Pub Date : 2024-09-09DOI: 10.1101/2024.09.09.611957
Robin Alexander Rothemann, Egor Pavlenko, Mritunjoy Mondal, Sarah Gerlich, Pavel Grobushkin, Sebastian Mostert, Julia Racho, Konstantin Weiss, Dylan Stobbe, Katharina Stillger, Kim Lapacz, Silja Lucia Salscheider, Carmelina Petrungaro, Dan Ehninger, Thi Hoang Doung Nguyen, Joern Dengjel, Ines Neundorf, Daniele Bano, Simon Poepsel, Jan Riemer
Apoptosis inducing factor 1 (AIFM1) is a flavoprotein essential for mitochondrial function and biogenesis. Its interaction with MIA40, the central component of the mitochondrial disulfide relay, accounts for some, but not all effects of AIFM1 loss. Our high-confidence AIFM1 interactome revealed novel interaction partners of AIFM1. For one of these interactors, adenylate kinase 2 (AK2), an essential enzyme maintaining cellular adenine nucleotide pools, AIFM1 binding specifically stabilized the isoform AK2A via interaction with its C-terminus. High resolution cryo-EM and biochemical analyses showed that both, MIA40 and AK2A bind AIFM1s C-terminal β-strand, enhancing NADH oxidoreductase activity by locking an active, dimer conformation and, in the case of MIA40, affecting the cofactor binding site. The AIFM1-AK2A interaction is crucial during respiratory conditions. We further identified ADP/ATP translocases and the ATP synthase as AIFM1 interactors, emphasizing its important regulatory role as a central, organizing platform in energy metabolism.
{"title":"Interaction with AK2A links AIFM1 to cellular energy metabolism","authors":"Robin Alexander Rothemann, Egor Pavlenko, Mritunjoy Mondal, Sarah Gerlich, Pavel Grobushkin, Sebastian Mostert, Julia Racho, Konstantin Weiss, Dylan Stobbe, Katharina Stillger, Kim Lapacz, Silja Lucia Salscheider, Carmelina Petrungaro, Dan Ehninger, Thi Hoang Doung Nguyen, Joern Dengjel, Ines Neundorf, Daniele Bano, Simon Poepsel, Jan Riemer","doi":"10.1101/2024.09.09.611957","DOIUrl":"https://doi.org/10.1101/2024.09.09.611957","url":null,"abstract":"Apoptosis inducing factor 1 (AIFM1) is a flavoprotein essential for mitochondrial function and biogenesis. Its interaction with MIA40, the central component of the mitochondrial disulfide relay, accounts for some, but not all effects of AIFM1 loss. Our high-confidence AIFM1 interactome revealed novel interaction partners of AIFM1. For one of these interactors, adenylate kinase 2 (AK2), an essential enzyme maintaining cellular adenine nucleotide pools, AIFM1 binding specifically stabilized the isoform AK2A via interaction with its C-terminus. High resolution cryo-EM and biochemical analyses showed that both, MIA40 and AK2A bind AIFM1s C-terminal β-strand, enhancing NADH oxidoreductase activity by locking an active, dimer conformation and, in the case of MIA40, affecting the cofactor binding site. The AIFM1-AK2A interaction is crucial during respiratory conditions. We further identified ADP/ATP translocases and the ATP synthase as AIFM1 interactors, emphasizing its important regulatory role as a central, organizing platform in energy metabolism.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"46 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175942","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 : 2024-09-09DOI: 10.1101/2024.09.08.611915
Lauren E Vostal, Noa E Dahan, Wenzhu Zhang, Matthew J Reynolds, Brian Chait, Tarun Mohan Kapoor
Errors in proteostasis, which requires regulated degradation and recycling of diverse proteins, are linked to aging, cancer and neurodegenerative disease. In particular, recycling proteins from multiprotein complexes, organelles and membranes is initiated by ubiquitylation, extraction and unfolding by the essential mechanoenzyme VCP, and ubiquitin removal by deubiquitinases (DUBs), a class of ~100 ubiquitin-specific proteases in humans. As VCP's substrate recognition requires ubiquitylation, the removal of ubiquitins from substrates for recycling must follow extraction and unfolding. How the activities of VCP and different DUBs are coordinated for protein recycling or other fates is unclear. Here, we employ a photochemistry-based approach to profile proteome-wide domain-specific VCP interactions in living cells. We identify DUBs that bind near the entry, exit, or both sites of VCP's central pore, the channel for ATP-dependent substrate translocation. From this set of DUBs, we focus on VCPIP1, required for organelle assembly and DNA repair, that our chemical proteomics workflow indicates binds the central pore's entry and exit sites. We determine a ~3Å cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer. VCPIP1's UBX-L domain binds VCP's N-domain in a 'down' conformation, linked to VCP's ADP-bound state, and the deubiquitinase domain is positioned at the central pore's exit site, poised to remove ubiquitin following substrate unfolding. We find that VCP stimulates VCPIP1's DUB activity and use mutagenesis and single-molecule mass photometry assays to test the structural model. Together, our data suggest that DUBs bind VCP at distinct sites and reveal how the two enzyme activities can be coordinated to achieve specific downstream outcomes for ubiquitylated proteins.
