Pub Date : 2024-09-15DOI: 10.1101/2024.09.11.612521
Kadidia Samassekou, Elisabeth E Garland-Kuntz, Vaani Ohri, Isaac J Fisher, Satchal K Erramilli, Kaushik Muralidharan, Livia M Bogdan, Abigail M Gick, Anthony A Kossiakoff, Angeline M Lyon
Phospholipase Cϵ (PLCϵ) increases intracellular Ca2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLCϵ to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 Å reconstruction of the largest fragment of PLCϵ to date in complex with an antigen binding fragment (Fab). The structure reveals that PLCϵ contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLCϵ and the first mechanistic insights into how it engages cellular membranes for activity.
磷脂酶 Cϵ（PLCϵ）在 G 蛋白偶联受体（GPCR）和受体酪氨酸激酶（RTK）的刺激下会增加心血管系统中的细胞内 Ca2+ 和蛋白激酶 C (PKC) 的活性。PLCϵ 能够对这些不同的输入做出反应，部分原因在于它具有多个构象动态调控域。然而，这种异质性也限制了对脂肪酶单个结构域或其催化核心的结构研究。在这里，我们报告了迄今为止最大的 PLCϵ 与抗原结合片段（Fab）复合片段的 3.9 Å 重建结构。该结构显示，PLCϵ包含一个pleckstrin homology（PH）结构域和四个串联的EF手，包括亚家族特异性插入和与催化核心的分子内相互作用。该结构以及全酶模型表明，N-末端和PH结构域的一部分形成了一个连续的表面，可与质膜和核膜周围的细胞质小叶接触，从而提高活性。对该表面的功能表征证实，它对最大的基础活性和 G 蛋白刺激活性至关重要。这项研究为了解 PLCϵ 的自抑制基础构象提供了新的视角，并首次从机理上揭示了 PLCϵ 是如何与细胞膜结合以发挥活性的。
{"title":"Cryo-EM Structure of Phospholipase Cϵ Defines N-terminal Domains and their Roles in Activity","authors":"Kadidia Samassekou, Elisabeth E Garland-Kuntz, Vaani Ohri, Isaac J Fisher, Satchal K Erramilli, Kaushik Muralidharan, Livia M Bogdan, Abigail M Gick, Anthony A Kossiakoff, Angeline M Lyon","doi":"10.1101/2024.09.11.612521","DOIUrl":"https://doi.org/10.1101/2024.09.11.612521","url":null,"abstract":"Phospholipase Cϵ (PLCϵ) increases intracellular Ca2+ and protein kinase C (PKC) activity in the cardiovascular system in response to stimulation of G protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs). The ability of PLCϵ to respond to these diverse inputs is due, in part, to multiple, conformationally dynamic regulatory domains. However, this heterogeneity has also limited structural studies of the lipase to either individual domains or its catalytic core. Here, we report the 3.9 &Aring reconstruction of the largest fragment of PLCϵ to date in complex with an antigen binding fragment (Fab). The structure reveals that PLCϵ contains a pleckstrin homology (PH) domain and four tandem EF hands, including subfamily-specific insertions and intramolecular interactions with the catalytic core. The structure, together with a model of the holoenzyme, suggest that part of the N-terminus and PH domain form a continuous surface that could engage cytoplasmic leaflets of the plasma and perinuclear membranes, contributing to activity. Functional characterization of this surface confirm it is critical for maximum basal and G protein-stimulated activities. This study provides new insights into the autoinhibited, basal conformation of PLCϵ and the first mechanistic insights into how it engages cellular membranes for activity.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254891","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 : 2024-09-14DOI: 10.1101/2024.09.13.612887
Christopher R. Fullenkamp, Shams Medhi, Christopher P Jones, Logan Tenney, Patricio Pichling, Peri Prestwood, Adrian Ferre-D'Amare, Pratyush S Tiwary, John S Schneekloth
The challenge of targeting RNA with small molecules necessitates a better understanding of RNA-ligand interaction mechanisms. However, the dynamic nature of nucleic acids, their ligand-induced stabilization, and how conformational changes influence gene expression pose significant difficulties for experimental investigation. This work employs a combination of computational and experimental methods to address these challenges. By integrating structure-informed design, crystallography, and machine learning-augmented all-atom molecular dynamics simulations (MD) we synthesized, biophysically and biochemically characterized, and studied the dissociation of a library of small molecule activators of the ZTP riboswitch, a ligand-binding RNA motif that regulates bacterial gene expression. We uncovered key interaction mechanisms, revealing valuable insights into the role of ligand binding kinetics on riboswitch activation. Further, we established that ligand on-rates determine activation potency as opposed to binding affinity and elucidated RNA structural differences, which provide mechanistic insights into the interplay of RNA structure on riboswitch activation.
