Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566605
Joseph Del Rosario, Stefano Coletta, Soon Ho Kim, Zach Mobille, Kayla Peelman, Brice Williams, Alan J. Otsuki, Alejandra Del Castillo Valerio, Kendell Worden, Lou T Blanpain, Lyndah Lovell, Hannah Choi, Bilal Haider
Lateral inhibition is a central principle for sensory system function. It is thought to operate by the activation of inhibitory neurons that restrict the spatial spread of sensory excitation. Much work on the role of inhibition in sensory systems has focused on visual cortex; however, the neurons, computations, and mechanisms underlying cortical lateral inhibition remain debated, and its importance for visual perception remains unknown. Here, we tested how lateral inhibition from PV or SST neurons in mouse primary visual cortex (V1) modulates neural and perceptual sensitivity to stimulus contrast. Lateral inhibition from PV neurons reduced neural and perceptual sensitivity to visual contrast in a uniform subtractive manner, whereas lateral inhibition from SST neurons more effectively changed the slope (or gain) of neural and perceptual contrast sensitivity. A neural circuit model identified spatially extensive lateral projections from SST neurons as the key factor, and we confirmed this with direct subthreshold measurements of a larger spatial footprint for SST versus PV lateral inhibition. Together, these results define cell-type specific computational roles for lateral inhibition in V1, and establish their unique consequences on sensitivity to contrast, a fundamental aspect of the visual world.
{"title":"Lateral inhibition in V1 controls neural & perceptual contrast sensitivity","authors":"Joseph Del Rosario, Stefano Coletta, Soon Ho Kim, Zach Mobille, Kayla Peelman, Brice Williams, Alan J. Otsuki, Alejandra Del Castillo Valerio, Kendell Worden, Lou T Blanpain, Lyndah Lovell, Hannah Choi, Bilal Haider","doi":"10.1101/2023.11.10.566605","DOIUrl":"https://doi.org/10.1101/2023.11.10.566605","url":null,"abstract":"Lateral inhibition is a central principle for sensory system function. It is thought to operate by the activation of inhibitory neurons that restrict the spatial spread of sensory excitation. Much work on the role of inhibition in sensory systems has focused on visual cortex; however, the neurons, computations, and mechanisms underlying cortical lateral inhibition remain debated, and its importance for visual perception remains unknown. Here, we tested how lateral inhibition from PV or SST neurons in mouse primary visual cortex (V1) modulates neural and perceptual sensitivity to stimulus contrast. Lateral inhibition from PV neurons reduced neural and perceptual sensitivity to visual contrast in a uniform subtractive manner, whereas lateral inhibition from SST neurons more effectively changed the slope (or gain) of neural and perceptual contrast sensitivity. A neural circuit model identified spatially extensive lateral projections from SST neurons as the key factor, and we confirmed this with direct subthreshold measurements of a larger spatial footprint for SST versus PV lateral inhibition. Together, these results define cell-type specific computational roles for lateral inhibition in V1, and establish their unique consequences on sensitivity to contrast, a fundamental aspect of the visual world.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"35 2","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992669","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566542
Sofie Skarup Kristensen, Henrik Jorntell
In cortical sensory processing, internal activity substantially impact the cortical response to any given input. But controlling for that internal activity is difficult since the thalamocortical network is high-dimensional, perpetually active and its state can change at a high pace. Here we report on a type of spontaneous local field potential sharp wave (LFP-SPW) in the somatosensory cortex (S1) and explore its impact on spiking responses evoked in S1 neurons by tactile stimulation. LFP-SPWs that preceded the tactile input could have major impacts, depressing the tactile responses in some neurons and enhancing them in others. In spontaneous data, in contrast, LFP-SPW events triggered short-lasting but massive neuronal activation in all neurons recorded with a subset of neurons initiating their activation up to 20 ms before the LFP-SPW onset. LFP-SPWs often coincided with ECoG-SPWs recorded at the cortical surface 2 mm away from the patch electrode, suggesting that the LFP-SPWs could be part of a more global cortical signal with a variable extent. When LFP-SPWs and ECoG-SPWs coincided, the impact of the LFP-SPW on the neuronal tactile response could change substantially, including inverting the impact to its opposite. Such diversified impacts of the SPWs on different S1 neurons are well in line with previous observations of cortical neurons displaying diverse, complementary, response types to given sensory inputs. These cortical SPWs had similar overall activity patterns as reported for hippocampal SPWs and may be a marker for a particular type of state change that possibly involves both hippocampus and neocortex.
