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Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.
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S-nitrosylation is a redox post-translational modification widely recognized to play an important role in cellular signaling as it can modulate protein function and conformation. At the physiological level, nitrosoglutathione (GSNO) is considered the major physiological NO-releasing compound due to its ability to transfer the NO moiety to protein thiols but the structural determinants regulating its redox specificity are not fully elucidated. In this study, we employed photosynthetic glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii (CrGAPA) to investigate the molecular mechanisms underlying GSNO-dependent thiol oxidation. We first observed that GSNO causes reversible enzyme inhibition by inducing S-nitrosylation. While the cofactor NADP+ partially protects the enzyme from GSNO-mediated S-nitrosylation, protein inhibition is not observed in the presence of the substrate 1,3-bisphosphoglycerate, indicating that the S-nitrosylation of the catalytic Cys149 is responsible for CrGAPA inactivation. The crystal structures of CrGAPA in complex with NADP+ and NAD+ reveal a general structural similarity with other photosynthetic GAPDH. Starting from the 3D structure, we carried out molecular dynamics simulations to identify the protein residues involved in GSNO binding. The reaction mechanism of GSNO with CrGAPA Cys149 was investigated by quantum mechanical/molecular mechanical calculations, which permitted to disclose the relative contribution of protein residues in modulating the activation barrier of the trans-nitrosylation reaction. Based on our findings, we provide functional and structural insights into the response of CrGAPA to GSNO-dependent regulation, possibly expanding the mechanistic features to other protein cysteines susceptible to be oxidatively modified by GSNO.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , S-Nitrosoglutationa , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , NADP/metabolismo , Óxido Nítrico/metabolismo , Oxirredução , Fotossíntese , S-Nitrosoglutationa/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Protein-protein interactions (PPIs) contribute to regulate many aspects of cell physiology and metabolism. Protein domains involved in PPIs are important building blocks for engineering genetic circuits through synthetic biology. These domains can be obtained from known proteins and rationally engineered to produce orthogonal scaffolds, or computationally designed de novo thanks to recent advances in structural biology and molecular dynamics prediction. Such circuits based on PPIs (or protein circuits) appear of particular interest, as they can directly affect transcriptional outputs, as well as induce behavioral/adaptational changes in cell metabolism, without the need for further protein synthesis. This last example was highlighted in recent works to enable the production of fast-responding circuits which can be exploited for biosensing and diagnostics. Notably, PPIs can also be engineered to develop new drugs able to bind specific intra- and extra-cellular targets. In this review, we summarize recent findings in the field of protein circuit design, with particular focus on the use of peptides as scaffolds to engineer these circuits.
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Rice coleoptile elongation under submergence guarantees fast seedling establishment in the field. We investigated the role of auxin in influencing the capacity of rice to produce a long coleoptile under water. In order to explore the complexity of auxin's role in coleoptile elongation, we used gene expression analysis, confocal microscopy of an auxin-responsive fluorescent reporter, gas chromatography coupled to tandem mass spectrometry (GC-MS/MS), and T-DNA insertional mutants of an auxin transport protein. We show that a higher auxin availability in the coleoptile correlates with the final coleoptile length under submergence. We also identified the auxin influx carrier AUX1 as a component influencing this trait under submergence. The coleoptile tip is involved in the final length of rice varieties harbouring a long coleoptile. Our experimental results indicate that auxin biosynthesis and transport underlies the differential elongation between short and long coleoptile-harbouring japonica rice varieties.
Assuntos
Oryza , Cotilédone , Ácidos Indolacéticos , Oryza/genética , Plântula , Espectrometria de Massas em TandemRESUMO
Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. Its kinetic properties were determined and the three-dimensional structures of the apo-, NAD+- and NAD+/GSNO-forms were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity and folding, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed.
