RESUMO
Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas.
Assuntos
Alelos , Sistemas CRISPR-Cas , Edição de Genes , Edição de Genes/métodos , Humanos , Polimorfismo de Nucleotídeo Único , Mutação , Software , Internet , Motivos de Nucleotídeos , Camelídeos Americanos/genéticaRESUMO
Root angle in crops represents a key trait for efficient capture of soil resources. Root angle is determined by competing gravitropic versus antigravitropic offset (AGO) mechanisms. Here we report a root angle regulatory gene termed ENHANCED GRAVITROPISM1 (EGT1) that encodes a putative AGO component, whose loss-of-function enhances root gravitropism. Mutations in barley and wheat EGT1 genes confer a striking root phenotype, where every root class adopts a steeper growth angle. EGT1 encodes an F-box and Tubby domain-containing protein that is highly conserved across plant species. Haplotype analysis found that natural allelic variation at the barley EGT1 locus impacts root angle. Gravitropic assays indicated that Hvegt1 roots bend more rapidly than wild-type. Transcript profiling revealed Hvegt1 roots deregulate reactive oxygen species (ROS) homeostasis and cell wall-loosening enzymes and cofactors. ROS imaging shows that Hvegt1 root basal meristem and elongation zone tissues have reduced levels. Atomic force microscopy measurements detected elongating Hvegt1 root cortical cell walls are significantly less stiff than wild-type. In situ analysis identified HvEGT1 is expressed in elongating cortical and stele tissues, which are distinct from known root gravitropic perception and response tissues in the columella and epidermis, respectively. We propose that EGT1 controls root angle by regulating cell wall stiffness in elongating root cortical tissue, counteracting the gravitropic machinery's known ability to bend the root via its outermost tissues. We conclude that root angle is controlled by EGT1 in cereal crops employing an antigravitropic mechanism.
Assuntos
Produtos Agrícolas , Gravitropismo , Hordeum , Proteínas de Plantas , Raízes de Plantas , Parede Celular/química , Produtos Agrícolas/química , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Gravitropismo/genética , Hordeum/química , Hordeum/genética , Hordeum/crescimento & desenvolvimento , Microscopia de Força Atômica , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Raízes de Plantas/química , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Espécies Reativas de Oxigênio/metabolismo , Transcrição GênicaRESUMO
MOTIVATION: Protein contact networks (PCNs) represent the 3D structure of a protein using network formalism. Inter-residue contacts are described as binary adjacency matrices, which are derived from the graph representation of residues (as α-carbons, ß-carbons or centroids) and Euclidean distances according to defined thresholds. Functional characterization algorithms are computed on binary adjacency matrices to unveil allosteric, dynamic, and interaction mechanisms in proteins. Such strategies are usually applied in a combinatorial manner, although rarely in seamless and user-friendly implementations. RESULTS: PyPCN is a plugin for PyMOL wrapping more than twenty PCN algorithms and metrics in an easy-to-use graphical user interface, to support PCN analysis. The plugin accepts 3D structures from the Protein Data Bank, user-provided PDBs, or precomputed adjacency matrices. The results are directly mapped to 3D protein structures and organized into interactive diagrams for their visualization. A dedicated graphical user interface combined with PyMOL visual support makes analysis more intuitive and easier, extending the applicability of PCNs. AVAILABILITY AND IMPLEMENTATION: https://github.com/pcnproject/PyPCN.