{"title":"Local field potential sharp waves with diversified impact on cortical neuronal encoding of haptic input","authors":"Sofie Skarup Kristensen, Henrik Jorntell","doi":"10.1101/2023.11.10.566542","DOIUrl":"https://doi.org/10.1101/2023.11.10.566542","url":null,"abstract":"In cortical sensory processing, internal activity substantially impact the cortical response to any given input. But controlling for that internal activity is difficult since the thalamocortical network is high-dimensional, perpetually active and its state can change at a high pace. Here we report on a type of spontaneous local field potential sharp wave (LFP-SPW) in the somatosensory cortex (S1) and explore its impact on spiking responses evoked in S1 neurons by tactile stimulation. LFP-SPWs that preceded the tactile input could have major impacts, depressing the tactile responses in some neurons and enhancing them in others. In spontaneous data, in contrast, LFP-SPW events triggered short-lasting but massive neuronal activation in all neurons recorded with a subset of neurons initiating their activation up to 20 ms before the LFP-SPW onset. LFP-SPWs often coincided with ECoG-SPWs recorded at the cortical surface 2 mm away from the patch electrode, suggesting that the LFP-SPWs could be part of a more global cortical signal with a variable extent. When LFP-SPWs and ECoG-SPWs coincided, the impact of the LFP-SPW on the neuronal tactile response could change substantially, including inverting the impact to its opposite. Such diversified impacts of the SPWs on different S1 neurons are well in line with previous observations of cortical neurons displaying diverse, complementary, response types to given sensory inputs. These cortical SPWs had similar overall activity patterns as reported for hippocampal SPWs and may be a marker for a particular type of state change that possibly involves both hippocampus and neocortex.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"34 22","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992673","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.12.566724
Ruth Jean Ae Kim, De Fan, Jiangman He, Keunhwa Kim, Juan Du, Meng Chen
Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear photobodies (PBs). However, the function of PBs remains frustratingly elusive. Here, we show that PHYB condensation enables the co-occurrence and competition of two antagonistic phase-separated signaling actions. We found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR5 (PIF5) to PBs and, surprisingly, that PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Our results support a model in which PHYB condensation stabilizes PIF5 in PBs to counteract PIF5 degradation in the surrounding nucleoplasm, thereby enabling an environmentally sensitive counterbalancing mechanism to titrate nucleoplasmic PIF5 and its transcriptional output. This PB-enabled signaling mechanism provides a framework for regulating a plethora of PHYB-interacting signaling molecules in diverse plant environmental responses. We propose that this function of PBs represents a general function of biomolecular condensates to allow distinct variations of a cellular process or signaling pathway to coexist and interact to generate dynamically adjustable integrated outputs within a single subcellular space.
{"title":"Photobodies enable the phase-separation and counterbalance of phytochrome B mediated PIF5 degradation and stabilization","authors":"Ruth Jean Ae Kim, De Fan, Jiangman He, Keunhwa Kim, Juan Du, Meng Chen","doi":"10.1101/2023.11.12.566724","DOIUrl":"https://doi.org/10.1101/2023.11.12.566724","url":null,"abstract":"Photoactivation of the plant photoreceptor and thermosensor phytochrome B (PHYB) triggers its condensation into subnuclear photobodies (PBs). However, the function of PBs remains frustratingly elusive. Here, we show that PHYB condensation enables the co-occurrence and competition of two antagonistic phase-separated signaling actions. We found that PHYB recruits PHYTOCHROME-INTERACTING FACTOR5 (PIF5) to PBs and, surprisingly, that PHYB exerts opposing roles in degrading and stabilizing PIF5. Perturbing PB size by overproducing PHYB provoked a biphasic PIF5 response: while a moderate increase in PHYB enhanced PIF5 degradation, further elevating the PHYB level stabilized PIF5 by retaining more of it in enlarged PBs. Our results support a model in which PHYB condensation stabilizes PIF5 in PBs to counteract PIF5 degradation in the surrounding nucleoplasm, thereby enabling an environmentally sensitive counterbalancing mechanism to titrate nucleoplasmic PIF5 and its transcriptional output. This PB-enabled signaling mechanism provides a framework for regulating a plethora of PHYB-interacting signaling molecules in diverse plant environmental responses. We propose that this function of PBs represents a general function of biomolecular condensates to allow distinct variations of a cellular process or signaling pathway to coexist and interact to generate dynamically adjustable integrated outputs within a single subcellular space.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"33 14","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134993578","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.10.566580
Naoshige Uchida, Sandra Romero Pinto
A hallmark of various psychiatric disorders is biased future predictions. Here we examined the mechanisms for biased value learning using reinforcement learning models incorporating recent findings on synaptic plasticity and opponent circuit mechanisms in the basal ganglia. We show that variations in tonic dopamine can alter the balance between learning from positive and negative reward prediction errors, leading to biased value predictions. This bias arises from the sigmoidal shapes of the dose-occupancy curves and distinct affinities of D1- and D2-type dopamine receptors: changes in tonic dopamine differentially alters the slope of the dose-occupancy curves of these receptors, thus sensitivities, at baseline dopamine concentrations. We show that this mechanism can explain biased value learning in both mice and humans and may also contribute to symptoms observed in psychiatric disorders. Our model provides a foundation for understanding the basal ganglia circuit and underscores the significance of tonic dopamine in modulating learning processes.
