Predicting free energy changes(DDG)is essential for enhancing our understanding of protein evolution and plays a pivotal role in protein engineering and pharmaceutical development.While traditional methods offer valua...Predicting free energy changes(DDG)is essential for enhancing our understanding of protein evolution and plays a pivotal role in protein engineering and pharmaceutical development.While traditional methods offer valuable insights,they are often constrained by computational speed and reliance on biased training datasets.These constraints become particularly evident when aiming for accurate DDG predictions across a diverse array of protein sequences.Herein,we introduce Pythia,a self-supervised graph neural network specifically designed for zero-shot DDG predictions.Our comparative benchmarks demonstrate that Pythia outperforms other self-supervised pretraining models and force field-based approaches while also exhibiting competitive performance with fully supervised models.Notably,Pythia shows strong correlations and achieves a remarkable increase in computational speed of up to 105-fold.We further validated Pythia’s performance in predicting the thermostabilizing mutations of limonene epoxide hydrolase,leading to higher experimental success rates.This exceptional efficiency has enabled us to explore 26 million high-quality protein structures,marking a significant advancement in our ability to navigate the protein sequence space and enhance our understanding of the relationships between protein genotype and phenotype.In addition,we established a web server at https://pythia.wulab.xyz to allow users to easily perform such predictions.展开更多
Plants have evolved a large number of transcription factors（TF）, which are enriched among duplicate genes,highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large pla...Plants have evolved a large number of transcription factors（TF）, which are enriched among duplicate genes,highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large plant TF family and participate in development and stress responses. To probe the conservation and divergence of AP2/EREBP genes,we analyzed the duplication patterns of this family in Brassicaceae and identified interacting proteins of representative Arabidopsis AP2/EREBP proteins. We found that many AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost, but many others were retained in all tested Brassicaceae species, suggesting early functional divergence followed by persistent conservation. In addition,the sequences of the AP2 domain and exon numbers were highly conserved in rosids. Furthermore, we used 16 A.thaliana AP2/EREBP proteins as baits in yeast screens and identified 1,970 potential AP2/EREBP-interacting proteins,with a small subset of interactions verified in planta. Many AP2 genes also exhibit reduced expression in an antherdefective mutant, providing a possible link to developmental regulation. The putative AP2-interacting proteins participate in many functions in development and stress responses,including photomorphogenesis, flower development, pathogenesis, drought and cold responses, abscisic acid and auxin signaling. Our results present the AP2/EREBP evolution patterns in Brassicaceae, and support a proposed interaction network of AP2/EREBP proteins and their putative interacting proteins for further study.展开更多
Abscisic acid(ABA)-based chemically induced proximity(CIP)is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1(PYL1)and the 2C-type protein phosphatase ABI1,which confers ABA-ind...Abscisic acid(ABA)-based chemically induced proximity(CIP)is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1(PYL1)and the 2C-type protein phosphatase ABI1,which confers ABA-induced proximity to their fusion proteins,and offers precise temporal control of a wide array of biological processes.However,broad application of ABA-based CIP has been limited by ABA response intensity.In this study,we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1(PYR1)and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells.We engineered PYR1-ABI1and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/d Cas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells.We further achieved ABA-induced RNA m6A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/d Cas13,successfully inhibiting tumor cell proliferation.We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution(PACE),successfully generating a PYR1 mutant(PYR1m)whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type.In addition,we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type.These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE,providing a new approach for the modification of other CIP systems.展开更多
High temperature requirement A1 （HtrA1） belongs to an ancient protein family that is linked to various human disorders. The pre- cise role of exon 1-encoded N-terminal domains and how these influence the biological ...High temperature requirement A1 （HtrA1） belongs to an ancient protein family that is linked to various human disorders. The pre- cise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrAZ remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is impticated in unfotded protein response. ｜n highly secre- tory cells of the retinal pigmented epithelia, endoplasmic reticulum （ER） stress upregulated HtrA1. HtrA1 co-localized with vimen- tin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions dis- played reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mam- malian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis.展开更多