{"title":"Distinct modes of coupling between VCP, an essential unfoldase, and deubiquitinases","authors":"Lauren E Vostal, Noa E Dahan, Wenzhu Zhang, Matthew J Reynolds, Brian Chait, Tarun Mohan Kapoor","doi":"10.1101/2024.09.08.611915","DOIUrl":"https://doi.org/10.1101/2024.09.08.611915","url":null,"abstract":"Errors in proteostasis, which requires regulated degradation and recycling of diverse proteins, are linked to aging, cancer and neurodegenerative disease. In particular, recycling proteins from multiprotein complexes, organelles and membranes is initiated by ubiquitylation, extraction and unfolding by the essential mechanoenzyme VCP, and ubiquitin removal by deubiquitinases (DUBs), a class of ~100 ubiquitin-specific proteases in humans. As VCP's substrate recognition requires ubiquitylation, the removal of ubiquitins from substrates for recycling must follow extraction and unfolding. How the activities of VCP and different DUBs are coordinated for protein recycling or other fates is unclear. Here, we employ a photochemistry-based approach to profile proteome-wide domain-specific VCP interactions in living cells. We identify DUBs that bind near the entry, exit, or both sites of VCP's central pore, the channel for ATP-dependent substrate translocation. From this set of DUBs, we focus on VCPIP1, required for organelle assembly and DNA repair, that our chemical proteomics workflow indicates binds the central pore's entry and exit sites. We determine a ~3Å cryo-EM structure of the VCP-VCPIP1 complex and find up to 3 VCPIP1 protomers interact with the VCP hexamer. VCPIP1's UBX-L domain binds VCP's N-domain in a 'down' conformation, linked to VCP's ADP-bound state, and the deubiquitinase domain is positioned at the central pore's exit site, poised to remove ubiquitin following substrate unfolding. We find that VCP stimulates VCPIP1's DUB activity and use mutagenesis and single-molecule mass photometry assays to test the structural model. Together, our data suggest that DUBs bind VCP at distinct sites and reveal how the two enzyme activities can be coordinated to achieve specific downstream outcomes for ubiquitylated proteins.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"53 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175915","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 : 2024-09-09DOI: 10.1101/2024.09.09.612087
Maria Dolores Moya-Garzon, Mengjie Wang, Veronica L Li, Wei Wei, Alan Sheng-Hwa Tung, Steffen H Raun, Meng Zhao, Laetitia Coassolo, Hashim Islam, Barbara Oliveira, Yuqin Dai, Jan Spaas, Antonio Delgado-Gonzalez, Kenyi Donoso, Aurora Alvarez-Buylla, Francisco Franco-Montalban, Anudari Letian, Catherine Ward, Lichao Liu, Katrin J Svensson, Emily L Goldberg, Christopher D Gardner, Jonathan P Little, Steven M Banik, Yong Xu, Jonathan Z Long
β-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.
