RESUMO
Ubiquitin E3 ligases control every aspect of eukaryotic biology by promoting protein ubiquitination and degradation. At the end of a three-enzyme cascade, ubiquitin ligases mediate the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to specific substrate proteins. Early investigations of E3s of the RING (really interesting new gene) and HECT (homologous to the E6AP carboxyl terminus) types shed light on their enzymatic activities, general architectures, and substrate degron-binding modes. Recent studies have provided deeper mechanistic insights into their catalysis, activation, and regulation. In this review, we summarize the current progress in structure-function studies of ubiquitin ligases as well as exciting new discoveries of novel classes of E3s and diverse substrate recognition mechanisms. Our increased understanding of ubiquitin ligase function and regulation has provided the rationale for developing E3-targeting therapeutics for the treatment of human diseases.
Assuntos
Proteínas de Bactérias/metabolismo , Células Eucarióticas/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Drogas em Investigação/síntese química , Células Eucarióticas/microbiologia , Células Eucarióticas/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Proteólise , Especificidade por Substrato , Ubiquitina/genética , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Proteolysis, including post-translational proteolytic processing as well as protein degradation and amino acid recycling, is an essential component of the growth and development of living organisms. In this article, experts in plant proteolysis pose and discuss compelling open questions in their areas of research. Topics covered include the role of proteolysis in the cell cycle, DNA damage response, mitochondrial function, the generation of N-terminal signals (degrons) that mark many proteins for degradation (N-terminal acetylation, the Arg/N-degron pathway, and the chloroplast N-degron pathway), developmental and metabolic signaling (photomorphogenesis, abscisic acid and strigolactone signaling, sugar metabolism, and post-harvest regulation), plant responses to environmental signals (endoplasmic-reticulum associated degradation, chloroplast-associated degradation, drought tolerance, the growth-defense tradeoff)), and the functional diversification of peptidases. We hope these thought-provoking discussions help to stimulate further research.
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KAI2 receptors, classified as plant α/ß hydrolase enzymes, are capable of perceiving smoke-derived butenolide signals and endogenous yet unidentified KAI2-ligands (KLs). While the number of functional KAI2 receptors varies among land plant species, rice has only one KAI2 gene. Rice, a significant crop and representative of grasses, relies on KAI2-mediated Arbuscular mycorrhiza (AM) symbioses to flourish in traditionally arid and nutrient-poor environments. This study presents the first crystal structure of an active rice (Oryza sativa, Os) KAI2 hydrolase receptor. Our structural and biochemical analyses uncover grass-unique pocket residues influencing ligand sensitivity and hydrolytic activity. Through structure-guided analysis, we identify a specific residue whose mutation enables the increase or decrease of ligand perception, catalytic activity, and signal transduction. Furthermore, we investigate OsKAI2-mediated signaling by examining its ability to form a complex with its binding partner, the F-box protein DWARF3 (D3) ubiquitin ligase and subsequent degradation of the target substrate OsSMAX1, demonstrating the significant role of hydrophobic interactions in the OsKAI2-D3 interface. This study provides new insights into the diverse and pivotal roles of the OsKAI2 signaling pathway in the plant kingdom, particularly in grasses.
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The strigolactones, a class of plant hormones, regulate many aspects of plant physiology. In the inhibition of shoot branching, the α/ß hydrolase D14-which metabolizes strigolactone-interacts with the F-box protein D3 to ubiquitinate and degrade the transcription repressor D53. Despite the fact that multiple modes of interaction between D14 and strigolactone have recently been determined, how the hydrolase functions with D3 to mediate hormone-dependent D53 ubiquitination remains unknown. Here we show that D3 has a C-terminal α-helix that can switch between two conformational states. The engaged form of this α-helix facilitates the binding of D3 and D14 with a hydrolysed strigolactone intermediate, whereas the dislodged form can recognize unmodified D14 in an open conformation and inhibits its enzymatic activity. The D3 C-terminal α-helix enables D14 to recruit D53 in a strigolactone-dependent manner, which in turn activates the hydrolase. By revealing the structural plasticity of the SCFD3-D14 ubiquitin ligase, our results suggest a mechanism by which the E3 coordinates strigolactone signalling and metabolism.