{"title":"Machine learning-augmented molecular dynamics simulations (MD) reveal insights into the disconnect between affinity and activation of ZTP riboswitch ligands","authors":"Christopher R. Fullenkamp, Shams Medhi, Christopher P Jones, Logan Tenney, Patricio Pichling, Peri Prestwood, Adrian Ferre-D'Amare, Pratyush S Tiwary, John S Schneekloth","doi":"10.1101/2024.09.13.612887","DOIUrl":"https://doi.org/10.1101/2024.09.13.612887","url":null,"abstract":"The challenge of targeting RNA with small molecules necessitates a better understanding of RNA-ligand interaction mechanisms. However, the dynamic nature of nucleic acids, their ligand-induced stabilization, and how conformational changes influence gene expression pose significant difficulties for experimental investigation. This work employs a combination of computational and experimental methods to address these challenges. By integrating structure-informed design, crystallography, and machine learning-augmented all-atom molecular dynamics simulations (MD) we synthesized, biophysically and biochemically characterized, and studied the dissociation of a library of small molecule activators of the ZTP riboswitch, a ligand-binding RNA motif that regulates bacterial gene expression. We uncovered key interaction mechanisms, revealing valuable insights into the role of ligand binding kinetics on riboswitch activation. Further, we established that ligand on-rates determine activation potency as opposed to binding affinity and elucidated RNA structural differences, which provide mechanistic insights into the interplay of RNA structure on riboswitch activation.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254896","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}
Ferredoxin is a small iron-sulfur protein and acts as an electron carrier. Low-potential ferredoxins harbor [4Fe-4S] cluster(s), which play(s) a crucial role as the redox center. Low-potential ferredoxins are able to cover a wide range of redox potentials (-700 to -200 mV); however, the mechanisms underlying the factors which control the redox potential are still enigmatic. Here, we determined the neutron structure of ferredoxin from Bacillus thermoproteolyticus, and experimentally revealed the exact hydrogen-bonding network involving the [4Fe-4S] cluster. The density functional theory calculations based on the hydrogen-bonding network revealed that protonation states of the sidechain of Asp64 close to the [4Fe-4S] cluster critically affected the stability of the reduced state in the cluster. These findings provide the first identification of the intrinsic control factor of redox potential for the [4Fe-4S] cluster in low-potential ferredoxins.
