Pub Date : 2021-03-12DOI: 10.1101/2021.03.12.434905
Rui Dong, Tural Aksel, Waipan Chan, R. Germain, R. Vale, Shawn M. Douglas
Significance It has been proposed that the spatial arrangement of ligands plays a key role in regulating downstream intracellular signals. Because of methodological limitations in precise ligand patterning, however, the relationship between spatial configuration of clusters and signaling dynamics remains poorly understood. By developing a DNA-based molecular “pegboard” for ligand patterning, we demonstrated that the nanometer arrangement of ligands plays significant roles in modulating signal transduction in T cells. Ligand clustering not only affects the triggering sensitivity but also determines the temporal dynamics of the intracellular signaling response. Our approach is highly translatable for studying various signaling pathways, and our results provide insights into biomolecular engineering for therapeutic uses. Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.
{"title":"DNA origami patterning of synthetic T cell receptors reveals spatial control of the sensitivity and kinetics of signal activation","authors":"Rui Dong, Tural Aksel, Waipan Chan, R. Germain, R. Vale, Shawn M. Douglas","doi":"10.1101/2021.03.12.434905","DOIUrl":"https://doi.org/10.1101/2021.03.12.434905","url":null,"abstract":"Significance It has been proposed that the spatial arrangement of ligands plays a key role in regulating downstream intracellular signals. Because of methodological limitations in precise ligand patterning, however, the relationship between spatial configuration of clusters and signaling dynamics remains poorly understood. By developing a DNA-based molecular “pegboard” for ligand patterning, we demonstrated that the nanometer arrangement of ligands plays significant roles in modulating signal transduction in T cells. Ligand clustering not only affects the triggering sensitivity but also determines the temporal dynamics of the intracellular signaling response. Our approach is highly translatable for studying various signaling pathways, and our results provide insights into biomolecular engineering for therapeutic uses. Receptor clustering plays a key role in triggering cellular activation, but the relationship between the spatial configuration of clusters and the elicitation of downstream intracellular signals remains poorly understood. We developed a DNA-origami–based system that is easily adaptable to other cellular systems and enables rich interrogation of responses to a variety of spatially defined inputs. Using a chimeric antigen receptor (CAR) T cell model system with relevance to cancer therapy, we studied signaling dynamics at single-cell resolution. We found that the spatial arrangement of receptors determines the ligand density threshold for triggering and encodes the temporal kinetics of signaling activities. We also showed that signaling sensitivity of a small cluster of high-affinity ligands is enhanced when surrounded by nonstimulating low-affinity ligands. Our results suggest that cells measure spatial arrangements of ligands, translate that information into distinct signaling dynamics, and provide insights into engineering immunotherapies.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"302 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82873998","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 : 2021-03-10DOI: 10.1101/2021.03.09.434618
Carley Snoznik, Valentina Medvedeva, J. Mojsilovic-Petrovic, Zenith D. Rudich, J. Oosten, R. Kalb, Todd Lamitina
Significance The G4C2 repeat expansion in the C9orf72 gene is a major cause of frontotemporal dementia and amyotrophic lateral sclerosis. Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins (DPRs), PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both Caenorhabditis elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through bromodomain proteins to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and “druggable” pathway that may represent a strategy for treating these currently incurable diseases. A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.
{"title":"The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity","authors":"Carley Snoznik, Valentina Medvedeva, J. Mojsilovic-Petrovic, Zenith D. Rudich, J. Oosten, R. Kalb, Todd Lamitina","doi":"10.1101/2021.03.09.434618","DOIUrl":"https://doi.org/10.1101/2021.03.09.434618","url":null,"abstract":"Significance The G4C2 repeat expansion in the C9orf72 gene is a major cause of frontotemporal dementia and amyotrophic lateral sclerosis. Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins (DPRs), PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both Caenorhabditis elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through bromodomain proteins to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and “druggable” pathway that may represent a strategy for treating these currently incurable diseases. A hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs PR50 and GR50 are highly toxic when expressed in Caenorhabditis elegans, and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNA interference (RNAi) screen for suppressors of PR50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by PR50 expression. All of these genes have vertebrate homologs, and 7 of 12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for GR50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that knockdown of bromodomain proteins in both C. elegans and mammalian neurons, which are known SPOP ubiquitination targets, suppresses the protective effect of SPOP inhibition. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent an entry point for therapeutic intervention to treat C9orf72 FTD/ALS.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"24 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85525784","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 : 2021-03-10DOI: 10.1101/2021.03.10.434820
C. Peters, Pin W. Liu, S. Morotti, Stephanie C. Gantz, E. Grandi, B. Bean, C. Proenza
Significance The funny current (If) is critical for spontaneous activity in cardiac pacemaker cells; however, its precise role remains enigmatic because it activates mostly outside the physiological voltage range and its kinetics are slow relative to the cardiac cycle. If is typically considered as an inward current; however, we show that If is persistently active in pacemaker cells. Once opened, the small fraction of ion channels that conduct If do not reclose. Consequently, If flows both inward and outward and, paradoxically, conducts a large fraction of the net charge movement. These results establish a new conceptual framework for pacemaking in which voltage-dependent gating of If is minimal and If contributes to spontaneous pacemaker activity by providing driving force in both directions. Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of −30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If. Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.