Assuntos
Compostos Heterocíclicos com 3 Anéis/metabolismo , Lactonas/metabolismo , Oryza/enzimologia , Oryza/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas Ligases SKP Culina F-Box/química , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Compostos Heterocíclicos com 3 Anéis/química , Lactonas/química , Modelos Moleculares , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Reguladores de Crescimento de Plantas/química , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Relação Estrutura-Atividade , Ubiquitina , UbiquitinaçãoRESUMO
Karrikins (KARs) are chemicals in smoke that can enhance germination of many plants. Lettuce (Lactuca sativa) cv. Grand Rapids germinates in response to nanomolar karrikinolide (KAR1). Lettuce is much less responsive to KAR2 or a mixture of synthetic strigolactone analogs, rac-GR24. We investigated the molecular basis of selective and sensitive KAR1 perception in lettuce. The lettuce genome contains two copies of KARRIKIN INSENSITIVE2 (KAI2), which in Arabidopsis (Arabidopsis thaliana) encodes a receptor that is required for KAR responses. LsKAI2b is more highly expressed than LsKAI2a in dry achenes and during early stages of imbibition. Through cross-species complementation assays in Arabidopsis, we found that an LsKAI2b transgene confers robust responses to KAR1, but LsKAI2a does not. Therefore, LsKAI2b likely mediates KAR1 responses in lettuce. We compared homology models of KAI2 proteins from lettuce and a fire-follower, whispering bells (Emmenanthe penduliflora). This identified pocket residues 96, 124, 139, and 161 as candidates that influence the ligand specificity of KAI2. Further support for the importance of these residues was found through a broader comparison of pocket residues among 281 KAI2 proteins from 184 asterid species. Almost all KAI2 proteins had either Tyr or Phe identity at position 124. Genes encoding Y124-type KAI2 are more broadly distributed in asterids than in F124-type KAI2. Substitutions at residues 96, 124, 139, and 161 in Arabidopsis KAI2 produced a broad array of responses to KAR1, KAR2, and rac-GR24. This suggests that the diverse ligand preferences observed among KAI2 proteins in plants could have evolved through relatively few mutations.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Furanos/metabolismo , Furanos/farmacologia , Germinação/genética , Hidrolases/genética , Lactuca/genética , Lactuca/metabolismo , Ligantes , Piranos , FumaçaRESUMO
Strigolactones (SLs) are a unique and novel class of phytohormones that regulate numerous processes of growth and development in plants. Besides their endogenous functions as hormones, SLs are exuded by plant roots to stimulate critical interactions with symbiotic fungi but can also be exploited by parasitic plants to trigger their seed germination. In the past decade, since their discovery as phytohormones, rapid progress has been made in understanding the SL biosynthesis and signaling pathway. Of particular interest are the diversification of natural SLs and their exact mode of perception, selectivity, and hydrolysis by their dedicated receptors in plants. Here we provide an overview of the emerging field of SL perception with a focus on the diversity of canonical, non-canonical, and synthetic SL probes. Moreover, this review offers useful structural insights into SL perception, the precise molecular adaptations that define receptor-ligand specificities, and the mechanisms of SL hydrolysis and its attenuation by downstream signaling components.
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Ubiquitylation (also known as ubiquitination) regulates essentially all of the intracellular processes in eukaryotes through highly specific modification of numerous cellular proteins, which is often tightly regulated in a spatial and temporal manner. Although most often associated with proteasomal degradation, ubiquitylation frequently serves non-proteolytic functions. In light of its central roles in cellular regulation, it has not been surprising to find that many of the components of the ubiquitin system itself are regulated by ubiquitylation. This observation has broad implications for pathophysiology.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Animais , Cadeia Alimentar , Humanos , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/fisiologia , Senso de Humor e Humor como AssuntoRESUMO
Plant ß-amylase (BAM) proteins play an essential role in growth, development, stress response, and hormone regulation. Despite their typical (ß/α)8 barrel structure as active catalysts in starch breakdown, catalytically inactive BAMs are implicated in diverse yet elusive functions in plants. The noncatalytic BAM7/8 contain N-terminal BZR1 domains and were shown to be involved in the regulation of brassinosteroid signaling and possibly serve as sensors of yet an uncharacterized metabolic signal. While the structures of several catalytically active BAMs have been reported, structural characterization of the catalytically inactive BZR1-type BAMs remain unknown. Here, we determine the crystal structure of ß-amylase domain of Zea mays BAM8/BES1/BZR1-5 and provide comprehensive insights into its noncatalytic adaptation. Using structural-guided comparison combined with biochemical analysis and molecular dynamics simulations, we revealed conformational changes in multiple distinct highly conserved regions resulting in rearrangement of the binding pocket. Altogether, this study adds a new layer of understanding to starch breakdown mechanism and elucidates the acquired adjustments of noncatalytic BZR1-type BAMs as putative regulatory domains and/or metabolic sensors in plants.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , beta-Amilase , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Plantas/metabolismo , Plantas , Amido/metabolismo , Zea mays/metabolismo , beta-Amilase/química , beta-Amilase/metabolismoRESUMO
Chloroplasts are ancient organelles responsible for photosynthesis and various biosynthetic functions essential to most life on Earth. Many of these functions require tightly controlled regulatory processes to maintain homeostasis at the protein level. One such regulatory mechanism is the ubiquitin-proteasome system whose fundamental role is increasingly emerging in chloroplasts. In particular, the role of E3 ubiquitin ligases as determinants in the ubiquitination and degradation of specific intra-chloroplast proteins. Here, we highlight recent advances in understanding the roles of plant E3 ubiquitin ligases SP1, COP1, PUB4, CHIP, and TT3.1 as well as the ubiquitin-dependent segregase CDC48 in chloroplast function.
