Pub Date : 2024-09-05DOI: 10.1101/2024.09.04.611219
Pei Li, Julia N. Faraone, Cheng Chih Hsu, Michelle Chamblee, Yajie Liu, Yi-Min Zheng, Yan Xu, Claire Carlin, Jeffrey C. Horowitz, Rama K. Mallampalli, Linda J. Saif, Eugene M. Oltz, Daniel Jones, Jianrong Li, Richard J. Gumina, Joseph S. Bednash, Kai Xu, Shan-Lu Liu
During the summer of 2024, COVID-19 cases surged globally, driven by variants derived from JN.1 subvariants of SARS-CoV-2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and stability of these subvariants-LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2, and increased resistance to elevated temperatures. Molecular modeling suggests that the DelS31 mutation induces a conformational change that stabilizes the NTD and strengthens the NTD-Receptor-Binding Domain (RBD) interaction, thus favoring the down conformation of RBD and reducing accessibility to both the ACE2 receptor and certain nAbs. Additionally, the DelS31 mutation introduces an N-linked glycan modification at N30, which shields the underlying NTD region from antibody recognition. Our data highlight the critical role of NTD mutations in the spike protein for nAb evasion, stability, and viral infectivity, and suggest consideration of updating COVID-19 vaccines with antigens containing DelS31.
{"title":"Neutralization and Stability of JN.1-derived LB.1, KP.2.3, KP.3 and KP.3.1.1 Subvariants","authors":"Pei Li, Julia N. Faraone, Cheng Chih Hsu, Michelle Chamblee, Yajie Liu, Yi-Min Zheng, Yan Xu, Claire Carlin, Jeffrey C. Horowitz, Rama K. Mallampalli, Linda J. Saif, Eugene M. Oltz, Daniel Jones, Jianrong Li, Richard J. Gumina, Joseph S. Bednash, Kai Xu, Shan-Lu Liu","doi":"10.1101/2024.09.04.611219","DOIUrl":"https://doi.org/10.1101/2024.09.04.611219","url":null,"abstract":"During the summer of 2024, COVID-19 cases surged globally, driven by variants derived from JN.1 subvariants of SARS-CoV-2 that feature new mutations, particularly in the N-terminal domain (NTD) of the spike protein. In this study, we report on the neutralizing antibody (nAb) escape, infectivity, fusion, and stability of these subvariants-LB.1, KP.2.3, KP.3, and KP.3.1.1. Our findings demonstrate that all of these subvariants are highly evasive of nAbs elicited by the bivalent mRNA vaccine, the XBB.1.5 monovalent mumps virus-based vaccine, or from infections during the BA.2.86/JN.1 wave. This reduction in nAb titers is primarily driven by a single serine deletion (DelS31) in the NTD of the spike, leading to a distinct antigenic profile compared to the parental JN.1 and other variants. We also found that the DelS31 mutation decreases pseudovirus infectivity in CaLu-3 cells, which correlates with impaired cell-cell fusion. Additionally, the spike protein of DelS31 variants appears more conformationally stable, as indicated by reduced S1 shedding both with and without stimulation by soluble ACE2, and increased resistance to elevated temperatures. Molecular modeling suggests that the DelS31 mutation induces a conformational change that stabilizes the NTD and strengthens the NTD-Receptor-Binding Domain (RBD) interaction, thus favoring the down conformation of RBD and reducing accessibility to both the ACE2 receptor and certain nAbs. Additionally, the DelS31 mutation introduces an N-linked glycan modification at N30, which shields the underlying NTD region from antibody recognition. Our data highlight the critical role of NTD mutations in the spike protein for nAb evasion, stability, and viral infectivity, and suggest consideration of updating COVID-19 vaccines with antigens containing DelS31.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212677","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 : 2024-09-04DOI: 10.1101/2024.09.03.611117
Joao Vitor Dutra Molino, Aaron Oliver, Harish Sethuram, Kalisa Kang, Barbara Saucedo, Crisandra Jade Diaz, Abhishek Gupta, Lee Jong Jen, Yasin Torres-tiji, Nora Hidasi, Amr Badary, Hunter Jenkins, Francis J. Fields, Stephen Mayfield
We present the comprehensive characterization of a newly identified microalga, Chlamydomonas pacifica, originally isolated from a soil sample in San Diego, CA, USA. This species showcases remarkable biological versatility, including a broad pH range tolerance (6-11.5), high thermal tolerance (up to 42 degree celsius), and salinity resilience (up to 2% NaCl). Its amenability to genetic manipulation and sexual reproduction via mating, particularly between the two opposing strains CC-5697 & CC-5699, now publicly available through the Chlamydomonas Resource Center, underscores its potential as a biotechnological chassis. The biological assessment of C. pacifica, revealed versatile metabolic capabilities, including diverse nitrogen assimilation capability, phototaxis, and motility. Genomic and transcriptomic analyses identified 