{"title":"Tonic dopamine and biases in value learning linked through a biologically inspired reinforcement learning model","authors":"Naoshige Uchida, Sandra Romero Pinto","doi":"10.1101/2023.11.10.566580","DOIUrl":"https://doi.org/10.1101/2023.11.10.566580","url":null,"abstract":"A hallmark of various psychiatric disorders is biased future predictions. Here we examined the mechanisms for biased value learning using reinforcement learning models incorporating recent findings on synaptic plasticity and opponent circuit mechanisms in the basal ganglia. We show that variations in tonic dopamine can alter the balance between learning from positive and negative reward prediction errors, leading to biased value predictions. This bias arises from the sigmoidal shapes of the dose-occupancy curves and distinct affinities of D1- and D2-type dopamine receptors: changes in tonic dopamine differentially alters the slope of the dose-occupancy curves of these receptors, thus sensitivities, at baseline dopamine concentrations. We show that this mechanism can explain biased value learning in both mice and humans and may also contribute to symptoms observed in psychiatric disorders. Our model provides a foundation for understanding the basal ganglia circuit and underscores the significance of tonic dopamine in modulating learning processes.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"22 9","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134953885","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.566837
Isabelle R Leo, Elena Kunold, Anastasia Audrey, Marianna Tampere, Jurgen Eirich, Rozbeh Jafari
Over the last decade, proteome-wide mapping of drug interactions has revealed that most targeted drugs bind to not only their intended targets, but additional proteins as well. However, the majority of these studies have focused on analyzing proteins as encoded by their genes, thus neglecting the fact that most proteins exist as dynamic populations of multiple proteoforms. Here, we addressed this problem by combining the use of thermal proteome profiling (TPP), a powerful method for proteome analysis, with proteoform detection to refine the target landscape of an approved drug, ibrutinib. We revealed that, in addition to known targets, ibrutinib exhibits an intricate network of interactions involving multiple different proteoforms. Notably, we discovered affinity for specific proteoforms that link ibrutinib to mechanisms in immunomodulation and cellular processes like Golgi trafficking, endosomal trafficking, and glycosylation. These insights provide a framework for interpreting clinically observed off-target and adverse events. More generally, our findings highlight the importance of proteoform-level deconvolution in understanding drug interactions and their functional impacts, and offer a critical perspective for drug mechanism studies and potential applications in precision medicine.
{"title":"Proteoform-level deconvolution reveals a broader spectrum of ibrutinib off-targets","authors":"Isabelle R Leo, Elena Kunold, Anastasia Audrey, Marianna Tampere, Jurgen Eirich, Rozbeh Jafari","doi":"10.1101/2023.11.14.566837","DOIUrl":"https://doi.org/10.1101/2023.11.14.566837","url":null,"abstract":"Over the last decade, proteome-wide mapping of drug interactions has revealed that most targeted drugs bind to not only their intended targets, but additional proteins as well. However, the majority of these studies have focused on analyzing proteins as encoded by their genes, thus neglecting the fact that most proteins exist as dynamic populations of multiple proteoforms. Here, we addressed this problem by combining the use of thermal proteome profiling (TPP), a powerful method for proteome analysis, with proteoform detection to refine the target landscape of an approved drug, ibrutinib. We revealed that, in addition to known targets, ibrutinib exhibits an intricate network of interactions involving multiple different proteoforms. Notably, we discovered affinity for specific proteoforms that link ibrutinib to mechanisms in immunomodulation and cellular processes like Golgi trafficking, endosomal trafficking, and glycosylation. These insights provide a framework for interpreting clinically observed off-target and adverse events. More generally, our findings highlight the importance of proteoform-level deconvolution in understanding drug interactions and their functional impacts, and offer a critical perspective for drug mechanism studies and potential applications in precision medicine.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"46 8","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991738","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.567096
Lev M.Z. Tsypin, Scott H Saunders, Allen W Chen, Dianne K. Newman
The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.