{"title":"A secondary β-hydroxybutyrate metabolic pathway linked to energy balance","authors":"Maria Dolores Moya-Garzon, Mengjie Wang, Veronica L Li, Wei Wei, Alan Sheng-Hwa Tung, Steffen H Raun, Meng Zhao, Laetitia Coassolo, Hashim Islam, Barbara Oliveira, Yuqin Dai, Jan Spaas, Antonio Delgado-Gonzalez, Kenyi Donoso, Aurora Alvarez-Buylla, Francisco Franco-Montalban, Anudari Letian, Catherine Ward, Lichao Liu, Katrin J Svensson, Emily L Goldberg, Christopher D Gardner, Jonathan P Little, Steven M Banik, Yong Xu, Jonathan Z Long","doi":"10.1101/2024.09.09.612087","DOIUrl":"https://doi.org/10.1101/2024.09.09.612087","url":null,"abstract":"β-hydroxybutyrate (BHB) is an abundant ketone body. To date, all known pathways of BHB metabolism involve interconversion of BHB and primary energy intermediates. Here we show that CNDP2 controls a previously undescribed secondary BHB metabolic pathway via enzymatic conjugation of BHB and free amino acids. This BHB-ylation reaction produces a family of endogenous ketone metabolites, the BHB-amino acids. Genetic ablation of CNDP2 in mice eliminates tissue amino acid BHB-ylation activity and reduces BHB-amino acid levels. Administration of BHB-Phe, the most abundant BHB-amino acid, to obese mice activates neural populations in the hypothalamus and brainstem and suppresses feeding and body weight. Conversely, CNDP2-KO mice exhibit increased food intake and body weight upon ketosis stimuli. CNDP2-dependent amino acid BHB-ylation and BHB-amino acid metabolites are also conserved in humans. Therefore, the metabolic pathways of BHB extend beyond primary metabolism and include secondary ketone metabolites linked to energy balance.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175913","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}
High urate levels in circulation lead to the accumulation of urate crystals in joints and ultimately inflammation and gout. The reabsorption process of urate in the kidney by the urate transporter URAT1 plays a pivotal role in controlling serum urate levels. Pharmacological inhibition of URAT1 by uricosuric drugs is a valid strategy for gout management. Despite the clinical significance of URAT1, its structure and mechanism remain elusive. Here, we report the structures of human URAT1 (hURAT1) in complex with substrate urate or inhibitors benzbromarone and verinurad at resolution ranges from 3.0 to 3.3 A. Urate-bound hURAT1 adopts the outward-facing conformation. Urate is wrapped in the center of hURAT1 by five phenylalanines and coordinated by two positively charged residues on each side. Uricosuric compounds benzbromarone and verinurad occupy the urate-binding site of hURAT1 in the inward-facing conformation. Structural comparison between different conformations of hURAT1 reveals the rocker-switch-like mechanism for urate transport. Benzbromarone and verinurad exert their inhibitory effect by blocking not only the binding of urate but also the structural isomerization of hURAT1.
{"title":"Mechanisms of urate transport and uricosuric drugs inhibition in human URAT1","authors":"Wenjun Guo, Miao Wei, Yunfeng Li, Jiaxuan Xu, Jiahe Zang, Yuezhou Chen, Lei Chen","doi":"10.1101/2024.09.08.611941","DOIUrl":"https://doi.org/10.1101/2024.09.08.611941","url":null,"abstract":"High urate levels in circulation lead to the accumulation of urate crystals in joints and ultimately inflammation and gout. The reabsorption process of urate in the kidney by the urate transporter URAT1 plays a pivotal role in controlling serum urate levels. Pharmacological inhibition of URAT1 by uricosuric drugs is a valid strategy for gout management. Despite the clinical significance of URAT1, its structure and mechanism remain elusive. Here, we report the structures of human URAT1 (hURAT1) in complex with substrate urate or inhibitors benzbromarone and verinurad at resolution ranges from 3.0 to 3.3 A. Urate-bound hURAT1 adopts the outward-facing conformation. Urate is wrapped in the center of hURAT1 by five phenylalanines and coordinated by two positively charged residues on each side. Uricosuric compounds benzbromarone and verinurad occupy the urate-binding site of hURAT1 in the inward-facing conformation. Structural comparison between different conformations of hURAT1 reveals the rocker-switch-like mechanism for urate transport. Benzbromarone and verinurad exert their inhibitory effect by blocking not only the binding of urate but also the structural isomerization of hURAT1.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"31 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175916","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 : 2024-09-09DOI: 10.1101/2024.09.09.612061
Susmita Das, Carmel L Keerthana, Saumya Ranjan, Gayathri Seenivasan, Nikhil Tuti, Unnikrishnan Shaji, Gargi Meur, Roy Anindya
Non-heme iron (FeII) and 2-oxoglutarate(2OG)-dependent dioxygenases catalyse a diverse array of biological reactions. These enzymes couple the oxidative decarboxylation of 2OG to the hydroxylation of the substrates. However, in the absence of the substrate, oxidative decarboxylation of 2OG generates succinate. We have determined succinate level by using succinyl-CoA synthetase to monitor this uncoupled decarboxylation of FeII/2OG-dependent dioxygenases and measured the uncoupled 2OG turnover of different FeII/2OG-dependent dioxygenases. We also performed comparative analysis and verified the functionality of human dioxygenase ALKBH6 with unknown substrate.