17,829 genes within a 121 Mb genome, featuring a slightly lower GC content (61%) than the model organism Chlamydomonas reinhardtii. The codon usage of C. pacifica closely mirrors that of C. reinhardtii, indicating a conserved genetic architecture that supports a trend in codon preference with minor variations. Phylogenetic analyses position C. pacifica within the core-Reinhardtinia clade yet distinct from known Volvocales species. Through metabolomic and lipidomic profiling, we observed a high carbohydrate content, approximately 30% of the dry cell weight (DCW), suggesting a substantial capacity for bioplastic precursor production. The lipidomic data revealed an abundance of triacylglycerols (TAGs), promising for biofuel applications and lipids for health-related benefits. Our investigation lays the groundwork for exploiting C. pacifica in biotechnological applications, spanning from biofuel generation to synthesizing biodegradable plastics, positioning it as a versatile host for future bioengineering endeavors.
{"title":"Description of a novel extremophile green algae, Chlamydomonas pacifica, and its potential as a biotechnology host","authors":"Joao Vitor Dutra Molino, Aaron Oliver, Harish Sethuram, Kalisa Kang, Barbara Saucedo, Crisandra Jade Diaz, Abhishek Gupta, Lee Jong Jen, Yasin Torres-tiji, Nora Hidasi, Amr Badary, Hunter Jenkins, Francis J. Fields, Stephen Mayfield","doi":"10.1101/2024.09.03.611117","DOIUrl":"https://doi.org/10.1101/2024.09.03.611117","url":null,"abstract":"We present the comprehensive characterization of a newly identified microalga, Chlamydomonas pacifica, originally isolated from a soil sample in San Diego, CA, USA. This species showcases remarkable biological versatility, including a broad pH range tolerance (6-11.5), high thermal tolerance (up to 42 degree celsius), and salinity resilience (up to 2% NaCl). Its amenability to genetic manipulation and sexual reproduction via mating, particularly between the two opposing strains CC-5697 & CC-5699, now publicly available through the Chlamydomonas Resource Center, underscores its potential as a biotechnological chassis. The biological assessment of C. pacifica, revealed versatile metabolic capabilities, including diverse nitrogen assimilation capability, phototaxis, and motility. Genomic and transcriptomic analyses identified 17,829 genes within a 121 Mb genome, featuring a slightly lower GC content (61%) than the model organism Chlamydomonas reinhardtii. The codon usage of C. pacifica closely mirrors that of C. reinhardtii, indicating a conserved genetic architecture that supports a trend in codon preference with minor variations. Phylogenetic analyses position C. pacifica within the core-Reinhardtinia clade yet distinct from known Volvocales species. Through metabolomic and lipidomic profiling, we observed a high carbohydrate content, approximately 30% of the dry cell weight (DCW), suggesting a substantial capacity for bioplastic precursor production. The lipidomic data revealed an abundance of triacylglycerols (TAGs), promising for biofuel applications and lipids for health-related benefits. Our investigation lays the groundwork for exploiting C. pacifica in biotechnological applications, spanning from biofuel generation to synthesizing biodegradable plastics, positioning it as a versatile host for future bioengineering endeavors.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"178 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212722","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 : 2024-09-04DOI: 10.1101/2024.09.03.610704
Florent Waltz, Ricardo D. Righetto, Xianjun Zhang, Ron Kelley, Martin Obr, Sagar Khavnekar, Abhay Kotecha, Benjamin D. Engel
Mitochondria produce energy through oxidative phosphorylation, carried out by five membrane-bound complexes collectively known as the respiratory chain. These complexes work in concert to transfer electrons and pump protons, leading to ATP regeneration. The precise organization of these complexes in native cells is debated, notably their assembly into higher-order supercomplexes called respirasomes. Here, we use in situ cryo-electron tomography to visualize the native structures and organization of several major mitochondrial complexes inside Chlamydomonas reinhardtii cells. ATP synthases and respiratory complexes are segregated into curved and flat crista membrane domains, respectively. Respiratory complexes I, III, and IV assemble into a single type of respirasome, from which we determined a native 5 Angstrom-resolution structure showing the binding of electron carrier cytochrome c. Combined with single-particle cryo-electron microscopy reconstruction at 2.4 Angstrom resolution, we assemble a detailed model of how the respiratory complexes interact with each other inside native mitochondria.