Assuntos
Cloroplastos , Ubiquitina-Proteína Ligases , Proteínas de Cloroplastos/metabolismo , Cloroplastos/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
MAIN CONCLUSION: Nutrient transporter genes could be a potential candidate for improving crop plants, with enhanced nutrient uptake leading to increased crop yield by providing tolerance against different biotic and abiotic stresses. The world's food supply is nearing a crisis in meeting the demands of an ever-growing global population, and an increase in both yield and nutrient value of major crops is vitally necessary to meet the increased population demand. Nutrients play an important role in plant metabolism as well as growth and development, and nutrient deficiency results in retarded plant growth and leads to reduced crop yield. A variety of cellular processes govern crop plant nutrient absorption from the soil. Among these, nutrient membrane transporters play an important role in the acquisition of nutrients from soil and transport of these nutrients to their target sites. In addition, as excess nutrient delivery has toxic effects on plant growth, these membrane transporters also play a significant role in the removal of excess nutrients in the crop plant. The key function provided by membrane transporters is the ability to supply the crop plant with an adequate level of tolerance against environmental stresses, such as soil acidity, alkalinity, salinity, drought, and pathogen attack. Membrane transporter genes have been utilized for the improvement of crop plants, with enhanced nutrient uptake leading to increased crop yield by providing tolerance against different biotic and abiotic stresses. Further understanding of the basic mechanisms of nutrient transport in crop plants could facilitate the advanced design of engineered plant crops to achieve increased yield and improve nutrient quality through the use of genetic technologies as well as molecular breeding. This review is focused on nutrient toxicity and tolerance mechanisms in crop plants to aid in understanding and addressing the anticipated global food demand.
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Produtos Agrícolas , Minerais , Estresse Fisiológico , Produtos Agrícolas/fisiologia , Minerais/metabolismo , Desenvolvimento Vegetal/fisiologia , Salinidade , Estresse Fisiológico/fisiologiaRESUMO
Hormonal cues regulate many aspects of plant growth and development, facilitating the plant's ability to systemically respond to environmental changes. Elucidating the molecular mechanisms governing these signaling pathways is crucial to understanding how plants function. Structural and functional biology methods have been essential in decoding plant genetic findings and revealing precise molecular actions at the protein level. Past studies of plant hormone signaling have uncovered mechanisms that involve highly coordinated protein turnover to elicit immediate cellular responses. Many phytohormone signaling pathways rely on the ubiquitin (Ub) proteasome system, specifically E3 Ub ligases, for perception and initiation of signaling transduction. In this review, we highlight structural aspects of plant hormone-sensing mechanisms by Ub ligases and discuss our current understanding of the emerging field of strigolactone signaling.
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Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismo , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismoRESUMO
The BES1/BZR1 transcription factors regulate the expression of genes responsive to brassinosteroids and play pivotal roles in plant development, but their role in regulating kernel development in maize remains unclear. In this study, we found that ZmBES1/BZR1-5 positively regulates kernel size. Association analysis of candidate genes in 513 diverse maize inbred lines indicated that three SNPs related to ZmBES1/BZR1-5 were significantly associated with kernel width and whilst four SNPs were related to 100-kernel weight. Overexpression of ZmBES1/BZR1-5 in Arabidopsis and rice both significantly increased seed size and weight, and smaller kernels were produced in maize Mu transposon insertion and EMS mutants. The ZmBES1/BZR1-5 protein locates in the nucleus, contains bHLH and BAM domains, and shows no transcriptional activity as a monomer but forms a homodimer through the BAM domain. ChIP-sequencing analysis, and yeast one-hybrid and dual-luciferase assays demonstrated that the protein binds to the promoters of AP2/EREBP genes (Zm00001d010676 and Zm00001d032077) and inhibits their transcription. cDNA library screening showed that ZmBES1/BZR1-5 interacts with casein kinase II subunit ß4 (ZmCKIIß4) and ferredoxin 2 (ZmFdx2) in vitro and in vivo, respectively. Taken together, our study suggests that ZmBES1/BZR1-5 positively regulates kernel size, and provides new insights into understanding the mechanisms of kernel development in maize.