细菌通过分泌代谢物进行细胞外电子转移的能力在自然、临床和工业环境中广泛存在。最近，我们发现了苯那嗪-1-羧酸(PCA)的生物氧化，这是自然产生的细胞外电子穿梭生物再生的第一个例子。然而，目前尚不清楚PCA氧化是如何催化的。本文通过对土壤分离物葡酸柠檬酸杆菌(Citrobacter portucalensis MBL)的支链电子传递链(ETC)进行遗传干扰，揭示了这一机制。生物PCA氧化与厌氧呼吸相结合，以硝酸盐、富马酸盐、二甲亚砜或三甲胺- n -氧化物作为终端电子受体。基因失活一个给定末端电子受体的所有冗余络合物的催化亚基可消除PCA氧化。在没有醌的情况下，PCA仍然可以向某些末端还原酶提供电子，尽管效率要低得多。在葡萄牙c.p orucalensis MBL中，PCA氧化主要由通过ETC的通量驱动，这表明任何具有可接近的细胞质膜的厌氧呼吸细菌都可能采用一种可推广的机制。这一模型得到了铜绿假单胞菌在硝酸盐呼吸过程中类似基因实验的支持。
{"title":"Genetically dissecting the electron transport chain of a soil bacterium reveals a generalizable mechanism for biological phenazine-1-carboxylic acid oxidation","authors":"Lev M.Z. Tsypin, Scott H Saunders, Allen W Chen, Dianne K. Newman","doi":"10.1101/2023.11.14.567096","DOIUrl":"https://doi.org/10.1101/2023.11.14.567096","url":null,"abstract":"The capacity for bacterial extracellular electron transfer via secreted metabolites is widespread in natural, clinical, and industrial environments. Recently, we discovered biological oxidation of phenazine-1-carboxylic acid (PCA), the first example of biological regeneration of a naturally produced extracellular electron shuttle. However, it remained unclear how PCA oxidation was catalyzed. Here, we report the mechanism, which we uncovered by genetically perturbing the branched electron transport chain (ETC) of the soil isolate Citrobacter portucalensis MBL. Biological PCA oxidation is coupled to anaerobic respiration with nitrate, fumarate, dimethyl sulfoxide, or trimethylamine-N-oxide as terminal electron acceptors. Genetically inactivating the catalytic subunits for all redundant complexes for a given terminal electron acceptor abolishes PCA oxidation. In the absence of quinones, PCA can still donate electrons to certain terminal reductases, albeit much less efficiently. In C. portucalensis MBL, PCA oxidation is largely driven by flux through the ETC, which suggests a generalizable mechanism that may be employed by any anaerobically respiring bacterium with an accessible cytoplasmic membrane. This model is supported by analogous genetic experiments during nitrate respiration by Pseudomonas aeruginosa.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"46 21","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991893","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.14.566657
Masayoshi Honda, Mortezaali Razzaghi, Paras Gaur, Eva Malacaria, Ludovica Di Biagi, Francesca Antonella Aiello, Emeleeta A Paintsil, Andrew Stanfield, Bailey J Deppe, Lokesh Gakhar, Nicholas J Schnicker, Michael Ashley Spies, Pietro Pichierri, Maria Spies
Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation. In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL1. The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.