{"title":"Protonation/deprotonation-driven switch for the redox stability of low-potential [4Fe-4S] ferredoxin","authors":"Kei Wada, Kenji Kobayashi, Iori Era, Yusuke Isobe, Taigo Kamimura, Masaki Marukawa, Takayuki Nagae, Kazuki Honjo, Noriko Kaseda, Yumiko Motoyama, Kengo Inoue, Masakazu Sugishima, Katsuhiro Kusaka, Naomine Yano, Keiichi Fukuyama, Masaki Mishima, Yasutaka Kitagawa, Masaki Unno","doi":"10.1101/2024.09.12.612615","DOIUrl":"https://doi.org/10.1101/2024.09.12.612615","url":null,"abstract":"Ferredoxin is a small iron-sulfur protein and acts as an electron carrier. Low-potential ferredoxins harbor [4Fe-4S] cluster(s), which play(s) a crucial role as the redox center. Low-potential ferredoxins are able to cover a wide range of redox potentials (-700 to -200 mV); however, the mechanisms underlying the factors which control the redox potential are still enigmatic. Here, we determined the neutron structure of ferredoxin from Bacillus thermoproteolyticus, and experimentally revealed the exact hydrogen-bonding network involving the [4Fe-4S] cluster. The density functional theory calculations based on the hydrogen-bonding network revealed that protonation states of the sidechain of Asp64 close to the [4Fe-4S] cluster critically affected the stability of the reduced state in the cluster. These findings provide the first identification of the intrinsic control factor of redox potential for the [4Fe-4S] cluster in low-potential ferredoxins.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254897","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 : 2024-09-14DOI: 10.1101/2024.09.13.612858
Caroline de Aquino Guerreiro, Leandro Dominiciano de Andrade, Layanne Nascimento Fraga, Tatiana Milena Marques, Samira Bernardino Ramos do Prado, Robert Jam Brummer, Joao Roberto Oliveira Nascimento, Victor Castro-Alves
It is well known that dietary fibers (DF) from plant-source foods can induce beneficial health effects through their physicochemical properties and utilization by the gut microbiota during fermentation, which is mainly explored with a focus on changes in the gut microbiota profile and the production of microbial-derived metabolites. Here, we characterized structural motifs (i.e., oligomers) produced during DF breakdown upon colonic fermentation and explored their interaction with toll-like receptors (TLRs) present on the surface of human intestinal and immune system cells. Firstly, a source of wheat arabinoxylan (WAX) was subjected to in vitro simulation of human colonic fermentation, followed by characterization and quantification of WAX structural motifs to explore their dynamics throughout fermentation. The identified structural motifs were further produced through enzymatic catalysis of WAX using carbohydrate-active enzymes and fractionated into six well-defined fractions of arabinoxylans and linear xylans. These fractions of structural motifs were then tested for interaction with TLR2 and TLR4 using a reporter cell assay. Results revealed structure-dependent effects, primarily with inhibition of TLR2 and activation of TLR4 depending on the degree of polymerization and branching of WAX structural motifs. The role of the fine structure of WAX structural motifs was confirmed by molecular docking, which revealed that minor structural changes substantially influence the interaction between structural motifs and TLRs. The results from in vitro and in silico studies also support the hypothesis that the direct effects of oligomers and polysaccharides on cell receptors are likely the result of complex interactions involving multiple cell surface receptors. Finally, in addition to highlighting that direct effects of structural motifs might play an important role in the overall effects of DF, this work suggests that enzymatic-tailoring design of DF can be a potential tool for producing functional ingredients with specific effects on human health.
{"title":"Production of bioactive structural motifs from wheat arabinoxylan via colonic fermentation and enzymatic catalysis: evidence of interaction with toll-like receptors from in vitro, in silico and functional analysis","authors":"Caroline de Aquino Guerreiro, Leandro Dominiciano de Andrade, Layanne Nascimento Fraga, Tatiana Milena Marques, Samira Bernardino Ramos do Prado, Robert Jam Brummer, Joao Roberto Oliveira Nascimento, Victor Castro-Alves","doi":"10.1101/2024.09.13.612858","DOIUrl":"https://doi.org/10.1101/2024.09.13.612858","url":null,"abstract":"It is well known that dietary fibers (DF) from plant-source foods can induce beneficial health effects through their physicochemical properties and utilization by the gut microbiota during fermentation, which is mainly explored with a focus on changes in the gut microbiota profile and the production of microbial-derived metabolites. Here, we characterized structural motifs (i.e., oligomers) produced during DF breakdown upon colonic fermentation and explored their interaction with toll-like receptors (TLRs) present on the surface of human intestinal and immune system cells. Firstly, a source of wheat arabinoxylan (WAX) was subjected to in vitro simulation of human colonic fermentation, followed by characterization and quantification of WAX structural motifs to explore their dynamics throughout fermentation. The identified structural motifs were further produced through enzymatic catalysis of WAX using carbohydrate-active enzymes and fractionated into six well-defined fractions of arabinoxylans and linear xylans. These fractions of structural motifs were then tested