线粒体通过氧化磷酸化产生能量，由五个膜结合复合体执行，统称为呼吸链。这些复合物协同工作，传递电子和泵送质子，从而实现 ATP 再生。关于这些复合物在原生细胞中的精确组织结构还存在争议，尤其是它们组装成更高阶的超级复合物--呼吸体。在这里，我们使用原位低温电子断层扫描技术来观察衣藻细胞内几种主要线粒体复合物的原生结构和组织。ATP 合成酶和呼吸复合体分别被分隔在弯曲和平坦的嵴膜域中。呼吸复合体 I、III 和 IV 组装成单一类型的呼吸体，我们从中确定了一个 5 埃分辨率的原生结构，该结构显示了电子载体细胞色素 c 的结合。
{"title":"In-cell architecture of the mitochondrial respiratory chain","authors":"Florent Waltz, Ricardo D. Righetto, Xianjun Zhang, Ron Kelley, Martin Obr, Sagar Khavnekar, Abhay Kotecha, Benjamin D. Engel","doi":"10.1101/2024.09.03.610704","DOIUrl":"https://doi.org/10.1101/2024.09.03.610704","url":null,"abstract":"Mitochondria produce energy through oxidative phosphorylation, carried out by five membrane-bound complexes collectively known as the respiratory chain. These complexes work in concert to transfer electrons and pump protons, leading to ATP regeneration. The precise organization of these complexes in native cells is debated, notably their assembly into higher-order supercomplexes called respirasomes. Here, we use in situ cryo-electron tomography to visualize the native structures and organization of several major mitochondrial complexes inside Chlamydomonas reinhardtii cells. ATP synthases and respiratory complexes are segregated into curved and flat crista membrane domains, respectively. Respiratory complexes I, III, and IV assemble into a single type of respirasome, from which we determined a native 5 Angstrom-resolution structure showing the binding of electron carrier cytochrome c. Combined with single-particle cryo-electron microscopy reconstruction at 2.4 Angstrom resolution, we assemble a detailed model of how the respiratory complexes interact with each other inside native mitochondria.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212676","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}
Cellular processes are governed by the regulation of gene expression, often at the level of translation control. The mechanisms of control have been shown to operate at various levels, but there is growing evidence to suggest that rRNA modification patterns play a key role in driving translational modulation of the ribosome. We investigated the intricate relationship between modification status and the decoding activity of the ribosome. We found that the level of 2'-O-methylation at specific nucleotides in the rRNA affects the properties of the ribosome, with consequences for both Saccharomyces. cerevisiae cell growth and antibiotic sensitivity. More precisely, we demonstrate that methylations within the peptide exit tunnel play an important role in nascent peptide folding. We also demonstrate the modulation of IRES-driven translation by variable methylation at the intersubunit surface of the 60S ribosomal subunit. These findings deepen our understanding of the mechanisms by which 2'-O-methylation confers functional specificity on the ribosome.