{"title":"Bidirectional flow of the funny current (If) during the pacemaking cycle in murine sinoatrial node myocytes","authors":"C. Peters, Pin W. Liu, S. Morotti, Stephanie C. Gantz, E. Grandi, B. Bean, C. Proenza","doi":"10.1101/2021.03.10.434820","DOIUrl":"https://doi.org/10.1101/2021.03.10.434820","url":null,"abstract":"Significance The funny current (If) is critical for spontaneous activity in cardiac pacemaker cells; however, its precise role remains enigmatic because it activates mostly outside the physiological voltage range and its kinetics are slow relative to the cardiac cycle. If is typically considered as an inward current; however, we show that If is persistently active in pacemaker cells. Once opened, the small fraction of ion channels that conduct If do not reclose. Consequently, If flows both inward and outward and, paradoxically, conducts a large fraction of the net charge movement. These results establish a new conceptual framework for pacemaking in which voltage-dependent gating of If is minimal and If contributes to spontaneous pacemaker activity by providing driving force in both directions. Sinoatrial node myocytes (SAMs) act as cardiac pacemaker cells by firing spontaneous action potentials (APs) that initiate each heartbeat. The funny current (If) is critical for the generation of these spontaneous APs; however, its precise role during the pacemaking cycle remains unresolved. Here, we used the AP-clamp technique to quantify If during the cardiac cycle in mouse SAMs. We found that If is persistently active throughout the sinoatrial AP, with surprisingly little voltage-dependent gating. As a consequence, it carries both inward and outward current around its reversal potential of −30 mV. Despite operating at only 2 to 5% of its maximal conductance, If carries a substantial fraction of both depolarizing and repolarizing net charge movement during the firing cycle. We also show that β-adrenergic receptor stimulation increases the percentage of net depolarizing charge moved by If, consistent with a contribution of If to the fight-or-flight increase in heart rate. These properties were confirmed by heterologously expressed HCN4 channels and by mathematical models of If. Modeling further suggested that the slow rates of activation and deactivation of the HCN4 isoform underlie the persistent activity of If during the sinoatrial AP. These results establish a new conceptual framework for the role of If in pacemaking, in which it operates at a very small fraction of maximal activation but nevertheless drives membrane potential oscillations in SAMs by providing substantial driving force in both inward and outward directions.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"30 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88759906","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}
Francis Mollica, Geoff Bacon, Noga Zaslavsky, Yang Xu, T. Regier, Charles Kemp
Significance Grammatical marking of features such as number, tense, and evidentiality varies widely across languages. Despite this variation, we show that grammatical markers support efficient information transfer from speakers to listeners. We apply a formal model of communication to data from dozens of languages and find that grammatical marking achieves a near-optimal balance between maximizing informativeness and minimizing code lengths. Our approach shows how general information-theoretic principles can capture variation in both form and meaning across languages. Functionalist accounts of language suggest that forms are paired with meanings in ways that support efficient communication. Previous work on grammatical marking suggests that word forms have lengths that enable efficient production, and work on the semantic typology of the lexicon suggests that word meanings represent efficient partitions of semantic space. Here we establish a theoretical link between these two lines of work and present an information-theoretic analysis that captures how communicative pressures influence both form and meaning. We apply our approach to the grammatical features of number, tense, and evidentiality and show that the approach explains both which systems of feature values are attested across languages and the relative lengths of the forms for those feature values. Our approach shows that general information-theoretic principles can capture variation in both form and meaning across languages.