for interaction with TLR2 and TLR4 using a reporter cell assay. Results revealed structure-dependent effects, primarily with inhibition of TLR2 and activation of TLR4 depending on the degree of polymerization and branching of WAX structural motifs. The role of the fine structure of WAX structural motifs was confirmed by molecular docking, which revealed that minor structural changes substantially influence the interaction between structural motifs and TLRs. The results from in vitro and in silico studies also support the hypothesis that the direct effects of oligomers and polysaccharides on cell receptors are likely the result of complex interactions involving multiple cell surface receptors. Finally, in addition to highlighting that direct effects of structural motifs might play an important role in the overall effects of DF, this work suggests that enzymatic-tailoring design of DF can be a potential tool for producing functional ingredients with specific effects on human health.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"17 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254892","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 : 2024-09-14DOI: 10.1101/2024.09.12.612633
Manuel Alejandro Herrera, Camille C Caldeira da Silva, Mauricio S Baptista, Alicia J Kowaltowski
The skin is our largest organ, and also the most exposed to solar radiation. As mitochondria within skin cells are rich in endophotosensitizers such as NADH, FADH2, and cytochromes, we studied the effects of different UV and visible light wavelengths on metabolic fluxes in keratinocytes, the main cell type in the epidermis. We find that 36 J/cm2 of UV light (λmax at 365 nm) leads to a complete inhibition of oxidative phosphorylation. The same light dose at other wavelengths (blue light, 450 nm, and green light, 517 nm) did not affect metabolic fluxes, but reduced cell viability, probably by photosensitizing oxidation. Strikingly, red light (660 nm) not only did not decrease cell viability, but also enhanced cell proliferation as well as basal and maximal oxygen consumption rates for up to two days after irradiation. To uncover the mechanisms in which this unexpected long-lived metabolic enhancement occurred, we measured quantities of oxidative phosphorylation-related proteins and oxygen consumption in permeabilized cells, which were unchanged. This result is indicative of a modulation of cytosolic metabolic processes by red light. Subsequently, we measured glycolytic, glutamine-dependent, and fatty-acid supported metabolic fluxes and determined that red light specifically activates fatty acid oxidation by mitochondria. Overall, our results demonstrate that light modulates oxidative phosphorylation with different effects at distinct wavelengths. Interestingly, we uncover a novel and highly specific effect of red light modulating fatty acid oxidation in keratinocytes, providing a novel mechanistic explanation for the metabolic effects of photobiomodulation.
{"title":"Mitochondrial Fatty Acid Oxidation is Stimulated by Red Light Irradiation","authors":"Manuel Alejandro Herrera, Camille C Caldeira da Silva, Mauricio S Baptista, Alicia J Kowaltowski","doi":"10.1101/2024.09.12.612633","DOIUrl":"https://doi.org/10.1101/2024.09.12.612633","url":null,"abstract":"The skin is our largest organ, and also the most exposed to solar radiation. As mitochondria within skin cells are rich in endophotosensitizers such as NADH, FADH2, and cytochromes, we studied the effects of different UV and visible light wavelengths on metabolic fluxes in keratinocytes, the main cell type in the epidermis. We find that 36 J/cm2 of UV light (λmax at 365 nm) leads to a complete inhibition of oxidative phosphorylation. The same light dose at other wavelengths (blue light, 450 nm, and green light, 517 nm) did not affect metabolic fluxes, but reduced cell viability, probably by photosensitizing oxidation. Strikingly, red light (660 nm) not only did not decrease cell viability, but also enhanced cell proliferation as well as basal and maximal oxygen consumption rates for up to two days after irradiation. To uncover the mechanisms in which this unexpected long-lived metabolic enhancement occurred, we measured quantities of oxidative phosphorylation-related proteins and oxygen consumption in permeabilized cells, which were unchanged. This result is indicative of a modulation of cytosolic metabolic processes by red light. Subsequently, we measured glycolytic, glutamine-dependent, and fatty-acid supported metabolic fluxes and determined that red light specifically activates fatty acid oxidation by mitochondria. Overall, our results demonstrate that light modulates oxidative phosphorylation with different effects at distinct wavelengths. Interestingly, we uncover a novel and highly specific effect of red light modulating fatty acid oxidation in keratinocytes, providing a novel mechanistic explanation for the metabolic effects of photobiomodulation.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"93 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254894","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}
Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-EM structures of post-B-act spliceosome intermediates from S. pombe primed for disassembly. We identify the DEAH-box helicase - G patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an over-stabilization of the U5 loop I interaction with the 5' exon leading to a single nucleotide insertion at the 5' splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.