{"title":"Variable rRNA 2'-O-methylation fine-tunes ribosome function in Saccharomyces cerevisiae","authors":"Sandra Gillot, Virginie Marchand, Yuri Motorin, Agnes Baudin-Baillieu, Olivier Namy","doi":"10.1101/2024.08.07.607065","DOIUrl":"https://doi.org/10.1101/2024.08.07.607065","url":null,"abstract":"Cellular processes are governed by the regulation of gene expression, often at the level of translation control. The mechanisms of control have been shown to operate at various levels, but there is growing evidence to suggest that rRNA modification patterns play a key role in driving translational modulation of the ribosome. We investigated the intricate relationship between modification status and the decoding activity of the ribosome. We found that the level of 2'-O-methylation at specific nucleotides in the rRNA affects the properties of the ribosome, with consequences for both Saccharomyces. cerevisiae cell growth and antibiotic sensitivity. More precisely, we demonstrate that methylations within the peptide exit tunnel play an important role in nascent peptide folding. We also demonstrate the modulation of IRES-driven translation by variable methylation at the intersubunit surface of the 60S ribosomal subunit. These findings deepen our understanding of the mechanisms by which 2'-O-methylation confers functional specificity on the ribosome.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941427","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 : 2024-08-11DOI: 10.1101/2024.08.11.606797
Alexander Kruse, Simon Schneider, Gina Esther Merges, Andreas Christian Froebius, Ignasi Forne, Axel Imhof, Hubert Schorle, Klaus Steger
Protamine 2 (Prm2/PRM2), together with Protamine 1 (Prm1/PRM1), constitute the two protamines found in both murine and human sperm. During spermiogenesis in haploid male germ cells, chromatin undergoes significant condensation, a phase in which most histones are replaced by a species-specific ratio of these two protamines. Altered PRM1/PRM2 ratios are associated with subfertility and infertility in both male mice and men. Notably, during histone-to-protamine exchange a small fraction of histones remains (ranging from 1% to 15%) bound to DNA. The regulatory roles of these residual histones, governed by post-translational modifications (PTMs), play a pivotal role in spermatogenesis, particularly in chromatin remodeling and epigenetic regulation of genes during sperm differentiation or even in early embryogenesis. In this study, utilizing a Prm2-deficient mouse model and conducting an analysis of sperm samples from men exhibiting either normozoospermia or atypical spermiograms, we observed alterations in the methylation and acetylation profiles of histones H3 and H4. Subsequent in- depth analysis revealed that discrepancies in protamine ratios do not significantly influence the post-translational modifications (PTMs) of histones in testicular sperm. In epididymal sperm these altered protamine ratios are associated with a reduction in the acetylation levels of histone H4 (H4ac), a phenomenon consistent across both murine and human samples. In particular, H4K5ac and H4K12ac were identified as the two modifications that appear to decrease as a result of reduced Prm2/PRM2 levels. Our findings reveal that Protamine 2 is necessary for the maintenance of specific histone PTMs, such as acetylation, which is essential for proper spermatogenesis and particularly for chromatin remodeling.