{"title":"The forms and meanings of grammatical markers support efficient communication","authors":"Francis Mollica, Geoff Bacon, Noga Zaslavsky, Yang Xu, T. Regier, Charles Kemp","doi":"10.17605/OSF.IO/S5B7H","DOIUrl":"https://doi.org/10.17605/OSF.IO/S5B7H","url":null,"abstract":"Significance Grammatical marking of features such as number, tense, and evidentiality varies widely across languages. Despite this variation, we show that grammatical markers support efficient information transfer from speakers to listeners. We apply a formal model of communication to data from dozens of languages and find that grammatical marking achieves a near-optimal balance between maximizing informativeness and minimizing code lengths. Our approach shows how general information-theoretic principles can capture variation in both form and meaning across languages. Functionalist accounts of language suggest that forms are paired with meanings in ways that support efficient communication. Previous work on grammatical marking suggests that word forms have lengths that enable efficient production, and work on the semantic typology of the lexicon suggests that word meanings represent efficient partitions of semantic space. Here we establish a theoretical link between these two lines of work and present an information-theoretic analysis that captures how communicative pressures influence both form and meaning. We apply our approach to the grammatical features of number, tense, and evidentiality and show that the approach explains both which systems of feature values are attested across languages and the relative lengths of the forms for those feature values. Our approach shows that general information-theoretic principles can capture variation in both form and meaning across languages.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"71 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82559571","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 : 2021-03-08DOI: 10.1101/2021.03.08.434340
C. Cox, Yixiao Zhang, Zijing Zhou, T. Walz, B. Martinac
Significance Mechanosensitive (MS) channels play a key role in the physiology of organisms from bacteria to man. Many prokaryotic and eukaryotic MS channels respond to membrane tension. Here, we show that cyclodextrin (CD)–mediated lipid removal induces membrane tension that activates not only the mechanosensitive channel of small conductance but the structurally unrelated mechanosensitive channel of large conductance, which gates at almost lytic membrane tensions. This finding suggests that for both functional and structural studies, provided that sufficient CD is added and enough lipids are removed, any tension-sensitive ion channel can be activated. Moreover, CDs may also prove useful for the in vitro study of other membrane proteins that are sensitive to mechanical forces. The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.
{"title":"Cyclodextrins increase membrane tension and are universal activators of mechanosensitive channels","authors":"C. Cox, Yixiao Zhang, Zijing Zhou, T. Walz, B. Martinac","doi":"10.1101/2021.03.08.434340","DOIUrl":"https://doi.org/10.1101/2021.03.08.434340","url":null,"abstract":"Significance Mechanosensitive (MS) channels play a key role in the physiology of organisms from bacteria to man. Many prokaryotic and eukaryotic MS channels respond to membrane tension. Here, we show that cyclodextrin (CD)–mediated lipid removal induces membrane tension that activates not only the mechanosensitive channel of small conductance but the structurally unrelated mechanosensitive channel of large conductance, which gates at almost lytic membrane tensions. This finding suggests that for both functional and structural studies, provided that sufficient CD is added and enough lipids are removed, any tension-sensitive ion channel can be activated. Moreover, CDs may also prove useful for the in vitro study of other membrane proteins that are sensitive to mechanical forces. The bacterial mechanosensitive channel of small conductance (MscS) has been extensively studied to understand how mechanical forces are converted into the conformational changes that underlie mechanosensitive (MS) channel gating. We showed that lipid removal by β-cyclodextrin can mimic membrane tension. Here, we show that all cyclodextrins (CDs) can activate reconstituted Escherichia coli MscS, that MscS activation by CDs depends on CD-mediated lipid removal, and that the CD amount required to gate MscS scales with the channel’s sensitivity to membrane tension. Importantly, cholesterol-loaded CDs do not activate MscS. CD-mediated lipid removal ultimately causes MscS desensitization, which we show is affected by the lipid environment. While many MS channels respond to membrane forces, generalized by the “force-from-lipids” principle, their different molecular architectures suggest that they use unique ways to convert mechanical forces into conformational changes. To test whether CDs can also be used to activate other MS channels, we chose to investigate the mechanosensitive channel of large conductance (MscL) and demonstrate that CDs can also activate this structurally unrelated channel. Since CDs can open the least tension-sensitive MS channel, MscL, they should be able to open any MS channel that responds to membrane tension. Thus, CDs emerge as a universal tool for the structural and functional characterization of unrelated MS channels.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"79911602","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 : 2021-03-06DOI: 10.1101/2021.03.05.433980
Catarina F. Almeida, D. G. Smith, T. Cheng, Christopher M. Harpur, Elena Batleska, C. Nguyen-Robertson, Tram Nguyen, Tamara Thelemann, S. Reddiex, Shihan Li, S. Eckle, I. van Rhijn, J. Rossjohn, Adam P. Uldrich, D. Moody, Spencer J. Williams, D. Pellicci, D. Godfrey
Significance Whereas T cells are known to recognize peptides, vitamin B metabolites, or lipid antigens, we identify several nonlipidic small molecules classified as pentamethylbenzofuransulfonates (PBFs) that activate a population of CD1d-restricted natural killer T (NKT) cells. This represents a breakthrough in the field of NKT cell biology. This study also reveals a previously unknown population of PBF-reactive NKT cells in healthy individuals with stereotyped receptors that paves the way for future studies of the role of these cells in immunity, including sulfa drug hypersensitivity. Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.