前 mRNA 剪接过程中的内含子去除异常复杂，其中断会导致人类多种遗传疾病。虽然通过结构-功能联合分析揭示了正常剪接体循环的关键步骤，但有关致力于过早解体的异常剪接体的结构信息尚不存在。在这里，我们报告了两种来自 S. pombe 的 B-act 后剪接体中间体的低温电子显微镜结构。我们确定了 DEAH-box 螺旋酶 - G patch 蛋白对（Gih35-Gpl1，与人类 DHX35-GPATCH1 同源），并展示了它如何维持催化休眠。在这两种结构中，Gpl1 都能识别因 U5 环 I 与 5'外显子相互作用过度稳定而导致 5'剪接位点单核苷酸插入所引入的重塑活性位点。重塑作用被传递到剪接体表面，介导解体的 Ntr1 复合物被招募。我们的数据为有针对性地分析剪接质量控制铺平了道路。
{"title":"Structures of aberrant spliceosome intermediates on their way to disassembly","authors":"Komal Soni, Attila Horvath, Olexandr Dybkov, Merli Schwan, Sasanan Trakansuebkul, Dirk Flemming, Klemens Wild, Henning Urlaub, Tamas Fischer, Irmgard Sinning","doi":"10.1101/2024.09.13.612651","DOIUrl":"https://doi.org/10.1101/2024.09.13.612651","url":null,"abstract":"Intron removal during pre-mRNA splicing is of extraordinary complexity and its disruption causes a vast number of genetic diseases in humans. While key steps of the canonical spliceosome cycle have been revealed by combined structure-function analyses, structural information on an aberrant spliceosome committed to premature disassembly is not available. Here, we report two cryo-EM structures of post-B-act spliceosome intermediates from S. pombe primed for disassembly. We identify the DEAH-box helicase - G patch protein pair (Gih35-Gpl1, homologous to human DHX35-GPATCH1) and show how it maintains catalytic dormancy. In both structures, Gpl1 recognizes a remodeled active site introduced by an over-stabilization of the U5 loop I interaction with the 5' exon leading to a single nucleotide insertion at the 5' splice site. Remodeling is communicated to the spliceosome surface and the Ntr1 complex that mediates disassembly is recruited. Our data pave the way for a targeted analysis of splicing quality control.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"4 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254895","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 : 2024-09-14DOI: 10.1101/2024.09.13.612636
Shih-En Chou, Vishal C Kalel, Ralf Erdmann
Leishmaniasis is a life-threatening neglected tropical disease caused by over 20 species of Leishmania parasites. Visceral leishmaniasis, also known as kala-azar, is particularly lethal, with a 95% mortality rate if left untreated. Currently, no vaccine is available, and chemotherapy remains the primary treatment option. However, these drugs have drawbacks such as high toxicities, the emergence of resistant strains, and high costs. Therefore, there is a need to develop new and safe treatments. Glycosomes are essential organelles for the survival of Leishmania parasites. They are maintained by peroxin (PEX) proteins, which are responsible for glycosome biogenesis, including targeting proteins to glycosomes. Previous studies have shown that blocking the interaction between the import receptor PEX19 and the docking factor PEX3 kills Trypanosoma brucei by disrupting glycosome biogenesis. In this study, we screened an FDA-approved drug repurposing library using an AlphaScreen based assay and identified inhibitors of LdPEX3-LdPEX19 interaction in vitro. The inhibitor effectively kills Leishmania parasites, including the challenging amastigote forms contained within the infected mammalian host cells. This study validates the inhibition of glycosome biogenesis in Leishmania as a potential approach for developing new anti-leishmanial therapies.