{"title":"An aberrant protamine ratio is associated with decreased H4ac levels in murine and human sperm","authors":"Alexander Kruse, Simon Schneider, Gina Esther Merges, Andreas Christian Froebius, Ignasi Forne, Axel Imhof, Hubert Schorle, Klaus Steger","doi":"10.1101/2024.08.11.606797","DOIUrl":"https://doi.org/10.1101/2024.08.11.606797","url":null,"abstract":"Protamine 2 (Prm2/PRM2), together with Protamine 1 (Prm1/PRM1), constitute the two protamines found in both murine and human sperm. During spermiogenesis in haploid male germ cells, chromatin undergoes significant condensation, a phase in which most histones are replaced by a species-specific ratio of these two protamines. Altered PRM1/PRM2 ratios are associated with subfertility and infertility in both male mice and men. Notably, during histone-to-protamine exchange a small fraction of histones remains (ranging from 1% to 15%) bound to DNA. The regulatory roles of these residual histones, governed by post-translational modifications (PTMs), play a pivotal role in spermatogenesis, particularly in chromatin remodeling and epigenetic regulation of genes during sperm differentiation or even in early embryogenesis. In this study, utilizing a Prm2-deficient mouse model and conducting an analysis of sperm samples from men exhibiting either normozoospermia or atypical spermiograms, we observed alterations in the methylation and acetylation profiles of histones H3 and H4. Subsequent in- depth analysis revealed that discrepancies in protamine ratios do not significantly influence the post-translational modifications (PTMs) of histones in testicular sperm. In epididymal sperm these altered protamine ratios are associated with a reduction in the acetylation levels of histone H4 (H4ac), a phenomenon consistent across both murine and human samples. In particular, H4K5ac and H4K12ac were identified as the two modifications that appear to decrease as a result of reduced Prm2/PRM2 levels. Our findings reveal that Protamine 2 is necessary for the maintenance of specific histone PTMs, such as acetylation, which is essential for proper spermatogenesis and particularly for chromatin remodeling.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941436","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 : 2024-08-11DOI: 10.1101/2024.08.11.607511
Parisa Koutsifeli, Lorna J Daniels, Joshua P.H. Neale, Sarah Fong, Upasna Varma, Marco Annandale, Xun Li, Yohanes Nursalim, James R Bell, Kate L Weeks, Aleksandr Stotland, David J Taylor, Roberta A Gottlieb, Lea M.D. Delbridge, Kimberley M Mellor
Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.
{"title":"Methods for detection of cardiac glycogen-autophagy","authors":"Parisa Koutsifeli, Lorna J Daniels, Joshua P.H. Neale, Sarah Fong, Upasna Varma, Marco Annandale, Xun Li, Yohanes Nursalim, James R Bell, Kate L Weeks, Aleksandr Stotland, David J Taylor, Roberta A Gottlieb, Lea M.D. Delbridge, Kimberley M Mellor","doi":"10.1101/2024.08.11.607511","DOIUrl":"https://doi.org/10.1101/2024.08.11.607511","url":null,"abstract":"Glycogen-autophagy ('glycophagy') is a selective autophagy process involved in delivering glycogen to the lysosome for bulk degradation. Glycophagy protein intermediaries include STBD1 as a glycogen tagging receptor, delivering the glycogen cargo into the forming phagosome by partnering with the Atg8 homolog, GABARAPL1. Glycophagy is emerging as a key process of energy metabolism and development of reliable tools for assessment of glycophagy activity is an important priority. Here we show that antibodies raised against the N terminus of the GABARAPL1 protein (but not the full-length protein) detected a specific endogenous GABARAPL1 immunoblot band at 18kDa. A stable GFP-GABARAPL1 cardiac cell line was used to quantify GABARAPL1 lysosomal flux via measurement of GFP puncta in response to lysosomal inhibition with bafilomycin. Endogenous glycophagy flux was quantified in primary rat ventricular myocytes by the extent of glycogen accumulation with bafilomycin combined with chloroquine treatment (no effect observed with bafilomycin or chloroquine alone). In wild-type isolated mouse hearts, bafilomycin alone and bafilomycin combined with chloroquine (but not chloroquine alone) elicited a significant increase in glycogen content signifying basal glycophagy flux. Collectively, these methodologies provide a comprehensive toolbox for tracking cardiac glycophagy activity to advance research into the role of glycophagy in health and disease.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941428","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 : 2024-08-10DOI: 10.1101/2024.08.09.607411
Madelaine M Usey, Anthony A Ruberto, Diego Huet
The production of energy in the form of ATP by the mitochondrial ATP synthase must be tightly controlled. One well-conserved form of regulation is mediated via ATPase inhibitory factor 1 (IF1), which governs ATP synthase activity and gene expression patterns through a cytoprotective process known as mitohormesis. In apicomplexans, the processes regulating ATP synthase activity are not fully elucidated. Using the model apicomplexan Toxoplasma gondii, we found that knockout and overexpression of TgIF1, the structural homolog of IF1, significantly affected gene expression. Additionally, TgIF1 overexpression resulted in the formation of a stable TgIF1 oligomer that increased the presence of higher order ATP synthase oligomers. We also show that parasites lacking TgIF1 exhibit reduced mitochondrial cristae density, and that while TgIF1 levels do not affect growth in conventional culture conditions, they are crucial for parasite survival under hypoxia. Interestingly, TgIF1 overexpression enhances recovery from oxidative stress, suggesting a mitohormetic function. In summary, while TgIF1 does not appear to play a role in metabolic regulation under conventional growth conditions, our work highlights its importance for adapting to stressors faced by T. gondii and other apicomplexans throughout their intricate life cycles.
