drastically reduced deaminase activity. As predicted by in silico analysis, the observed reduction of deaminase activity has been related to the drastic alterations of hRIDA structure inferred by K-succinylation. The role of hRIDA and the importance of its K-succinylation in cell metabolism, especially in cancer biology, have been discussed.

Plant transporters involved in combating boron toxicity: beyond 3D structures.

Membrane transporters control the movement and distribution of solutes, including the disposal or compartmentation of toxic substances that accumulate in plants under adverse environmental conditions. In this minireview, in the light of the approaching 100th anniversary of unveiling the significance of boron to plants (K. Warington, 1923; Ann. Bot.37, 629) we discuss the current state of the knowledge on boron transport systems that plants utilise to combat boron toxicity. These transport proteins include: (i) nodulin-26-like intrinsic protein-types of aquaporins, and (ii) anionic efflux (borate) solute carriers. We describe the recent progress made on the structure-function relationships of these transport proteins and point out that this progress is integral to quantitative considerations of the transporter's roles in tissue boron homeostasis. Newly acquired knowledge at the molecular level has informed on the transport mechanics and conformational states of boron transport systems that can explain their impact on cell biology and whole plant physiology. We expect that this information will form the basis for engineering transporters with optimised features to alleviate boron toxicity tolerance in plants exposed to suboptimal soil conditions for sustained food production.

Severe epidermolysis bullosa simplex phenotype caused by codominant mutations p.Ile377Thr in keratin 14 and p.Gly138Glu in keratin 5.

Epidermolysis bullosa simplex (EBS) is a rare skin disease usually inherited in an autosomal dominant pattern. EBS is resulting from mutations in keratin 5 (KRT5) and keratin 14 (KRT14) genes encoding the keratins 5 and 14 proteins expressed in the keratinocytes of the basal layer of the epidermis. To date, seven pathogenic mutations have been reported to be responsible for EBS in the Canadian population from the province of Quebec: p.Pro25Leu, p.Leu150Pro, p.Met327Thr and p.Arg559X in KRT5; p.Arg125Ser, p.Ile377Thr and p.Ile412Phe in KRT14. Here, we present a novel French-Canadian patient diagnosed with EBS confined to the soles but presenting a severe complication form including blisters, hyperkeratosis, skin erosions and toenail abnormalities. Mutation screening was performed by direct sequencing of the entire coding regions of KRT5 and KRT14 genes and revealed the previously reported missense heterozygous mutation c. 1130T > C in KRT14 (p.Ile377Thr). Furthermore, this patient is carrying a second mutation in KRT5, c.413G > A (p.Gly138Glu), which has been linked to an increased risk of basal cell carcinoma in the literature. We suspect an impact of the p.Gly138Glu variant on the EBS phenotype severity of the studied patient. The pathogenicity and consequences of both genetic variations were simulated by in silico tools.

LEGO-based generalized set of two linear algebraic 3D bio-macro-molecular descriptors: Theory and validation by QSARs.

Novel 3D protein descriptors based on bilinear, quadratic and linear algebraic maps in Rn are proposed. The latter employs the kth 2-tuple (dis) similarity matrix to codify information related to covalent and non-covalent interactions in these biopolymers. The calculation of the inter-amino acid distances is generalized by using several dis-similarity coefficients, where normalization procedures based on the simple stochastic and mutual probability schemes are applied. A new local-fragment approach based on amino acid-types and amino acid-groups is proposed to characterize regions of interest in proteins. Topological and geometric macromolecular cutoffs are defined using local and total indices to highlight non-covalent interactions existing between the side-chains of each amino acid. Moreover, local and total indices calculations are generalized considering a LEGO approach, by using several aggregation operators. Collinearity and variability analyses are performed to evaluate every generalizing component applied to the definition of these novel indices. These experiments are oriented to reduce the number of MDs obtained for performing prediction models. The predictive power of the proposed indices was evaluated using two benchmark datasets, folding rate and secondary structural classification of proteins. The proposed MDs are modeled using the following strategies: Multiple Linear Regression (MLR) and Support Vector Machine (SVM), respectively. The best regression model developed for the folding rate of proteins yields a cross-validation coefficient of 0.875 (Test Set) and the best model developed for secondary structural classification obtained 98% of instances correctly classified (Test Set). These statistical parameters are superior to the ones obtained with existing MDs reported in the literature. Overall, the new theoretical generalization enhanced the information extraction into the MDs, allowing a better correlation between these two evaluated benchmark datasets and the proposed indices. The optimal theoretical configurations defined for the calculation of these MDs consider low collinearity and less information redundancy among them. These theoretical configurations and the software are available at http://tomocomd.com/mulims-mcompas.