Assuntos
Algoritmos , Proteínas , Proteínas/química , SoftwareRESUMO
Barley (Hordeum vulgare) is an important global cereal crop and a model in genetic studies. Despite advances in characterising barley genomic resources, few mutant studies have identified genes controlling root architecture and anatomy, which plays a critical role in capturing soil resources. Our phenotypic screening of a TILLING mutant collection identified line TM5992 exhibiting a short-root phenotype compared with wild-type (WT) Morex background. Outcrossing TM5992 with barley variety Proctor and subsequent SNP array-based bulk segregant analysis, fine mapped the mutation to a cM scale. Exome sequencing pinpointed a mutation in the candidate gene HvPIN1a, further confirming this by analysing independent mutant alleles. Detailed analysis of root growth and anatomy in Hvpin1a mutant alleles exhibited a slower growth rate, shorter apical meristem and striking vascular patterning defects compared to WT. Expression and mutant analyses of PIN1 members in the closely related cereal brachypodium (Brachypodium distachyon) revealed that BdPIN1a and BdPIN1b were redundantly expressed in root vascular tissues but only Bdpin1a mutant allele displayed root vascular defects similar to Hvpin1a. We conclude that barley PIN1 genes have sub-functionalised in cereals, compared to Arabidopsis (Arabidopsis thaliana), where PIN1a sequences control root vascular patterning.
RESUMO
The root growth angle defines how roots grow toward the gravity vector and is among the most important determinants of root system architecture. It controls water uptake capacity, nutrient use efficiency, stress resilience, and, as a consequence, yield of crop plants. We demonstrated that the egt2 (enhanced gravitropism 2) mutant of barley exhibits steeper root growth of seminal and lateral roots and an auxin-independent higher responsiveness to gravity compared to wild-type plants. We cloned the EGT2 gene by a combination of bulked-segregant analysis and whole genome sequencing. Subsequent validation experiments by an independent CRISPR/Cas9 mutant allele demonstrated that egt2 encodes a STERILE ALPHA MOTIF domain-containing protein. In situ hybridization experiments illustrated that EGT2 is expressed from the root cap to the elongation zone. We demonstrated the evolutionary conserved role of EGT2 in root growth angle control between barley and wheat by knocking out the EGT2 orthologs in the A and B genomes of tetraploid durum wheat. By combining laser capture microdissection with RNA sequencing, we observed that seven expansin genes were transcriptionally down-regulated in the elongation zone. This is consistent with a role of EGT2 in this region of the root where the effect of gravity sensing is executed by differential cell elongation. Our findings suggest that EGT2 is an evolutionary conserved regulator of root growth angle in barley and wheat that could be a valuable target for root-based crop improvement strategies in cereals.
Assuntos
Gravitropismo , Hordeum/fisiologia , Proteínas de Plantas/fisiologia , Raízes de Plantas/crescimento & desenvolvimento , Motivo Estéril alfa , Triticum/fisiologia , Parede Celular/metabolismo , Sequência Conservada , Evolução Molecular , Técnicas de Inativação de Genes , Genes de Plantas , Hordeum/genética , Hordeum/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Triticum/genética , Triticum/crescimento & desenvolvimentoRESUMO
Recent advances in protein structure prediction, driven by AlphaFold 2 and machine learning, demonstrate proficiency in static structures but encounter challenges in capturing essential dynamic features crucial for understanding biological function. In this context, homology-based modeling emerges as a cost-effective and computationally efficient alternative. The MODELLER (version 10.5, accessed on 30 November 2023) algorithm can be harnessed for this purpose since it computes intermediate models during simulated annealing, enabling the exploration of attainable configurational states and energies while minimizing its objective function. There have been a few attempts to date to improve the models generated by its algorithm, and in particular, there is no literature regarding the implementation of an averaging procedure involving the intermediate models in the MODELLER algorithm. In this study, we examined MODELLER's output using 225 target-template pairs, extracting the best representatives of intermediate models. Applying an averaging procedure to the selected intermediate structures based on statistical potentials, we aimed to determine: (1) whether averaging improves the quality of structural models during the building phase; (2) if ranking by statistical potentials reliably selects the best models, leading to improved final model quality; (3) whether using a single template versus multiple templates affects the averaging approach; (4) whether the "ensemble" nature of the MODELLER building phase can be harnessed to capture low-energy conformations in holo structures modeling. Our findings indicate that while improvements typically fall short of a few decimal points in the model evaluation metric, a notable fraction of configurations exhibit slightly higher similarity to the native structure than MODELLER's proposed final model. The averaging-building procedure proves particularly beneficial in (1) regions of low sequence identity between the target and template(s), the most challenging aspect of homology modeling; (2) holo protein conformations generation, an area in which MODELLER and related tools usually fall short of the expected performance.