Assuntos
Chlamydomonas reinhardtii , Oxirredutases , Aldeído Oxirredutases/genética , Chlamydomonas reinhardtii/genética , Cisteína , Óxido Nítrico , S-NitrosoglutationaRESUMO
Hypoxic conditions often arise from waterlogging and flooding, affecting several aspects of plant metabolism, including the uptake of nutrients. We identified a member of the CALCINEURIN ß-LIKE INTERACTING PROTEIN KINASE (CIPK) family in Arabidopsis, CIPK25, which is induced in the root endodermis under low-oxygen conditions. A cipk25 mutant exhibited higher sensitivity to anoxia in conditions of potassium limitation, suggesting that this kinase is involved in the regulation of potassium uptake. Interestingly, we found that CIPK25 interacts with AKT1, the major inward rectifying potassium channel in Arabidopsis. Under anoxic conditions, cipk25 mutant seedlings were unable to maintain potassium concentrations at wild-type levels, suggesting that CIPK25 likely plays a role in modulating potassium homeostasis under low-oxygen conditions. In addition, cipk25 and akt1 mutants share similar developmental defects under waterlogging, further supporting an interplay between CIPK25 and AKT1.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Oxigênio , Potássio/metabolismo , Proteínas Serina-Treonina Quinases , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Calcineurina , Homeostase , Raízes de Plantas/metabolismo , Canais de Potássio/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Tomato (Solanum lycopersicum) fruits are typically red at ripening, with high levels of carotenoids and a low content in flavonoids. Considerable work has been done to enrich the spectrum of their health-beneficial phytochemicals, and interspecific crosses with wild species have successfully led to purple anthocyanin-colored fruits. The Aft (Anthocyanin fruit) tomato accession inherited from Solanum chilense the ability to accumulate anthocyanins in fruit peel through the introgression of loci controlling anthocyanin pigmentation, including four R2R3 MYB transcription factor-encoding genes. Here, we carried out a comparative functional analysis of these transcription factors in wild-type and Aft plants, and tested their ability to take part in the transcriptional complexes that regulate the biosynthetic pathway and their efficiency in inducing anthocyanin pigmentation. Significant differences emerged for SlAN2like, both in the expression level and protein functionality, with splicing mutations determining a complete loss of function of the wild-type protein. This transcription factor thus appears to play a key role in the anthocyanin fruit pigmentation. Our data provide new clues to the long-awaited genetic basis of the Aft phenotype and contribute to understand why domesticated tomato fruits display a homogeneous red coloration without the typical purple streaks observed in wild tomato species.
Assuntos
Antocianinas/biossíntese , Frutas/genética , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Fatores de Transcrição/genética , Processamento Alternativo , Antocianinas/genética , Frutas/metabolismo , Regulação da Expressão Gênica de Plantas , Introgressão Genética , Solanum lycopersicum/metabolismo , Proteínas de Plantas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
Unexpected and increasingly frequent extreme precipitation events result in soil flooding or waterlogging. Legumes have the capacity to establish a symbiotic relationship with endosymbiotic atmospheric dinitrogen-fixing rhizobia, thus contributing to natural nitrogen soil enrichment and reducing the need for chemical fertilization. The impact of waterlogging on nitrogen fixation and legume productivity needs to be considered for crop improvement. This review focuses on the legumes-rhizobia symbiotic models. We aim to summarize the mechanisms underlying symbiosis establishment, nodule development and functioning under waterlogging. The mechanisms of oxygen sensing of the host plant and symbiotic partner are considered in view of recent scientific advances.
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Rice is unique among cereals for its ability to germinate not only when submerged but also under anoxic conditions. Rice germination under submergence or anoxia is characterized by a longer coleoptile and delay in radicle emergence. A panel of temperate and tropical japonica rice accessions showing a large variability in coleoptile length was used to investigate genetic factors involved in this developmental process. The ability of the Khao Hlan On rice landrace to vigorously germinate when submerged has been previously associated with the presence of the trehalose 6 phosphate phosphatase 7 (TPP7) gene. In this study, we found that, in the presence of TPP7, polymorphisms and transcriptional variations of the gene in coleoptile tissue were not related to differences in the final coleoptile length under submergence. In order to find new chromosomal regions associated with the different ability of rice to elongate the coleoptile under submergence, we used genome-wide association study analysis on a panel of 273 japonica rice accessions. We discovered 11 significant marker-trait associations and identified candidate genes potentially involved in coleoptile length. Candidate gene expression analyses indicated that japonica rice genotypes possess complex genetic elements that control final coleoptile length under low oxygen.