Structure of the UspA1 protein fragment from Moraxella catarrhalis responsible for C3d binding.

The gram-negative bacterium Moraxella catarrhalis infects humans exclusively, causing various respiratory tract diseases, including acute otitis media in children, septicaemia or meningitis in adults, and pneumonia in the elderly. To do so, M. catarrhalis expresses virulence factors facilitating its entry and survival in the host. Among them are the ubiquitous surface proteins (Usps): A1, A2, and A2H, which all belong to the trimeric autotransporter adhesin family. They bind extracellular matrix molecules and inhibit the classical and alternative pathways of the complement cascade by recruiting complement regulators C3d and C4b binding protein. Here, we report the 2.5 Šresolution X-ray structure of UspA1299-452, which previous work had suggested contained the canonical C3d binding site found in both UspA1 and UspA2. We show that this fragment of the passenger domain contains part of the long neck domain (residues 299-336) and a fragment of the stalk (residues 337-452). The coiled-coil stalk is left-handed, with 7 polar residues from each chain facing the core and coordinating chloride ions or water molecules. Despite the previous reports of tight binding in serum-based assays, we were not able to demonstrate binding between C3d and UspA1299-452 using ELISA or biolayer interferometry, and the two proteins run separately on size-exclusion chromatography. Microscale thermophoresis suggested that the dissociation constant was 140.5 ±â€¯8.4 µM. We therefore suggest that full-length proteins or other additional factors are important in UspA1-C3d interactions. Other molecules on the bacterial surface or present in serum may enhance binding of those two molecules.

Automated shape-based clustering of 3D immunoglobulin protein structures in chronic lymphocytic leukemia.

BACKGROUND: Although the etiology of chronic lymphocytic leukemia (CLL), the most common type of adult leukemia, is still unclear, strong evidence implicates antigen involvement in disease ontogeny and evolution. Primary and 3D structure analysis has been utilised in order to discover indications of antigenic pressure. The latter has been mostly based on the 3D models of the clonotypic B cell receptor immunoglobulin (BcR IG) amino acid sequences. Therefore, their accuracy is directly dependent on the quality of the model construction algorithms and the specific methods used to compare the ensuing models. Thus far, reliable and robust methods that can group the IG 3D models based on their structural characteristics are missing. RESULTS: Here we propose a novel method for clustering a set of proteins based on their 3D structure focusing on 3D structures of BcR IG from a large series of patients with CLL. The method combines techniques from the areas of bioinformatics, 3D object recognition and machine learning. The clustering procedure is based on the extraction of 3D descriptors, encoding various properties of the local and global geometrical structure of the proteins. The descriptors are extracted from aligned pairs of proteins. A combination of individual 3D descriptors is also used as an additional method. The comparison of the automatically generated clusters to manual annotation by experts shows an increased accuracy when using the 3D descriptors compared to plain bioinformatics-based comparison. The accuracy is increased even more when using the combination of 3D descriptors. CONCLUSIONS: The experimental results verify that the use of 3D descriptors commonly used for 3D object recognition can be effectively applied to distinguishing structural differences of proteins. The proposed approach can be applied to provide hints for the existence of structural groups in a large set of unannotated BcR IG protein files in both CLL and, by logical extension, other contexts where it is relevant to characterize BcR IG structural similarity. The method does not present any limitations in application and can be extended to other types of proteins.

The wheat TabZIP2 transcription factor is activated by the nutrient starvation-responsive SnRK3/CIPK protein kinase.