{"title":"Benzofuran sulfonates and small self-lipid antigens activate type II NKT cells via CD1d","authors":"Catarina F. Almeida, D. G. Smith, T. Cheng, Christopher M. Harpur, Elena Batleska, C. Nguyen-Robertson, Tram Nguyen, Tamara Thelemann, S. Reddiex, Shihan Li, S. Eckle, I. van Rhijn, J. Rossjohn, Adam P. Uldrich, D. Moody, Spencer J. Williams, D. Pellicci, D. Godfrey","doi":"10.1101/2021.03.05.433980","DOIUrl":"https://doi.org/10.1101/2021.03.05.433980","url":null,"abstract":"Significance Whereas T cells are known to recognize peptides, vitamin B metabolites, or lipid antigens, we identify several nonlipidic small molecules classified as pentamethylbenzofuransulfonates (PBFs) that activate a population of CD1d-restricted natural killer T (NKT) cells. This represents a breakthrough in the field of NKT cell biology. This study also reveals a previously unknown population of PBF-reactive NKT cells in healthy individuals with stereotyped receptors that paves the way for future studies of the role of these cells in immunity, including sulfa drug hypersensitivity. Natural killer T (NKT) cells detect lipids presented by CD1d. Most studies focus on type I NKT cells that express semi-invariant αβ T cell receptors (TCR) and recognize α-galactosylceramides. However, CD1d also presents structurally distinct lipids to NKT cells expressing diverse TCRs (type II NKT cells), but our knowledge of the antigens for type II NKT cells is limited. An early study identified a nonlipidic NKT cell agonist, phenyl pentamethyldihydrobenzofuransulfonate (PPBF), which is notable for its similarity to common sulfa drugs, but its mechanism of NKT cell activation remained unknown. Here, we demonstrate that a range of pentamethylbenzofuransulfonates (PBFs), including PPBF, activate polyclonal type II NKT cells from human donors. Whereas these sulfa drug–like molecules might have acted pharmacologically on cells, here we demonstrate direct contact between TCRs and PBF-treated CD1d complexes. Further, PBF-treated CD1d tetramers identified type II NKT cell populations expressing αβTCRs and γδTCRs, including those with variable and joining region gene usage (TRAV12-1–TRAJ6) that was conserved across donors. By trapping a CD1d–type II NKT TCR complex for direct mass-spectrometric analysis, we detected molecules that allow the binding of CD1d to TCRs, finding that both selected PBF family members and short-chain sphingomyelin lipids are present in these complexes. Furthermore, the combination of PPBF and short-chain sphingomyelin enhances CD1d tetramer staining of PPBF-reactive T cell lines over either molecule alone. This study demonstrates that nonlipidic small molecules, which resemble sulfa drugs implicated in systemic hypersensitivity and drug allergy reactions, are targeted by a polyclonal population of type II NKT cells in a CD1d-restricted manner.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"9 2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83461964","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 : 2021-03-06DOI: 10.1101/2021.03.06.434200
A. Taguchi, J. Page, H. Tsui, M. Winkler, S. Walker
Significance Bacteria are protected from their surrounding environment by the peptidoglycan cell wall, which is a major target for antibiotics. Counterintuitively, cell wall assembly requires enzymes that cleave newly built peptidoglycan chains. Here, using nascent peptidoglycan we assembled in vitro, we characterized two membrane-bound glycosidases that are vital for proper cell division and elongation in Streptococcus pneumoniae. These enzymes were proposed to perform different chemical reactions. Instead, we show that they perform the same chemical reaction but cut the peptidoglycan backbone at different sites. We identify the mechanistic basis for cleavage site selection and also identify an amino acid switch that alters the cleavage chemistry. This work advances our understanding of how peptidoglycan glycosidases help build the cell wall. The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.