Assuntos
Proteínas de Plantas/metabolismo , Sementes/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Zea mays/genética , Brassinosteroides , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Fatores de Transcrição/genética , Zea mays/metabolismoRESUMO
Creating true-breeding lines is a critical step in plant breeding. Novel, completely homozygous true-breeding lines can be generated by doubled haploid technology in single generation. Haploid induction through modification of the centromere-specific histone 3 variant (CENH3), including chimeric proteins, expression of non-native CENH3 and single amino acid substitutions, has been shown to induce, on outcrossing to wild type, haploid progeny possessing only the genome of the wild-type parent, in Arabidopsis thaliana. Here, we report the characterization of 31 additional EMS-inducible amino acid substitutions in CENH3 for their ability to complement a knockout in the endogenous CENH3 gene and induce haploid progeny when pollinated by the wild type. We also tested the effect of double amino acid changes, which might be generated through a second round of EMS mutagenesis. Finally, we report on the effects of CRISPR/Cas9-mediated in-frame deletions in the αN helix of the CENH3 histone fold domain. Remarkably, we found that complete deletion of the αN helix, which is conserved throughout angiosperms, results in plants which exhibit normal growth and fertility while acting as excellent haploid inducers when pollinated by wild-type pollen. Both of these technologies, CRISPR mutagenesis and EMS mutagenesis, represent non-transgenic approaches to the generation of haploid inducers.
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A polyubiquitin chain anchored to the substrate has been the hallmark of proteasomal recognition. However, the degradation signal appears to be more complex and to contain also a substrate's unstructured region. Recent reports have shown that the proteasome can degrade also monoubiquitylated proteins, which adds an additional layer of complexity to the signal. Here, we demonstrate that the size of the substrate is an important determinant in its extent of ubiquitylation: a single ubiquitin moiety fused to a tail of up to â¼150 residues derived from either short artificial repeats or from naturally occurring proteins, is sufficient to target them for proteasomal degradation. Importantly, chemically synthesized adducts, where ubiquitin is attached to the substrate via a naturally occurring isopeptide bond, display similar characteristics. Taken together, these findings suggest that the ubiquitin proteasomal signal is adaptive, and is not always made of a long polyubiquitin chain.
Assuntos
Poliubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitinação/fisiologia , Sequência de Aminoácidos , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de Aminoácidos , Especificidade por Substrato , Ubiquitina/metabolismoRESUMO
Strigolactones (SLs), a newly discovered class of carotenoid-derived phytohormones, are essential for developmental processes that shape plant architecture and interactions with parasitic weeds and symbiotic arbuscular mycorrhizal fungi. Despite the rapid progress in elucidating the SL biosynthetic pathway, the perception and signalling mechanisms of SL remain poorly understood. Here we show that DWARF 53 (D53) acts as a repressor of SL signalling and that SLs induce its degradation. We find that the rice (Oryza sativa) d53 mutant, which produces an exaggerated number of tillers compared to wild-type plants, is caused by a gain-of-function mutation and is insensitive to exogenous SL treatment. The D53 gene product shares predicted features with the class I Clp ATPase proteins and can form a complex with the α/ß hydrolase protein DWARF 14 (D14) and the F-box protein DWARF 3 (D3), two previously identified signalling components potentially responsible for SL perception. We demonstrate that, in a D14- and D3-dependent manner, SLs induce D53 degradation by the proteasome and abrogate its activity in promoting axillary bud outgrowth. Our combined genetic and biochemical data reveal that D53 acts as a repressor of the SL signalling pathway, whose hormone-induced degradation represents a key molecular link between SL perception and responses.
Assuntos
Lactonas/metabolismo , Oryza/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/metabolismo , Proteólise , Proteínas Ligases SKP Culina F-Box/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutação/genética , Oryza/genética , Fenótipo , Proteínas de Plantas/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação ProteicaRESUMO
Plants utilize the ubiquitin proteasome system (UPS) to orchestrate numerous essential cellular processes, including the rapid responses required to cope with abiotic and biotic stresses. The 26S proteasome serves as the central catalytic component of the UPS that allows for the proteolytic degradation of ubiquitin-conjugated proteins in a highly specific manner. Despite the increasing number of studies employing cell-free degradation assays to dissect the pathways and target substrates of the UPS, the precise extraction methods of highly potent tissues remain unexplored. Here, we utilize a fluorogenic reporting assay using two extraction methods to survey proteasomal activity in different Arabidopsis thaliana tissues. This study provides new insights into the enrichment of activity and varied presence of proteasomes in specific plant tissues.