Macromolecular assemblies such as protein complexes and protein/RNA condensates are involved in most fundamental cellular processes.The arrangement of subunits within these nano-assemblies is critical for their biolog...Macromolecular assemblies such as protein complexes and protein/RNA condensates are involved in most fundamental cellular processes.The arrangement of subunits within these nano-assemblies is critical for their biological function and is determined by the topology of physical contacts within and between the subunits forming the complex.Describing the spatial arrangement of these interactions is of central importance to understand their functional and stability consequences.In this concept article,we propose a circuit topology-based formalism to define the topology of a complex consisting of linear polymeric chains with interand intrachain interactions.We apply our method to a system of model polymer chains as well as protein assemblies.We show that circuit topology can categorize different forms of chain assemblies.Our multi-chain circuit topology should aid analysis and predictions of mechanistic and evolutionary principles in the design of macromolecular assemblies.展开更多
基金supported by the National Key R&D Program of China(grant no.2023YFA0916000)the National Natural Science Foundation of China(32225002,32170033,and 32422001)+2 种基金the Key Research Program of Frontier Sciences(ZDBS-LYSM014)the Biological Resources Program(KFJ-BRP-009 and KFJ-BRP-017-58)from the Chinese Academy of Sciences,the Informatization Plan of Chinese Academy of Sciences(CAS-WX2021SF-0111)the Youth Innovation Promotion Association CAS(2022086).
文摘Predicting free energy changes(DDG)is essential for enhancing our understanding of protein evolution and plays a pivotal role in protein engineering and pharmaceutical development.While traditional methods offer valuable insights,they are often constrained by computational speed and reliance on biased training datasets.These constraints become particularly evident when aiming for accurate DDG predictions across a diverse array of protein sequences.Herein,we introduce Pythia,a self-supervised graph neural network specifically designed for zero-shot DDG predictions.Our comparative benchmarks demonstrate that Pythia outperforms other self-supervised pretraining models and force field-based approaches while also exhibiting competitive performance with fully supervised models.Notably,Pythia shows strong correlations and achieves a remarkable increase in computational speed of up to 105-fold.We further validated Pythia’s performance in predicting the thermostabilizing mutations of limonene epoxide hydrolase,leading to higher experimental success rates.This exceptional efficiency has enabled us to explore 26 million high-quality protein structures,marking a significant advancement in our ability to navigate the protein sequence space and enhance our understanding of the relationships between protein genotype and phenotype.In addition,we established a web server at https://pythia.wulab.xyz to allow users to easily perform such predictions.
基金financial support from the National Natural Science Foundation of China (91131007)funds from Fudan University
文摘Plants have evolved a large number of transcription factors（TF）, which are enriched among duplicate genes,highlighting their roles in complex regulatory networks. The APETALA2/EREBP-like genes constitute a large plant TF family and participate in development and stress responses. To probe the conservation and divergence of AP2/EREBP genes,we analyzed the duplication patterns of this family in Brassicaceae and identified interacting proteins of representative Arabidopsis AP2/EREBP proteins. We found that many AP2/EREBP duplicates generated early in Brassicaceae history were quickly lost, but many others were retained in all tested Brassicaceae species, suggesting early functional divergence followed by persistent conservation. In addition,the sequences of the AP2 domain and exon numbers were highly conserved in rosids. Furthermore, we used 16 A.thaliana AP2/EREBP proteins as baits in yeast screens and identified 1,970 potential AP2/EREBP-interacting proteins,with a small subset of interactions verified in planta. Many AP2 genes also exhibit reduced expression in an antherdefective mutant, providing a possible link to developmental regulation. The putative AP2-interacting proteins participate in many functions in development and stress responses,including photomorphogenesis, flower development, pathogenesis, drought and cold responses, abscisic acid and auxin signaling. Our results present the AP2/EREBP evolution patterns in Brassicaceae, and support a proposed interaction network of AP2/EREBP proteins and their putative interacting proteins for further study.