{"title":"Biochemical reconstitution defines new functions for membrane-bound glycosidases in assembly of the bacterial cell wall","authors":"A. Taguchi, J. Page, H. Tsui, M. Winkler, S. Walker","doi":"10.1101/2021.03.06.434200","DOIUrl":"https://doi.org/10.1101/2021.03.06.434200","url":null,"abstract":"Significance Bacteria are protected from their surrounding environment by the peptidoglycan cell wall, which is a major target for antibiotics. Counterintuitively, cell wall assembly requires enzymes that cleave newly built peptidoglycan chains. Here, using nascent peptidoglycan we assembled in vitro, we characterized two membrane-bound glycosidases that are vital for proper cell division and elongation in Streptococcus pneumoniae. These enzymes were proposed to perform different chemical reactions. Instead, we show that they perform the same chemical reaction but cut the peptidoglycan backbone at different sites. We identify the mechanistic basis for cleavage site selection and also identify an amino acid switch that alters the cleavage chemistry. This work advances our understanding of how peptidoglycan glycosidases help build the cell wall. The peptidoglycan cell wall is a macromolecular structure that encases bacteria and is essential for their survival. Proper assembly of the cell wall requires peptidoglycan synthases as well as membrane-bound cleavage enzymes that control where new peptidoglycan is made and inserted. Previous studies have shown that two membrane-bound proteins in Streptococcus pneumoniae, here named MpgA and MpgB, are important in maintaining cell wall integrity. MpgA was predicted to be a lytic transglycosylase based on its homology to Escherichia coli MltG, while the enzymatic activity of MpgB was unclear. Using nascent peptidoglycan substrates synthesized in vitro from the peptidoglycan precursor Lipid II, we report that both MpgA and MpgB are muramidases. We show that replacing a single amino acid in E. coli MltG with the corresponding amino acid from MpgA results in muramidase activity, allowing us to predict from the presence of this amino acid that other putative lytic transglycosylases actually function as muramidases. Strikingly, we report that MpgA and MpgB cut nascent peptidoglycan at different positions along the sugar backbone relative to the reducing end, with MpgA producing much longer peptidoglycan oligomers. We show that the cleavage site selectivity of MpgA is controlled by the LysM-like subdomain, which is required for its full functionality in cells. We propose that MltG’s ability to complement the loss of MpgA in S. pneumoniae despite performing different cleavage chemistry is because it can cleave nascent peptidoglycan at the same distance from the lipid anchor.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"122 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84151055","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 : 2021-03-04DOI: 10.1101/2021.03.03.433580
M. Moulton, Scott Barish, Isha Ralhan, Jinlan Chang, Lindsey D. Goodman, Jake G. Harland, Paul C. Marcogliese, J. Johansson, Maria S. Ioannou, H. Bellen
Significance Multiple studies have implicated dozens of risk loci that may be associated with Alzheimer’s disease (AD), but common mechanisms underlying how they may contribute to disease onset or progression remain elusive. This study identifies cell-specific roles for Drosophila orthologs of AD risk genes in lipid droplet formation that, when disrupted, lead to neurodegeneration. Our work reinforces a critical role for the sequestration of peroxidated lipids in glia, and places Apolipoprotein E ε4 (APOE4) with other AD risk factors in the transfer process of lipids from neurons to glia to form lipid droplets. A growing list of Alzheimer’s disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. We have previously shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, we tested the role of AD risk genes in ROS-induced LD formation and demonstrate that several genes impact neuroprotective LD formation, including homologs of human ABCA1, ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP. Our data also show that ROS enhances Aβ42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.