Assuntos
Algoritmos , Proteínas , Proteínas/química , Conformação Proteica , Simulação de Dinâmica Molecular , Modelos Químicos , SoftwareRESUMO
MOTIVATION: The primary strategy for predicting the binding mode of small molecules to their receptors and for performing receptor-based virtual screening studies is protein-ligand docking, which is undoubtedly the most popular and successful approach in computer-aided drug discovery. The increased popularity of docking has resulted in the development of different docking algorithms and scoring functions. Nonetheless, it is unlikely that a single approach outperforms the others in terms of reproducibility and precision. In this ground, consensus docking techniques are taking hold. RESULTS: We have developed DockingPie, an open source PyMOL plugin for individual, as well as consensus docking analyses. Smina, AutoDock Vina, ADFR and RxDock are the four docking engines that DockingPie currently supports in an easy and extremely intuitive way, thanks to its integrated docking environment and its GUI, fully integrated within PyMOL. AVAILABILITY AND IMPLEMENTATION: https://github.com/paiardin/DockingPie. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Proteínas , Software , Consenso , Reprodutibilidade dos Testes , Ligantes , Proteínas/química , Simulação de Acoplamento MolecularRESUMO
Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds.
Assuntos
Oxirredutases , Piridoxaminafosfato Oxidase , Humanos , Sítio Alostérico , Oxirredutases/metabolismo , Fosfatos , Fosfato de Piridoxal/metabolismo , Piridoxaminafosfato Oxidase/genética , Piridoxaminafosfato Oxidase/metabolismoRESUMO
Over the past few decades, the number of available structural bioinformatics pipelines, libraries, plugins, web resources and software has increased exponentially and become accessible to the broad realm of life scientists. This expansion has shaped the field as a tangled network of methods, algorithms and user interfaces. In recent years PyMOL, widely used software for biomolecules visualization and analysis, has started to play a key role in providing an open platform for the successful implementation of expert knowledge into an easy-to-use molecular graphics tool. This review outlines the plugins and features that make PyMOL an eligible environment for supporting structural bioinformatics analyses.
Assuntos
Algoritmos , Software , Biologia Computacional/métodosRESUMO
Protein-peptide interactions (PpIs) are a subset of the overall protein-protein interaction (PPI) network in the living cell and are pivotal for the majority of cell processes and functions. High-throughput methods to detect PpIs and PPIs usually require time and costs that are not always affordable. Therefore, reliable in silico predictions represent a valid and effective alternative. In this work, a new algorithm is described, implemented in a freely available tool, i.e., "PepThreader", to carry out PPIs and PpIs prediction and analysis. PepThreader threads multiple fragments derived from a full-length protein sequence (or from a peptide library) onto a second template peptide, in complex with a protein target, "spotting" the potential binding peptides and ranking them according to a sequence-based and structure-based threading score. The threading algorithm first makes use of a scoring function that is based on peptides sequence similarity. Then, a rerank of the initial hits is performed, according to structure-based scoring functions. PepThreader has been benchmarked on a dataset of 292 protein-peptide complexes that were collected from existing databases of experimentally determined protein-peptide interactions. An accuracy of 80%, when considering the top predicted 25 hits, was achieved, which performs in a comparable way with the other state-of-art tools in PPIs and PpIs modeling. Nonetheless, PepThreader is unique in that it is able at the same time to spot a binding peptide within a full-length sequence involved in PPI and model its structure within the receptor. Therefore, PepThreader adds to the already-available tools supporting the experimental PPIs and PpIs identification and characterization.