{"title":"An inhibitor targeting glycosome membrane biogenesis kills Leishmania parasites","authors":"Shih-En Chou, Vishal C Kalel, Ralf Erdmann","doi":"10.1101/2024.09.13.612636","DOIUrl":"https://doi.org/10.1101/2024.09.13.612636","url":null,"abstract":"Leishmaniasis is a life-threatening neglected tropical disease caused by over 20 species of <em>Leishmania</em> parasites. Visceral leishmaniasis, also known as kala-azar, is particularly lethal, with a 95% mortality rate if left untreated. Currently, no vaccine is available, and chemotherapy remains the primary treatment option. However, these drugs have drawbacks such as high toxicities, the emergence of resistant strains, and high costs. Therefore, there is a need to develop new and safe treatments. Glycosomes are essential organelles for the survival of <em>Leishmania</em> parasites. They are maintained by peroxin (PEX) proteins, which are responsible for glycosome biogenesis, including targeting proteins to glycosomes. Previous studies have shown that blocking the interaction between the import receptor PEX19 and the docking factor PEX3 kills <em>Trypanosoma brucei</em> by disrupting glycosome biogenesis. In this study, we screened an FDA-approved drug repurposing library using an AlphaScreen based assay and identified inhibitors of <em>Ld</em>PEX3-<em>Ld</em>PEX19 interaction <em>in vitro</em>. The inhibitor effectively kills Leishmania parasites, including the challenging amastigote forms contained within the infected mammalian host cells. This study validates the inhibition of glycosome biogenesis in <em>Leishmania</em> as a potential approach for developing new anti-leishmanial therapies.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"13 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254899","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 : 2024-09-14DOI: 10.1101/2024.09.13.611920
Amanda Dwikarina, Mohamed Bayati, Novianus Efrat, Anuradha Roy, Zhentian Lei, Khanh-Van Ho, Lloyd W. Sumner, Andrew L. Thomas, Wendy Applequist, Michael Michael Greenlief, Andrew Townesmith, Chung-Ho Lin
American elderberry (Sambucus nigra subsp. canadensis) is a rapidly emerging new perennial crop for Missouri, recognized for its high level of bioactive compounds with significant health benefits, including antimicrobial, antiviral, and antioxidant properties. A high-throughput screening assay combined with untargeted metabolomics analysis was utilized on American elderberry juice from 21 genotypes to explore and characterize these bioactive compounds. Our metabolomics study has identified 32 putative bioactive compounds in the American Elderberry juices. An array of high-throughput screening bioassays evaluated 1) total antioxidant capacity, 2) activation of antioxidant response elements (ARE), 3) antiviral activity, and 4) antibacterial activity of the putatively identified compounds. Our results revealed that 14 of the 32 American elderberry compounds exhibited strong antioxidant activity. Four compounds (isorhamnetin 3-O-glucoside, kaempferol, quercetin, and naringenin) activated ARE activity and were found to be non-cytotoxic to cells. Notably, six of the 32 compounds demonstrated significant antiviral activity in an in vitro TZM-bl assay against two strains of HIV-1 virus, CXCR4-dependent NL4-3 virus and CCR5-dependent BaL virus. Luteolin showed the most potent anti-HIV activity in an in vitro TZM-bl assay against the NL4-3 virus (IC50 = 1.49 microM), followed by isorhamnetin (IC50 = 1.67 microM). The most potent anti-HIV compound against the BaL virus was myricetin (IC50 = 1.14 microM), followed by luteolin (IC50 = 4.38 microM). Additionally, six compounds were found to have antibacterial activity against gram-positive bacteria S. aureus, with cyanidin 3-O-rutinoside having the most potent antibacterial activity in vitro (IC50 = 2.9 microM), followed by cyanidin 3-O-glucoside (IC50 = 3.7 microM). These findings support and validate the potential health benefits of compounds found in American elderberry juices, and highlight their potential for use in dietary supplements as well as innovative applications in health and medicine.