{"title":"A double-ring of human RAD52 remodels replication forks restricting fork reversal","authors":"Masayoshi Honda, Mortezaali Razzaghi, Paras Gaur, Eva Malacaria, Ludovica Di Biagi, Francesca Antonella Aiello, Emeleeta A Paintsil, Andrew Stanfield, Bailey J Deppe, Lokesh Gakhar, Nicholas J Schnicker, Michael Ashley Spies, Pietro Pichierri, Maria Spies","doi":"10.1101/2023.11.14.566657","DOIUrl":"https://doi.org/10.1101/2023.11.14.566657","url":null,"abstract":"Human RAD52 is a multifunctional DNA repair protein involved in several cellular events that support genome stability including protection of stalled DNA replication forks from excessive degradation. In its gatekeeper role, RAD52 binds to and stabilizes stalled replication forks during replication stress protecting them from reversal by SMARCAL1. The structural and molecular mechanism of the RAD52-mediated fork protection remains elusive. Here, using P1 nuclease sensitivity, biochemical and single-molecule analyses we show that RAD52 dynamically remodels replication forks through its strand exchange activity. The presence of the ssDNA binding protein RPA at the fork modulates the kinetics of the strand exchange without impeding the reaction outcome. Mass photometry and single-particle cryo-electron microscopy show that the replication fork promotes a unique nucleoprotein structure containing head-to-head arrangement of two undecameric RAD52 rings with an extended positively charged surface that accommodates all three arms of the replication fork. We propose that the formation and continuity of this surface is important for the strand exchange reaction and for competition with SMARCAL1.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"46 16","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134991898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566922
Gordon Rix, Rory L. Williams, Hansen Spinner, Vincent J. Hu, Debora S. Marks, Chang C. Liu
When nature maintains or evolves a gene's function over millions of years at scale, it produces a diversity of homologous sequences whose patterns of conservation and change contain rich structural, functional, and historical information about the gene. However, natural gene diversity likely excludes vast regions of functional sequence space and includes phylogenetic and evolutionary eccentricities, limiting what information we can extract. We introduce an accessible experimental approach for compressing long-term gene evolution to laboratory timescales, allowing for the direct observation of extensive adaptation and divergence followed by inference of structural, functional, and environmental constraints for any selectable gene. To enable this approach, we developed a new orthogonal DNA replication (OrthoRep) system that durably hypermutates chosen genes at a rate of >10^-4 substitutions per base in vivo. When OrthoRep was used to evolve a conditionally essential maladapted enzyme, we obtained thousands of unique multi-mutation sequences with many pairs >60 amino acids apart (>15% divergence), revealing known and new factors influencing enzyme adaptation. The fitness of evolved sequences was not predictable by advanced machine learning models trained on natural variation. We suggest that OrthoRep supports the prospective and systematic discovery of constraints shaping gene evolution, uncovering of new regions in fitness landscapes, and general applications in biomolecular engineering.
{"title":"Continuous evolution of user-defined genes at 1-million-times the genomic mutation rate","authors":"Gordon Rix, Rory L. Williams, Hansen Spinner, Vincent J. Hu, Debora S. Marks, Chang C. Liu","doi":"10.1101/2023.11.13.566922","DOIUrl":"https://doi.org/10.1101/2023.11.13.566922","url":null,"abstract":"When nature maintains or evolves a gene's function over millions of years at scale, it produces a diversity of homologous sequences whose patterns of conservation and change contain rich structural, functional, and historical information about the gene. However, natural gene diversity likely excludes vast regions of functional sequence space and includes phylogenetic and evolutionary eccentricities, limiting what information we can extract. We introduce an accessible experimental approach for compressing long-term gene evolution to laboratory timescales, allowing for the direct observation of extensive adaptation and divergence followed by inference of structural, functional, and environmental constraints for any selectable gene. To enable this approach, we developed a new orthogonal DNA replication (OrthoRep) system that durably hypermutates chosen genes at a rate of >10^-4 substitutions per base in vivo. When OrthoRep was used to evolve a conditionally essential maladapted enzyme, we obtained thousands of unique multi-mutation sequences with many pairs >60 amino acids apart (>15% divergence), revealing known and new factors influencing enzyme adaptation. The fitness of evolved sequences was not predictable by advanced machine learning models trained on natural variation. We suggest that OrthoRep supports the prospective and systematic discovery of constraints shaping gene evolution, uncovering of new regions in fitness landscapes, and general applications in biomolecular engineering.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"48 2","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.11.566699
Kuo Hao Lee, Lei Shi
Effective rational drug discovery targeting a specific protein hinges on understanding their functional states and distinguishing it from homologs. However, for the G protein coupled receptors, both the activation-related conformational changes (ACCs) and the intrinsic divergence among receptors can be misled or obscured by ligand-induced conformational changes (LCCs). Here, we unraveled ACCs and intrinsic divergence from LCCs of the dopamine D3 and D2 receptors (D3R and D2R), by analyzing their experimentally determined structures and the molecular dynamics simulation results of the receptors bound with different ligands. In addition to the ACCs common to other aminergic receptors, we revealed unique ACCs for these two receptors including TM5e and TM6e shifting away from TM2e and TM3e, with a subtle rotation of TM5e. In identifying intrinsic divergence, we found pronounced outward tilting of TM6e in the D2R compared to the D3R in both experimental structures and simulations with ligands in different scaffolds. This tilting was drastically reduced in the simulations of the receptors bound with nonselective full agonist quinpirole, suggesting a misleading impact of LCCs. Further, in the quinpirole-bound simulations, TM1 showed a greater disparity between these receptors, indicating that LCCs may obscure intrinsic divergence. In addition, our analysis showed that the impact of the nonconserved TM1 propagated to conserved Trp7.40 and Glu2.65, both are ligand binding residues. We also found that the D2R exhibited heightened flexibility compared to the D3R in the extracellular portions of TMs 5, 6, and 7, potentially associated with its greater ligand binding site plasticity. Our results lay the groundwork for crafting ligands specifically targeting D2R or D3R with more precise pharmacological profiles.