Assuntos
Peptídeos , Mapeamento de Interação de Proteínas , Sequência de Aminoácidos , Biblioteca de Peptídeos , Peptídeos/química , Mapeamento de Interação de Proteínas/métodos , SoftwareRESUMO
Maize ear fasciation originates from excessive or abnormal proliferation of the ear meristem and usually manifests as flattened multiple-tipped ear and/or disordered kernel arrangement. Ear prolificacy expresses as multiple ears per plant or per node. Both ear fasciation and prolificacy can affect grain yield. The genetic control of the two traits was studied using two recombinant inbred line populations (B73 × Lo1016 and Lo964 × Lo1016) with Lo1016 and Lo964 as donors of ear fasciation and prolificacy, respectively. Ear fasciation-related traits, number of kernel rows (KRN), ear prolificacy and number of tillers were phenotyped in multi-year field experiments. Ear fasciation traits and KRN showed relatively high heritability (h 2 > 0.5) except ratio of ear diameters. For all ear fasciation-related traits, fasciation level positively correlated with KRN (0.30 ≤ r ≤ 0.68). Prolificacy and tillering were not correlated and their h 2 ranged from 0.41 to 0.78. QTL mapping identified four QTLs for ear fasciation, on chromosomes 1 (two QTLs), 5 and 7, the latter two overlapping with QTLs for number of kernel rows. Notably, at these QTLs, the Lo1016 alleles increased both ear fasciation and KRN across populations, thus showing potential breeding applicability. Four and five non-overlapping QTLs were mapped for ear prolificacy and tillering, respectively. Two ear fasciation QTLs, qFas1.2 and qFas7, overlapped with fasciation QTLs mapped in other studies and spanned compact plant2 and ramosa1 candidate genes. Our study identified novel ear fasciation loci and alleles positively affecting grain yield components, and ear prolificacy and tillering loci which are unexpectedly still segregating in elite maize materials, contributing useful information for genomics-assisted breeding programs.
RESUMO
Disease lesion mimic (DLM) or necrotic mutants display necrotic lesions in the absence of pathogen infections. They can show improved resistance to some pathogens and their molecular dissection can contribute to revealing components of plant defense pathways. Although forward-genetics strategies to find genes causal to mutant phenotypes are available in crops, these strategies require the production of experimental cross populations, mutagenesis, or gene editing and are time- and resource-consuming or may have to deal with regulated plant materials. In this study, we described a collection of 34 DLM mutants in barley (Hordeum vulgare L.) and applied a novel method called complementation by sequencing (CBS), which enables the identification of the gene responsible for a mutant phenotype given the availability of two or more chemically mutagenized individuals showing the same phenotype. Complementation by sequencing relies on the feasibility to obtain all induced mutations present in chemical mutants and on the low probability that different individuals share the same mutated genes. By CBS, we identified a cytochrome P450 CYP71P1 gene as responsible for orange blotch DLM mutants, including the historical barley nec3 locus. By comparative phylogenetic analysis we showed that CYP71P1 gene family emerged early in angiosperm evolution but has been recurrently lost in some lineages including Arabidopsis thaliana (L.) Heynh. Complementation by sequencing is a straightforward cost-effective approach to clone genes controlling phenotypes in a chemically mutagenized collection. The TILLMore (TM) collection will be instrumental for understanding the molecular basis of DLM phenotypes and to contribute knowledge about mechanisms of host-pathogen interaction.
Assuntos
Arabidopsis , Hordeum , Arabidopsis/genética , Clonagem Molecular , Genes de Plantas , Hordeum/genética , Mutação , FilogeniaRESUMO
In the evolution of land plants, the plant immune system has experienced expansion in immune receptor and signaling pathways. Lineage-specific expansions have been observed in diverse gene families that are potentially involved in immunity but lack causal association. Here, we show that Rps8-mediated resistance in barley to the pathogen Puccinia striiformis f. sp. tritici (wheat stripe rust) is conferred by a genetic module: Pur1 and Exo70FX12, which are together necessary and sufficient. Pur1 encodes a leucine-rich repeat receptor kinase and is the ortholog of rice Xa21, and Exo70FX12 belongs to the Poales-specific Exo70FX clade. The Exo70FX clade emerged after the divergence of the Bromeliaceae and Poaceae and comprises from 2 to 75 members in sequenced grasses. These results demonstrate the requirement of a lineage-specific Exo70FX12 in Pur1-mediated immunity and suggest that the Exo70FX clade may have evolved a specialized role in receptor kinase signaling.
RESUMO
The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release.