{"title":"Exploring American Elderberry Compounds for Antioxidant, Antiviral, and Antibacterial Properties Through High-Throughput Screening Assays Combined with Untargeted Metabolomics","authors":"Amanda Dwikarina, Mohamed Bayati, Novianus Efrat, Anuradha Roy, Zhentian Lei, Khanh-Van Ho, Lloyd W. Sumner, Andrew L. Thomas, Wendy Applequist, Michael Michael Greenlief, Andrew Townesmith, Chung-Ho Lin","doi":"10.1101/2024.09.13.611920","DOIUrl":"https://doi.org/10.1101/2024.09.13.611920","url":null,"abstract":"American elderberry (Sambucus nigra subsp. canadensis) is a rapidly emerging new perennial crop for Missouri, recognized for its high level of bioactive compounds with significant health benefits, including antimicrobial, antiviral, and antioxidant properties. A high-throughput screening assay combined with untargeted metabolomics analysis was utilized on American elderberry juice from 21 genotypes to explore and characterize these bioactive compounds. Our metabolomics study has identified 32 putative bioactive compounds in the American Elderberry juices. An array of high-throughput screening bioassays evaluated 1) total antioxidant capacity, 2) activation of antioxidant response elements (ARE), 3) antiviral activity, and 4) antibacterial activity of the putatively identified compounds. Our results revealed that 14 of the 32 American elderberry compounds exhibited strong antioxidant activity. Four compounds (isorhamnetin 3-O-glucoside, kaempferol, quercetin, and naringenin) activated ARE activity and were found to be non-cytotoxic to cells. Notably, six of the 32 compounds demonstrated significant antiviral activity in an in vitro TZM-bl assay against two strains of HIV-1 virus, CXCR4-dependent NL4-3 virus and CCR5-dependent BaL virus. Luteolin showed the most potent anti-HIV activity in an in vitro TZM-bl assay against the NL4-3 virus (IC50 = 1.49 microM), followed by isorhamnetin (IC50 = 1.67 microM). The most potent anti-HIV compound against the BaL virus was myricetin (IC50 = 1.14 microM), followed by luteolin (IC50 = 4.38 microM). Additionally, six compounds were found to have antibacterial activity against gram-positive bacteria S. aureus, with cyanidin 3-O-rutinoside having the most potent antibacterial activity in vitro (IC50 = 2.9 microM), followed by cyanidin 3-O-glucoside (IC50 = 3.7 microM). These findings support and validate the potential health benefits of compounds found in American elderberry juices, and highlight their potential for use in dietary supplements as well as innovative applications in health and medicine.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254900","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 : 2024-09-14DOI: 10.1101/2024.09.13.612482
Johana M Lambert, Anna Kovilakath, Maryam Jamil, Yolander Valentine, Andrea Anderson, David Montefusco, Lauren Ashley Cowart
Sphingosine kinase 1 (SphK1) plays a crucial role in regulating metabolic pathways within adipocytes and is elevated in the adipose tissue of obese mice. While previous studies have reported both pro- and inhibitory effects of SphK1 and its product, sphingosine-1-phosphate (S1P), on adipogenesis, the precise mechanisms remain unclear. This study explores the timing and downstream effects of SphK1/S1P expression and activation during in vitro adipogenesis. We demonstrate that the synthetic glucocorticoid dexamethasone robustly induces SphK1 expression, suggesting its involvement in glucocorticoid-dependent signaling during adipogenesis. Notably, the activation of C/EBPδ, a key gene in early adipogenesis and a target of glucocorticoids, is diminished in SphK1-/- adipose-derived stem cells (ADSCs). Furthermore, glucocorticoid administration promotes adipose tissue expansion via SphK1 in a depot-specific manner. Although adipose expansion still occurs in SphK1-/- mice, it is significantly reduced. These findings indicate that while SphK1 is not essential for adipogenesis, it enhances early gene activation, thereby facilitating adipose tissue expansion.