{"title":"Neuronal ROS-induced glial lipid droplet formation is altered by loss of Alzheimer’s disease–associated genes","authors":"M. Moulton, Scott Barish, Isha Ralhan, Jinlan Chang, Lindsey D. Goodman, Jake G. Harland, Paul C. Marcogliese, J. Johansson, Maria S. Ioannou, H. Bellen","doi":"10.1101/2021.03.03.433580","DOIUrl":"https://doi.org/10.1101/2021.03.03.433580","url":null,"abstract":"Significance Multiple studies have implicated dozens of risk loci that may be associated with Alzheimer’s disease (AD), but common mechanisms underlying how they may contribute to disease onset or progression remain elusive. This study identifies cell-specific roles for Drosophila orthologs of AD risk genes in lipid droplet formation that, when disrupted, lead to neurodegeneration. Our work reinforces a critical role for the sequestration of peroxidated lipids in glia, and places Apolipoprotein E ε4 (APOE4) with other AD risk factors in the transfer process of lipids from neurons to glia to form lipid droplets. A growing list of Alzheimer’s disease (AD) genetic risk factors is being identified, but the contribution of each variant to disease mechanism remains largely unknown. We have previously shown that elevated levels of reactive oxygen species (ROS) induces lipid synthesis in neurons leading to the sequestration of peroxidated lipids in glial lipid droplets (LD), delaying neurotoxicity. This neuron-to-glia lipid transport is APOD/E-dependent. To identify proteins that modulate these neuroprotective effects, we tested the role of AD risk genes in ROS-induced LD formation and demonstrate that several genes impact neuroprotective LD formation, including homologs of human ABCA1, ABCA7, VLDLR, VPS26, VPS35, AP2A, PICALM, and CD2AP. Our data also show that ROS enhances Aβ42 phenotypes in flies and mice. Finally, a peptide agonist of ABCA1 restores glial LD formation in a humanized APOE4 fly model, highlighting a potentially therapeutic avenue to prevent ROS-induced neurotoxicity. This study places many AD genetic risk factors in a ROS-induced neuron-to-glia lipid transfer pathway with a critical role in protecting against neurotoxicity.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83538933","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 : 2021-03-04DOI: 10.1101/2021.03.04.433973
Nguyen-Hung Le, Victor Pinedo, Juvenal Lopez, Felipe Cava, M. Feldman
Significance Previous studies have indicated that Gram-positive bacteria are not affected by type VI secretion serum (T6SS) intoxication. However, here we show that Acinetobacter baumannii employs its T6SS to kill different Gram-positive bacteria. Furthermore, we determined that killing was dependent on Tse4, a bifunctional effector possessing lytic transglycosylase and endopeptidase activities. Tse4 represents a broad family of modularly organized T6SS peptidoglycan-degrading effectors. In addition, we show that secretion of D-lysine by A. baumannii results in a pH increase, which greatly enhances Tse4 activity. These results expand the range of T6SS-mediated interbacterial interactions that may shape the composition of bacterial communities in the context of the human microbiota and polymicrobial infections. The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.
{"title":"Killing of Gram-negative and Gram-positive bacteria by a bifunctional cell wall-targeting T6SS effector","authors":"Nguyen-Hung Le, Victor Pinedo, Juvenal Lopez, Felipe Cava, M. Feldman","doi":"10.1101/2021.03.04.433973","DOIUrl":"https://doi.org/10.1101/2021.03.04.433973","url":null,"abstract":"Significance Previous studies have indicated that Gram-positive bacteria are not affected by type VI secretion serum (T6SS) intoxication. However, here we show that Acinetobacter baumannii employs its T6SS to kill different Gram-positive bacteria. Furthermore, we determined that killing was dependent on Tse4, a bifunctional effector possessing lytic transglycosylase and endopeptidase activities. Tse4 represents a broad family of modularly organized T6SS peptidoglycan-degrading effectors. In addition, we show that secretion of D-lysine by A. baumannii results in a pH increase, which greatly enhances Tse4 activity. These results expand the range of T6SS-mediated interbacterial interactions that may shape the composition of bacterial communities in the context of the human microbiota and polymicrobial infections. The type VI secretion system (T6SS) is a powerful tool deployed by Gram-negative bacteria to antagonize neighboring organisms. Here, we report that Acinetobacter baumannii ATCC 17978 (Ab17978) secretes D-lysine (D-Lys), increasing the extracellular pH and enhancing the peptidoglycanase activity of the T6SS effector Tse4. This synergistic effect of D-Lys on Tse4 activity enables Ab17978 to outcompete Gram-negative bacterial competitors, demonstrating that bacteria can modify their microenvironment to increase their fitness during bacterial warfare. Remarkably, this lethal combination also results in T6SS-mediated killing of Gram-positive bacteria. Further characterization revealed that Tse4 is a bifunctional enzyme consisting of both lytic transglycosylase and endopeptidase activities, thus representing a family of modularly organized T6SS peptidoglycan-degrading effectors with an unprecedented impact in antagonistic bacterial interactions.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"76756983","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 : 2021-03-03DOI: 10.5194/EGUSPHERE-EGU21-1737