KEY MESSAGE: The understanding of roles of bZIP factors in biological processes during plant development and under abiotic stresses requires the detailed mechanistic knowledge of behaviour of TFs. Basic leucine zipper (bZIP) transcription factors (TFs) play key roles in the regulation of grain development and plant responses to abiotic stresses. We investigated the role and molecular mechanisms of function of the TabZIP2 gene isolated from drought-stressed wheat plants. Molecular characterisation of TabZIP2 and derived protein included analyses of gene expression and its target promoter, and the influence of interacting partners on the target promoter activation. Two interacting partners of TabZIP2, the 14-3-3 protein, TaWIN1 and the bZIP transcription factor TaABI5L, were identified in a Y2H screen. We established that under elevated ABA levels the activity of TabZIP2 was negatively regulated by the TaWIN1 protein and positively regulated by the SnRK3/CIPK protein kinase WPK4, reported previously to be responsive to nutrient starvation. The physical interaction between the TaWIN1 and the WPK4 was detected. We also compared the influence of homo- and hetero-dimerisation of TabZIP2 and TaABI5L on DNA binding. TabZIP2 gene functional analyses were performed using drought-inducible overexpression of TabZIP2 in transgenic wheat. Transgenic plants grown under moderate drought during flowering, were smaller than control plants, and had fewer spikes and seeds per plant. However, a single seed weight was increased compared to single seed weights of control plants in three of four evaluated transgenic lines. The observed phenotypes of transgenic plants and the regulation of TabZIP2 activity by nutrient starvation-responsive WPK4, suggest that the TabZIP2 could be the part of a signalling pathway, which controls the rearrangement of carbohydrate and nutrient flows in plant organs in response to drought.

Overexpression of the class I homeodomain transcription factor TaHDZipI-5 increases drought and frost tolerance in transgenic wheat.

Characterization of the function of stress-related genes helps to understand the mechanisms of plant responses to environmental conditions. The findings of this work defined the role of the wheat TaHDZipI-5 gene, encoding a stress-responsive homeodomain-leucine zipper class I (HD-Zip I) transcription factor, during the development of plant tolerance to frost and drought. Strong induction of TaHDZipI-5 expression by low temperatures, and the elevated TaHDZipI-5 levels of expression in flowers and early developing grains in the absence of stress, suggests that TaHDZipI-5 is involved in the regulation of frost tolerance at flowering. The TaHDZipI-5 protein behaved as an activator in a yeast transactivation assay, and the TaHDZipI-5 activation domain was localized to its C-terminus. The TaHDZipI-5 protein homo- and hetero-dimerizes with related TaHDZipI-3, and differences between DNA interactions in both dimers were specified at 3D molecular levels. The constitutive overexpression of TaHDZipI-5 in bread wheat significantly enhanced frost and drought tolerance of transgenic wheat lines with the appearance of undesired phenotypic features, which included a reduced plant size and biomass, delayed flowering and a grain yield decrease. An attempt to improve the phenotype of transgenic wheat by the application of stress-inducible promoters with contrasting properties did not lead to the elimination of undesired phenotype, apparently due to strict spatial requirements for TaHDZipI-5 overexpression.

Structure and Assembly of Clathrin Cages.

The unusual structure of clathrin, combined with its ability to assemble and disassemble rapidly in cells provides a model system for us to learn about the ways in which proteins can contribute mechanically to a functioning cell. In this article, we discuss the structural properties of clathrin cages and the triskelions which assemble to form them. The function of clathrin depends on the structure of these triskelions and the interactions they make both with each other during assembly and with the adaptor protein network that drives coated vesicle formation. The atomic resolution structure of clathrin domains has been revealed by X-ray crystallography while scattering studies have enabled the shape of a triskelion in solution to be deduced. Cryo-electron microscopy maps have shown the secondary structure of entire cages, how individual triskelion legs are arranged to form a cage and enabled some bound adaptor proteins to be located. Cage formation itself is energetically finely balanced and requires specific interactions between triskelion legs to be productive, as biochemical studies and in silico modeling have shown. Theoretical, structural and cell biological investigations over many years have contributed to our knowledge of clathrin structure and assembly. It now remains to determine the precise nature of the interactions which occur between clathrin triskelions, light chain and heavy chain and the adaptor protein network.