{"title":"Comparative analysis of uncoupled succinate production by the FeII/2-oxoglutarate-dependent dioxygenases","authors":"Susmita Das, Carmel L Keerthana, Saumya Ranjan, Gayathri Seenivasan, Nikhil Tuti, Unnikrishnan Shaji, Gargi Meur, Roy Anindya","doi":"10.1101/2024.09.09.612061","DOIUrl":"https://doi.org/10.1101/2024.09.09.612061","url":null,"abstract":"Non-heme iron (FeII) and 2-oxoglutarate(2OG)-dependent dioxygenases catalyse a diverse array of biological reactions. These enzymes couple the oxidative decarboxylation of 2OG to the hydroxylation of the substrates. However, in the absence of the substrate, oxidative decarboxylation of 2OG generates succinate. We have determined succinate level by using succinyl-CoA synthetase to monitor this uncoupled decarboxylation of FeII/2OG-dependent dioxygenases and measured the uncoupled 2OG turnover of different FeII/2OG-dependent dioxygenases. We also performed comparative analysis and verified the functionality of human dioxygenase ALKBH6 with unknown substrate.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"8 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175914","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 : 2024-09-08DOI: 10.1101/2024.09.05.611418
Edward CT Waters, Friedrich Baark, Matthew R. Orton, Michael J. Shattock, Richard Southworth, Thomas R Eykyn
We present a compartmental modelling approach to analyse radioactive time activity curves for first pass kinetics of [99mTc]sestamibi in the heart. Reparametrizing the kinetic equations using the Nernst membrane-potential equation provides a novel means of non-invasively estimating the sarcolemmal (Em) and mitochondrial (ΔΨm) membrane potentials in the heart. A Markov Chain Monte Carlo (MCMC) fitting approach was applied to data derived from established interventions in Langendorff perfused rat hearts where the sarcolemmal membrane was depolarised using hyperkalaemic Krebs Henseleit buffers; the mitochondrial membrane was depolarised using carbonylcyanide-3-chlorophenylhydrazone (CCCP); or both membranes were depolarised using their combination. Translating this approach to single photon emission planar scintigraphy kinetics from healthy rats allowed an estimate of these membrane potentials (voltages) in vivo for the first time; the values were Em =-62 ± 5 mV and ΔΨm = -151 ± 5 mV (n = 4, mean ± SD).
{"title":"Sarcolemmal and mitochondrial membrane potentials measured ex vivo and in vivo in the heart by pharmacokinetic modelling of [99mTc]sestamibi","authors":"Edward CT Waters, Friedrich Baark, Matthew R. Orton, Michael J. Shattock, Richard Southworth, Thomas R Eykyn","doi":"10.1101/2024.09.05.611418","DOIUrl":"https://doi.org/10.1101/2024.09.05.611418","url":null,"abstract":"We present a compartmental modelling approach to analyse radioactive time activity curves for first pass kinetics of [<sup>99m</sup>Tc]sestamibi in the heart. Reparametrizing the kinetic equations using the Nernst membrane-potential equation provides a novel means of non-invasively estimating the sarcolemmal (<em>E<sub>m</sub></em>) and mitochondrial (ΔΨ<sub>m</sub>) membrane potentials in the heart. A Markov Chain Monte Carlo (MCMC) fitting approach was applied to data derived from established interventions in Langendorff perfused rat hearts where the sarcolemmal membrane was depolarised using hyperkalaemic Krebs Henseleit buffers; the mitochondrial membrane was depolarised using carbonylcyanide-3-chlorophenylhydrazone (CCCP); or both membranes were depolarised using their combination. Translating this approach to single photon emission planar scintigraphy kinetics from healthy rats allowed an estimate of these membrane potentials (voltages) in vivo for the first time; the values were <em>E<sub>m</sub></em> =-62 ± 5 mV and ΔΨ<sub>m</sub> = -151 ± 5 mV (n = 4, mean ± SD).","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"11 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175947","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}
Chronic kidney disease (CKD) presents a pressing global health challenge, characterized by progressive renal function decline and heightened morbidity and mortality. The interplay between CKD and hypothyroidism, particularly through the non-thyroidal low triiodothyronine (T3) syndrome, exacerbates disease progression and elevates mortality rates. Perturbations in the hypothalamic-pituitary-thyroidal (HPT) axis contribute to this scenario, while fibrotic kidneys exhibit diminished levels of the protective protein Klotho due to abnormal activation of the Wnt/β-catenin pathway. Leveraging our previous findings showcasing T3's ability to downregulate aberrant Wnt/β-catenin pathway activity by enhancing Klotho expression, both in vitro and in vivo, and Baicalein's direct inhibition of the Wnt pathway in C. elegans, we investigated Klotho's potential as a molecular link between CKD and hypothyroidism. Through experiments utilizing an adenine-induced