线粒体 ATP 合成酶以 ATP 形式产生能量的过程必须受到严格控制。其中一种保存完好的调控形式是通过 ATP 酶抑制因子 1（IF1）介导的，它通过一种称为有丝分裂的细胞保护过程来调控 ATP 合成酶的活性和基因表达模式。在 apicomplexans 中，调节 ATP 合成酶活性的过程尚未完全阐明。我们利用模式 apicomplexan 弓形虫（Toxoplasma gondii）发现，IF1 的结构同源物 TgIF1 的敲除和过表达会显著影响基因表达。此外，TgIF1 的过度表达导致形成稳定的 TgIF1 寡聚体，从而增加了高阶 ATP 合成酶寡聚体的存在。我们还发现，缺乏 TgIF1 的寄生虫线粒体嵴密度降低，虽然 TgIF1 水平不会影响寄生虫在常规培养条件下的生长，但它们对寄生虫在缺氧条件下的存活至关重要。有趣的是，TgIF1 的过表达增强了从氧化应激中恢复的能力，这表明它具有丝裂激素功能。总之，虽然 TgIF1 在常规生长条件下似乎并不发挥新陈代谢调节作用，但我们的工作凸显了它在冈底斯淋球菌和其他类囊体错综复杂的生命周期中适应压力的重要性。
{"title":"The Toxoplasma gondii homolog of ATPase inhibitory factor 1 is critical for mitochondrial cristae maintenance and stress response","authors":"Madelaine M Usey, Anthony A Ruberto, Diego Huet","doi":"10.1101/2024.08.09.607411","DOIUrl":"https://doi.org/10.1101/2024.08.09.607411","url":null,"abstract":"The production of energy in the form of ATP by the mitochondrial ATP synthase must be tightly controlled. One well-conserved form of regulation is mediated via ATPase inhibitory factor 1 (IF1), which governs ATP synthase activity and gene expression patterns through a cytoprotective process known as mitohormesis. In apicomplexans, the processes regulating ATP synthase activity are not fully elucidated. Using the model apicomplexan Toxoplasma gondii, we found that knockout and overexpression of TgIF1, the structural homolog of IF1, significantly affected gene expression. Additionally, TgIF1 overexpression resulted in the formation of a stable TgIF1 oligomer that increased the presence of higher order ATP synthase oligomers. We also show that parasites lacking TgIF1 exhibit reduced mitochondrial cristae density, and that while TgIF1 levels do not affect growth in conventional culture conditions, they are crucial for parasite survival under hypoxia. Interestingly, TgIF1 overexpression enhances recovery from oxidative stress, suggesting a mitohormetic function. In summary, while TgIF1 does not appear to play a role in metabolic regulation under conventional growth conditions, our work highlights its importance for adapting to stressors faced by T. gondii and other apicomplexans throughout their intricate life cycles.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941437","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 : 2024-08-10DOI: 10.1101/2024.08.09.607355
Joseph D. DeAngelo, Maxim I. Maron, Jacob S. Roth, Aliza M. Silverstein, Varun Gupta, Stephanie Stransky, Joey Azofeifa, Simone Sidoli, Matthew J. Gamble, David Shechter
Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A), but not type I PRMT inhibition, leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.