{"title":"Unraveling activation-related rearrangements and intrinsic divergence from ligand-induced conformational changes of the dopamine D3 and D2 receptors","authors":"Kuo Hao Lee, Lei Shi","doi":"10.1101/2023.11.11.566699","DOIUrl":"https://doi.org/10.1101/2023.11.11.566699","url":null,"abstract":"Effective rational drug discovery targeting a specific protein hinges on understanding their functional states and distinguishing it from homologs. However, for the G protein coupled receptors, both the activation-related conformational changes (ACCs) and the intrinsic divergence among receptors can be misled or obscured by ligand-induced conformational changes (LCCs). Here, we unraveled ACCs and intrinsic divergence from LCCs of the dopamine D3 and D2 receptors (D3R and D2R), by analyzing their experimentally determined structures and the molecular dynamics simulation results of the receptors bound with different ligands. In addition to the ACCs common to other aminergic receptors, we revealed unique ACCs for these two receptors including TM5e and TM6e shifting away from TM2e and TM3e, with a subtle rotation of TM5e. In identifying intrinsic divergence, we found pronounced outward tilting of TM6e in the D2R compared to the D3R in both experimental structures and simulations with ligands in different scaffolds. This tilting was drastically reduced in the simulations of the receptors bound with nonselective full agonist quinpirole, suggesting a misleading impact of LCCs. Further, in the quinpirole-bound simulations, TM1 showed a greater disparity between these receptors, indicating that LCCs may obscure intrinsic divergence. In addition, our analysis showed that the impact of the nonconserved TM1 propagated to conserved Trp7.40 and Glu2.65, both are ligand binding residues. We also found that the D2R exhibited heightened flexibility compared to the D3R in the extracellular portions of TMs 5, 6, and 7, potentially associated with its greater ligand binding site plasticity. Our results lay the groundwork for crafting ligands specifically targeting D2R or D3R with more precise pharmacological profiles.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"44 14","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992319","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-14DOI: 10.1101/2023.11.13.566957
na wei, zhen bin liu, si yuan li, yuan Gao
Background: Myocardial ischemia-reperfusion injury (MIRI) can significantly aggravate myocardial injury in patients with ST-segment elevation myocardial infarction (STEMI). At present, there are few effective treatments for MIRI. The Shexiang Baoxin Pill (SBP) can reduce MIRI. The PI3K/Akt/eNOS signaling pathway, inflammation, oxidative stress, and apoptosis are all involved in the regulation of MIRI. SBP has multi-component, multi-target, and synergistic effects, but its mechanism of action on MIRI has not been reported. Purpose: We sought to explore whether SBP exerts a protective mechanism by inhibiting the inflammatory reaction, oxidative stress, and apoptosis, reducing MIRI through the PI3K/Akt/eNOS signal pathway. Materials and methods: Hypoxia-reoxygenation (H/R) H9c2 cardiomyocytes were used as an in vitro model of MIRI. The active components of Shexiang Baoxin pills were extracted with water. The levels of phosphorylated proteins and genes related to the PI3K/Akt/eNOS pathway were measured by Western blotting and real-time fluorescence quantitative PCR. Cell viability, apoptosis rates, and apoptosis-related proteins (Bcl-2, Bax, Caspase-3) were detected by CCK-8, flow cytometry, and Western blotting. The expression of reactive oxygen species (ROS), homocysteine (Hcy), malondialdehyde (MDA), and gp91phox was detected by fluorescence probe, ELISA, TBA, and Western blotting. The levels of inflammatory factors (TNF-α, IL-6, IL-18) were measured by an ELISA method. Results: SBP increased the cell survival rate of H/R cardiomyocytes, reduced the injury to H/R cardiomyocytes, and increased the protein phosphorylation levels of p-PI3KY607, p-AktSer473, p-eNOSSer1177, and mRNA of H/R cardiomyocytes. In addition, SBP increased the level of Bcl-2 protein and the Bcl-2/Bax ratio and decreased the apoptosis rate and Bax and Caspase-3 expression. It reduced the levels of oxidative stress indexes (ROS, HCY, MDA, and gp91phox) and inflammatory factors (TNF-γ, IL-6, IL-18) and enhanced antioxidant stress, anti-apoptosis, and an anti-inflammatory reaction. The above effects were attenuated after the inhibition of the PI3K/Akt/eNOS signal pathway. Conclusion: We established that SBP extract inhibited oxidative stress, inflammatory response, and apoptosis through the PI3K/Akt/eNOS signal pathway and alleviated the injury of H9c2 cells induced by hypoxia-reoxygenation.