{"title":"Sphingosine kinase 1 is induced by glucocorticoids in adipose derived stem cells and enhances glucocorticoid mediated signaling in adipose expansion.","authors":"Johana M Lambert, Anna Kovilakath, Maryam Jamil, Yolander Valentine, Andrea Anderson, David Montefusco, Lauren Ashley Cowart","doi":"10.1101/2024.09.13.612482","DOIUrl":"https://doi.org/10.1101/2024.09.13.612482","url":null,"abstract":"Sphingosine kinase 1 (SphK1) plays a crucial role in regulating metabolic pathways within adipocytes and is elevated in the adipose tissue of obese mice. While previous studies have reported both pro- and inhibitory effects of SphK1 and its product, sphingosine-1-phosphate (S1P), on adipogenesis, the precise mechanisms remain unclear. This study explores the timing and downstream effects of SphK1/S1P expression and activation during in vitro adipogenesis. We demonstrate that the synthetic glucocorticoid dexamethasone robustly induces SphK1 expression, suggesting its involvement in glucocorticoid-dependent signaling during adipogenesis. Notably, the activation of C/EBPδ, a key gene in early adipogenesis and a target of glucocorticoids, is diminished in SphK1-/- adipose-derived stem cells (ADSCs). Furthermore, glucocorticoid administration promotes adipose tissue expansion via SphK1 in a depot-specific manner. Although adipose expansion still occurs in SphK1-/- mice, it is significantly reduced. These findings indicate that while SphK1 is not essential for adipogenesis, it enhances early gene activation, thereby facilitating adipose tissue expansion.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"18 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254893","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 : 2024-09-14DOI: 10.1101/2024.09.13.612428
Sian Catherine Allerton, Marina Kuimova, Francesco Antonio Aprile
α-synuclein is an intrinsically disordered protein forming amyloids in Parkinson's disease. Currently, detection methods predominantly report on the formation of mature amyloids but are poorly sensitive to the early-stage, toxic oligomers. Molecular rotors are fluorophores that sense changes in the viscosity of their local environment. Here, we monitor α-synuclein oligomer formation, based on fluorescence lifetime of molecular rotors. We detected oligomer formation and conversion into amyloids for wild type and two α-synuclein variants; the pathological mutant A30P and ΔP1 α-synuclein, which lacks a master regulator region of aggregation (residues 36-42). We report that A30P α-synuclein showed a similar rate of oligomer formation compared to wild type α-synuclein, whereas ΔP1 α-synuclein showed delayed oligomer formation. Additionally, both variants demonstrated a slower conversion of oligomers to amyloids. Our method provides a quantitative approach to unveiling the complex mechanism of α-synuclein aggregation which is key to understanding the pathology of Parkinson's disease.
{"title":"Molecular rotors provide insight into the mechanism of formation and conversion of α-synuclein aggregates","authors":"Sian Catherine Allerton, Marina Kuimova, Francesco Antonio Aprile","doi":"10.1101/2024.09.13.612428","DOIUrl":"https://doi.org/10.1101/2024.09.13.612428","url":null,"abstract":"α-synuclein is an intrinsically disordered protein forming amyloids in Parkinson's disease. Currently, detection methods predominantly report on the formation of mature amyloids but are poorly sensitive to the early-stage, toxic oligomers. Molecular rotors are fluorophores that sense changes in the viscosity of their local environment. Here, we monitor α-synuclein oligomer formation, based on fluorescence lifetime of molecular rotors. We detected oligomer formation and conversion into amyloids for wild type and two α-synuclein variants; the pathological mutant A30P and ΔP1 α-synuclein, which lacks a master regulator region of aggregation (residues 36-42). We report that A30P α-synuclein showed a similar rate of oligomer formation compared to wild type α-synuclein, whereas ΔP1 α-synuclein showed delayed oligomer formation. Additionally, both variants demonstrated a slower conversion of oligomers to amyloids. Our method provides a quantitative approach to unveiling the complex mechanism of α-synuclein aggregation which is key to understanding the pathology of Parkinson's disease.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142254898","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}
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