CKD aged mouse model, we aimed to enhance Klotho expression via exogenous T3 administration and bidirectional Wnt pathway blockade using Baicalein (BAI). Our results demonstrate a significant synergistic upregulation of Klotho expression with combined T3 and BAI treatment, surpassing the effects of individual treatments. Moreover, this combination therapy effectively suppressed aberrant signaling molecules such as transforming growth factor beta (TGF), nuclear factor kappa B (NFκB), and glycogen synthase kinase 3 (GSK3), thus mitigating renal fibrosis. Improvements were observed in CKD-induced complications including cardiovascular disorders, dyslipidemia, and alterations in bone and serum markers. This unique bidirectional approach, targeting Klotho biology directly enhanced by T3 and sustained by T3 coupled with Wnt pathway blockade using BAI, presents a promising strategy for CKD management. Particularly relevant for elderly CKD patients with hypothyroidism, this approach holds potential to ameliorate renal degradation. Our findings underscore the substantial therapeutic promise of hormone and natural chemical interventions in CKD management.
{"title":"A Dual intervention of Triiodothyronine and Baicalein bi-directionally upregulates Klotho with attenuation of chronic kidney disease and its complications in aged BALB/c mice","authors":"Saswat Kumar Mohanty, Vikas Kumar Sahu, Bhanu Pratap Singh, Kitlangki Suchiang","doi":"10.1101/2024.09.08.611868","DOIUrl":"https://doi.org/10.1101/2024.09.08.611868","url":null,"abstract":"Chronic kidney disease (CKD) presents a pressing global health challenge, characterized by progressive renal function decline and heightened morbidity and mortality. The interplay between CKD and hypothyroidism, particularly through the non-thyroidal low triiodothyronine (T3) syndrome, exacerbates disease progression and elevates mortality rates. Perturbations in the hypothalamic-pituitary-thyroidal (HPT) axis contribute to this scenario, while fibrotic kidneys exhibit diminished levels of the protective protein Klotho due to abnormal activation of the Wnt/β-catenin pathway. Leveraging our previous findings showcasing T3's ability to downregulate aberrant Wnt/β-catenin pathway activity by enhancing Klotho expression, both in vitro and in vivo, and Baicalein's direct inhibition of the Wnt pathway in C. elegans, we investigated Klotho's potential as a molecular link between CKD and hypothyroidism. Through experiments utilizing an adenine-induced CKD aged mouse model, we aimed to enhance Klotho expression via exogenous T3 administration and bidirectional Wnt pathway blockade using Baicalein (BAI). Our results demonstrate a significant synergistic upregulation of Klotho expression with combined T3 and BAI treatment, surpassing the effects of individual treatments. Moreover, this combination therapy effectively suppressed aberrant signaling molecules such as transforming growth factor beta (TGF), nuclear factor kappa B (NFκB), and glycogen synthase kinase 3 (GSK3), thus mitigating renal fibrosis. Improvements were observed in CKD-induced complications including cardiovascular disorders, dyslipidemia, and alterations in bone and serum markers. This unique bidirectional approach, targeting Klotho biology directly enhanced by T3 and sustained by T3 coupled with Wnt pathway blockade using BAI, presents a promising strategy for CKD management. Particularly relevant for elderly CKD patients with hypothyroidism, this approach holds potential to ameliorate renal degradation. Our findings underscore the substantial therapeutic promise of hormone and natural chemical interventions in CKD management.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"19 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175943","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 : 2024-09-08DOI: 10.1101/2024.09.08.611895
Jungsan Sohn, Jacob Lueck, Alexander Strom, Shuai Wu, Hannah Wendorff
Cyclic G/AMP Synthase (cGAS) initiates inflammatory responses against pathogenic double-stranded (ds)DNA. Although it is well established that cGAS forms phase-separated condensates with dsDNA, their function remains poorly defined. We report here that the dimerization of cGAS on dsDNA creates a mesh-like network, leading to gel-like condensate formation. Although cGAS binds to and forms condensates with various nucleic acids, only dsDNA permits the dimerization necessary for activation and gelation. cGAS co-condenses dsDNA and other nucleic acids but retains a distinct dsDNA-mediated gel-like substate, which single-stranded RNA can dissolve and deactivate the enzyme. Moreover, gel-like, but not liquid-like, condensation not only protects bound dsDNA from exonucleases, but also limits the mobility of NTPs and the dinucleotide intermediate for efficient cGAMP synthesis. Together, our results show that enzymes can finetune surrounding microenvironments to regulate their signaling activities.