A. Di Chiara, L. Tauxe, T. Levy, M. Najjar, F. Florindo, E. Ben‐Yosef
Significance The Earth’s magnetic field has changed significantly in the past with implications for related phenomena, such as deep-Earth processes and evolution of life. Accurate datasets of its past behavior also provide a dating tool. We present data from Neolithic ceramics and flint from Jordan. Our results are among the oldest in the Levant, covering a period of major changes in human history. The data help in refining the resolution of the archaeomagnetic curve, in turn enhancing its use as a dating tool and for understanding past field behavior. Moreover, we demonstrate the potential for the use of flint material, the most common raw material for the manufacturing of tools in the entire Paleolithic and younger periods, for archaeointensity investigations. Constraining secular variation of the Earth’s magnetic field strength in the past is fundamental to understanding short-term processes of the geodynamo. Such records also constitute a powerful and independent dating tool for archaeological sites and geological formations. In this study, we present 11 robust archaeointensity results from Pre-Pottery to Pottery Neolithic Jordan that are based on both clay and flint (chert) artifacts. Two of these results constitute the oldest archaeointensity data for the entire Levant, ancient Egypt, Turkey, and Mesopotamia, extending the archaeomagnetic reference curve for the Holocene. Virtual Axial Dipole Moments (VADMs) show that the Earth’s magnetic field in the Southern Levant was weak (about two-thirds the present field) at around 7600 BCE, recovering its strength to greater than the present field around 7000 BCE, and gradually weakening again around 5200 BCE. In addition, successful results obtained from burnt flint demonstrate the potential of this very common, and yet rarely used, material in archaeomagnetic research, in particular for prehistoric periods from the first use of fire to the invention of pottery.
{"title":"The strength of the Earth’s magnetic field from Pre-Pottery to Pottery Neolithic, Jordan","authors":"A. Di Chiara, L. Tauxe, T. Levy, M. Najjar, F. Florindo, E. Ben‐Yosef","doi":"10.5194/EGUSPHERE-EGU21-1737","DOIUrl":"https://doi.org/10.5194/EGUSPHERE-EGU21-1737","url":null,"abstract":"Significance The Earth’s magnetic field has changed significantly in the past with implications for related phenomena, such as deep-Earth processes and evolution of life. Accurate datasets of its past behavior also provide a dating tool. We present data from Neolithic ceramics and flint from Jordan. Our results are among the oldest in the Levant, covering a period of major changes in human history. The data help in refining the resolution of the archaeomagnetic curve, in turn enhancing its use as a dating tool and for understanding past field behavior. Moreover, we demonstrate the potential for the use of flint material, the most common raw material for the manufacturing of tools in the entire Paleolithic and younger periods, for archaeointensity investigations. Constraining secular variation of the Earth’s magnetic field strength in the past is fundamental to understanding short-term processes of the geodynamo. Such records also constitute a powerful and independent dating tool for archaeological sites and geological formations. In this study, we present 11 robust archaeointensity results from Pre-Pottery to Pottery Neolithic Jordan that are based on both clay and flint (chert) artifacts. Two of these results constitute the oldest archaeointensity data for the entire Levant, ancient Egypt, Turkey, and Mesopotamia, extending the archaeomagnetic reference curve for the Holocene. Virtual Axial Dipole Moments (VADMs) show that the Earth’s magnetic field in the Southern Levant was weak (about two-thirds the present field) at around 7600 BCE, recovering its strength to greater than the present field around 7000 BCE, and gradually weakening again around 5200 BCE. In addition, successful results obtained from burnt flint demonstrate the potential of this very common, and yet rarely used, material in archaeomagnetic research, in particular for prehistoric periods from the first use of fire to the invention of pottery.","PeriodicalId":20595,"journal":{"name":"Proceedings of the National Academy of Sciences","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2021-03-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73246541","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：