基金supported by grants from China Postdoctoral Science Foundation (2022TQ0120,2022M721320)the Science and Technology Research Project of the Education Department of Jilin Province,China (JJKH20221036KJ)+1 种基金the Open Project of State Key Laboratory of Supramolecular Structure and Materials (sklssm2023033)Natural Science Foundation of Jilin Province (20240305059YY)。
文摘Abscisic acid(ABA)-based chemically induced proximity(CIP)is primarily mediated by the interaction of the ABA receptor pyrabactin resistance 1-like 1(PYL1)and the 2C-type protein phosphatase ABI1,which confers ABA-induced proximity to their fusion proteins,and offers precise temporal control of a wide array of biological processes.However,broad application of ABA-based CIP has been limited by ABA response intensity.In this study,we demonstrated that ABA-induced interaction between another ABA receptor pyrabactin resistance 1(PYR1)and ABI1 exhibited higher ABA response intensity than that between PYL1 and ABI1 in HEK293T cells.We engineered PYR1-ABI1and PYL1-ABI1 into ABA-induced transcriptional activation tools in mammalian cells by integration with CRISPR/d Cas9 and found that the tool based on PYR1-ABI1 demonstrated better ABA response intensity than that based on PYL1-ABI1 for both exogenous and endogenous genes in mammalian cells.We further achieved ABA-induced RNA m6A modification installation and erasure by combining ABA-induced PYR1-ABI1 interaction with CRISPR/d Cas13,successfully inhibiting tumor cell proliferation.We subsequently improved the interaction of PYR1-ABI1 through phage-assisted continuous evolution(PACE),successfully generating a PYR1 mutant(PYR1m)whose interaction with ABI1 exhibited a higher ABA response intensity than that of the wild-type.In addition,we tested the transcriptional activation tool based on PYRm-ABI1 and found that it also showed a higher ABA response intensity than that of the wild type.These results demonstrate that we have developed a novel ABA-based CIP and further improved upon it using PACE,providing a new approach for the modification of other CIP systems.
文摘High temperature requirement A1 （HtrA1） belongs to an ancient protein family that is linked to various human disorders. The pre- cise role of exon 1-encoded N-terminal domains and how these influence the biological functions of human HtrAZ remain elusive. In this study, we traced the evolutionary origins of these N-terminal domains to a single gene fusion event in the most recent common ancestor of vertebrates. We hypothesized that human HtrA1 is impticated in unfotded protein response. ｜n highly secre- tory cells of the retinal pigmented epithelia, endoplasmic reticulum （ER） stress upregulated HtrA1. HtrA1 co-localized with vimen- tin intermediate filaments in highly arborized fashion. Upon ER stress, HtrA1 tracked along intermediate filaments, which collapsed and bundled in an aggresome at the microtubule organizing center. Gene silencing of HtrA1 altered the schedule and amplitude of adaptive signaling and concomitantly resulted in apoptosis. Restoration of wild-type HtrA1, but not its protease inactive mutant, was necessary and sufficient to protect from apoptosis. A variant of HtrA1 that harbored exon 1 substitutions dis- played reduced efficacy in rescuing cells from proteotoxicity. Our results illuminate the integration of HtrA1 in the toolkit of mam- malian cells against protein misfolding and the implications of defects in HtrA1 in proteostasis.
文摘Macromolecular assemblies such as protein complexes and protein/RNA condensates are involved in most fundamental cellular processes.The arrangement of subunits within these nano-assemblies is critical for their biological function and is determined by the topology of physical contacts within and between the subunits forming the complex.Describing the spatial arrangement of these interactions is of central importance to understand their functional and stability consequences.In this concept article,we propose a circuit topology-based formalism to define the topology of a complex consisting of linear polymeric chains with interand intrachain interactions.We apply our method to a system of model polymer chains as well as protein assemblies.We show that circuit topology can categorize different forms of chain assemblies.Our multi-chain circuit topology should aid analysis and predictions of mechanistic and evolutionary principles in the design of macromolecular assemblies.