{"title":"The Shexiang Baoxin Pill Protects Myocardial Cells from Multiple Targets of MIRI through the PI3K/Akt/eNOS Signal Pathway","authors":"na wei, zhen bin liu, si yuan li, yuan Gao","doi":"10.1101/2023.11.13.566957","DOIUrl":"https://doi.org/10.1101/2023.11.13.566957","url":null,"abstract":"Background: Myocardial ischemia-reperfusion injury (MIRI) can significantly aggravate myocardial injury in patients with ST-segment elevation myocardial infarction (STEMI). At present, there are few effective treatments for MIRI. The Shexiang Baoxin Pill (SBP) can reduce MIRI. The PI3K/Akt/eNOS signaling pathway, inflammation, oxidative stress, and apoptosis are all involved in the regulation of MIRI. SBP has multi-component, multi-target, and synergistic effects, but its mechanism of action on MIRI has not been reported. Purpose: We sought to explore whether SBP exerts a protective mechanism by inhibiting the inflammatory reaction, oxidative stress, and apoptosis, reducing MIRI through the PI3K/Akt/eNOS signal pathway. Materials and methods: Hypoxia-reoxygenation (H/R) H9c2 cardiomyocytes were used as an in vitro model of MIRI. The active components of Shexiang Baoxin pills were extracted with water. The levels of phosphorylated proteins and genes related to the PI3K/Akt/eNOS pathway were measured by Western blotting and real-time fluorescence quantitative PCR. Cell viability, apoptosis rates, and apoptosis-related proteins (Bcl-2, Bax, Caspase-3) were detected by CCK-8, flow cytometry, and Western blotting. The expression of reactive oxygen species (ROS), homocysteine (Hcy), malondialdehyde (MDA), and gp91phox was detected by fluorescence probe, ELISA, TBA, and Western blotting. The levels of inflammatory factors (TNF-α, IL-6, IL-18) were measured by an ELISA method. Results: SBP increased the cell survival rate of H/R cardiomyocytes, reduced the injury to H/R cardiomyocytes, and increased the protein phosphorylation levels of p-PI3KY607, p-AktSer473, p-eNOSSer1177, and mRNA of H/R cardiomyocytes. In addition, SBP increased the level of Bcl-2 protein and the Bcl-2/Bax ratio and decreased the apoptosis rate and Bax and Caspase-3 expression. It reduced the levels of oxidative stress indexes (ROS, HCY, MDA, and gp91phox) and inflammatory factors (TNF-γ, IL-6, IL-18) and enhanced antioxidant stress, anti-apoptosis, and an anti-inflammatory reaction. The above effects were attenuated after the inhibition of the PI3K/Akt/eNOS signal pathway. Conclusion: We established that SBP extract inhibited oxidative stress, inflammatory response, and apoptosis through the PI3K/Akt/eNOS signal pathway and alleviated the injury of H9c2 cells induced by hypoxia-reoxygenation.","PeriodicalId":486943,"journal":{"name":"bioRxiv (Cold Spring Harbor Laboratory)","volume":"24 4","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134992855","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
京公网安备 11010802042870号
京ICP备2023020795号-1
扫码关注我们
求助内容：
应助结果提醒方式：