{"title":"Dimerization-dependent gel-like condensation with dsDNA underpins the activation of human cGAS","authors":"Jungsan Sohn, Jacob Lueck, Alexander Strom, Shuai Wu, Hannah Wendorff","doi":"10.1101/2024.09.08.611895","DOIUrl":"https://doi.org/10.1101/2024.09.08.611895","url":null,"abstract":"Cyclic G/AMP Synthase (cGAS) initiates inflammatory responses against pathogenic double-stranded (ds)DNA. Although it is well established that cGAS forms phase-separated condensates with dsDNA, their function remains poorly defined. We report here that the dimerization of cGAS on dsDNA creates a mesh-like network, leading to gel-like condensate formation. Although cGAS binds to and forms condensates with various nucleic acids, only dsDNA permits the dimerization necessary for activation and gelation. cGAS co-condenses dsDNA and other nucleic acids but retains a distinct dsDNA-mediated gel-like substate, which single-stranded RNA can dissolve and deactivate the enzyme. Moreover, gel-like, but not liquid-like, condensation not only protects bound dsDNA from exonucleases, but also limits the mobility of NTPs and the dinucleotide intermediate for efficient cGAMP synthesis. Together, our results show that enzymes can finetune surrounding microenvironments to regulate their signaling activities.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"2015 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175944","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 : 2024-09-08DOI: 10.1101/2024.09.08.611893
Brianna M Woodbury, Rebecca L Newcomer, Andrei T Alexandrescu, Carolyn M Teschke
The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a three-stranded intertwined spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec1-98) was used to demonstrate that the stable globular OB-fold domain folds independently of the C-terminal residues. However, Dec1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, however, implying Dec trimer assembly in vivo is templated by the phage capsid. The Thermodynamic Hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.
134 个残基的噬菌体 L 装饰蛋白（Dec）形成了一种噬菌体稳定同源三聚体，当它与噬菌体 L 的噬菌体结合时，具有不对称的三脚架状结构。三聚体亚基的 N 端由空间上分离的球状 OB 折叠结构域组成，可与噬菌体 L 或相关噬菌体 P22 的病毒相互作用。三聚体的 C 端形成了一个三链交织的尖峰结构，几乎所有的相互作用都是通过该结构来稳定三聚体的。一个删除了尖峰残基 99-134 的 Dec 突变体（Dec1-98）被用来证明稳定的球状 OB 折叠结构域的折叠与 C 端残基无关。然而，Dec1-98 无法与噬菌体 P22 病毒结合，这表明 C 端尖峰对稳定的囊膜相互作用至关重要。将全长 Dec 三聚体酸化至 pH 值为 2 时，可将其分解为单体。这些单体保留了折叠的球状 OB 折叠结构域，但尖峰被展开。将 Dec 单体溶液的 pH 值提高到 pH 值 6，可使三聚体在体外缓慢形成数天。然而，噬菌体 L 的感染周期仅为一小时左右，这意味着 Dec 三聚体在体内的组装是由噬菌体外壳模板化的。热力学假说认为，蛋白质的折叠是由氨基酸序列决定的。噬菌体是一个不寻常的例子，它的低聚物折叠步骤在动力学上被病毒噬菌体模板加速。噬菌体模板机制可以满足Dec适应成熟噬菌体L噬菌体上不寻常的准对称结合位点所需的灵活性。
{"title":"Templated trimerization of the phage L decoration protein on capsids","authors":"Brianna M Woodbury, Rebecca L Newcomer, Andrei T Alexandrescu, Carolyn M Teschke","doi":"10.1101/2024.09.08.611893","DOIUrl":"https://doi.org/10.1101/2024.09.08.611893","url":null,"abstract":"The 134-residue phage L decoration protein (Dec) forms a capsid-stabilizing homotrimer that has an asymmetric tripod-like structure when bound to phage L capsids. The N-termini of the trimer subunits consist of spatially separated globular OB-fold domains that interact with the virions of phage L or the related phage P22. The C-termini of the trimer form a three-stranded intertwined spike structure that accounts for nearly all the interactions that stabilize the trimer. A Dec mutant with the spike residues 99-134 deleted (Dec1-98) was used to demonstrate that the stable globular OB-fold domain folds independently of the C-terminal residues. However, Dec1-98 was unable to bind phage P22 virions, indicating the C-terminal spike is essential for stable capsid interaction. The full-length Dec trimer is disassembled into monomers by acidification to pH <2. These monomers retain the folded globular OB-fold domain structure, but the spike is unfolded. Increasing the pH of the Dec monomer solution to pH 6 allowed for slow trimer formation in vitro over the course of days. The infectious cycle of phage L is only around an hour, however, implying Dec trimer assembly in vivo is templated by the phage capsid. The Thermodynamic Hypothesis holds that protein folding is determined by the amino acid sequence. Dec serves as an unusual example of an oligomeric folding step that is kinetically accelerated by a viral capsid template. The capsid templating mechanism could satisfy the flexibility needed for Dec to adapt to the unusual quasi-symmetric binding site on the mature phage L capsid.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175945","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 : 2024-09-08DOI: 10.1101/2024.09.08.611884
Lulu Yin, Ke Shi, Yanjun Chen, Reuben S Harris, Hideki Aihara
DNA deaminase toxins are involved in interbacterial antagonism and the generation of genetic diversity in surviving bacterial populations. These enzymes have also been adopted as genome engineering tools. The single-stranded (ss)DNA deaminase SsdA represents the bacterial deaminase toxin family-2 (BaDTF2) and it deaminates ssDNA cytosines with little sequence context dependence, which contrasts with the AID/APOBEC family of sequence-selective ssDNA cytosine deaminases. Here we report the crystal structure of SsdA in complex with a ssDNA substrate. The structure reveals a unique mode of substrate binding, in which a cluster of aromatic residues of SsdA engages ssDNA in a V-shaped conformation sharply bent across the target cytosine. The bases 5' or 3' to the target cytosine are stacked linearly and make few sequence-specific protein contacts, thus explaining the broad substrate selectivity of SsdA. Unexpectedly, SsdA contains a β-amino acid isoaspartate, which is important for enzymatic activity and may contribute to the stability of SsdA as a toxin. Structure-function studies helped to design SsdA mutants active in human cells, which could lead to future applications in genome engineering.
{"title":"Structural basis for sequence context-independent single-stranded DNA cytosine deamination by the bacterial toxin SsdA","authors":"Lulu Yin, Ke Shi, Yanjun Chen, Reuben S Harris, Hideki Aihara","doi":"10.1101/2024.09.08.611884","DOIUrl":"https://doi.org/10.1101/2024.09.08.611884","url":null,"abstract":"DNA deaminase toxins are involved in interbacterial antagonism and the generation of genetic diversity in surviving bacterial populations. These enzymes have also been adopted as genome engineering tools. The single-stranded (ss)DNA deaminase SsdA represents the bacterial deaminase toxin family-2 (BaDTF2) and it deaminates ssDNA cytosines with little sequence context dependence, which contrasts with the AID/APOBEC family of sequence-selective ssDNA cytosine deaminases. Here we report the crystal structure of SsdA in complex with a ssDNA substrate. The structure reveals a unique mode of substrate binding, in which a cluster of aromatic residues of SsdA engages ssDNA in a V-shaped conformation sharply bent across the target cytosine. The bases 5' or 3' to the target cytosine are stacked linearly and make few sequence-specific protein contacts, thus explaining the broad substrate selectivity of SsdA. Unexpectedly, SsdA contains a β-amino acid isoaspartate, which is important for enzymatic activity and may contribute to the stability of SsdA as a toxin. Structure-function studies helped to design SsdA mutants active in human cells, which could lead to future applications in genome engineering.","PeriodicalId":501147,"journal":{"name":"bioRxiv - Biochemistry","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142175946","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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