{"title":"Productive mRNA Chromatin Escape is Promoted by PRMT5 Methylation of SNRPB","authors":"Joseph D. DeAngelo, Maxim I. Maron, Jacob S. Roth, Aliza M. Silverstein, Varun Gupta, Stephanie Stransky, Joey Azofeifa, Simone Sidoli, Matthew J. Gamble, David Shechter","doi":"10.1101/2024.08.09.607355","DOIUrl":"https://doi.org/10.1101/2024.08.09.607355","url":null,"abstract":"Protein Arginine Methyltransferase 5 (PRMT5) regulates RNA splicing and transcription by symmetric dimethylation of arginine residues (Rme2s/SDMA) in many RNA binding proteins. However, the mechanism by which PRMT5 couples splicing to transcriptional output is unknown. Here, we demonstrate that a major function of PRMT5 activity is to promote chromatin escape of a novel, large class of mRNAs that we term Genomically Retained Incompletely Processed Polyadenylated Transcripts (GRIPPs). Using nascent and total transcriptomics, spike-in controlled fractionated cell transcriptomics, and total and fractionated cell proteomics, we show that PRMT5 inhibition and knockdown of the PRMT5 SNRP (Sm protein) adapter protein pICln (CLNS1A), but not type I PRMT inhibition, leads to gross detention of mRNA, SNRPB, and SNRPD3 proteins on chromatin. Compared to most transcripts, these chromatin-trapped polyadenylated RNA transcripts have more introns, are spliced slower, and are enriched in detained introns. Using a combination of PRMT5 inhibition and inducible isogenic wildtype and arginine-mutant SNRPB, we show that arginine methylation of these snRNPs is critical for mediating their homeostatic chromatin and RNA interactions. Overall, we conclude that a major role for PRMT5 is in controlling transcript processing and splicing completion to promote chromatin escape and subsequent nuclear export.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"79 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941441","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 : 2024-08-10DOI: 10.1101/2024.08.09.607297
Anna Hoyle, Joan Chang, Marie FA Cutiongco, Ronan O'Cualain, Stacey Warwood, David Knight, Qing-Jun Meng, Karl E Kadler, Joe Swift
Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix (ECM), assembled during development, stays with us for life. However, recent work suggests that type-I collagen in ECM-rich mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Despite this, the underlying regulation of such distinct pools is not understood. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives. Furthermore, the properties of the matrix pools are altered during development and ageing. Tail tendon tissue was harvested from mice fed on a heavy-lysine diet; protein was then extracted for analysis using a sequential two-step protocol. The first, soluble fraction (F1) was found to contain intracellular proteins, and a range of core and associated extracellular matrix proteins, including a pool of type-I collagen shown to be circadian-regulated. The remaining fraction (F2) contained primarily collagens, including type-I collagen which did not show rhythmicity. In adult mice, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 ± 2 days in F1, compared to 700 ± 100 days in F2. Circadian-regulated matrix proteins were found to have significantly faster turnover than non-circadian in adult mice, but this distinction was lost in older animals. This work identifies protein turnover as the underlying mechanism for the circadian/permanent model of tendon matrix, and suggests a loss of circadian regulation as a characteristic of ECM ageing.
在核弹试验时代长大的人的肌腱中的重碳同位素表明，在发育过程中形成的细胞外基质（ECM）会伴随我们一生。然而，最近的研究表明，在富含 ECM 的小鼠肌腱中，I 型胶原蛋白存在于两个池中：一个是永久性基质，另一个是可溶性更强、受昼夜节律调节的基质。尽管如此，人们仍不了解这种不同池的基本调控机制。在这里，我们利用稳定同位素标记和质谱蛋白质组学证明，昼夜节律基质池和永久基质池的半衰期明显不同。此外，基质池的特性在发育和老化过程中会发生改变。从小鼠尾肌腱组织中获取重赖氨酸饮食，然后采用连续两步法提取蛋白质进行分析。第一个可溶性部分（F1）含有细胞内蛋白质、一系列核心蛋白质和相关细胞外基质蛋白质，其中包括一个Ⅰ型胶原蛋白池，该蛋白池显示受昼夜节律调控。其余部分（F2）主要含有胶原蛋白，包括未显示节律性的 I 型胶原蛋白。在成年小鼠体内，F1池中提取的基质蛋白的半衰期明显短于F2，其中I型胶原蛋白在F1中的半衰期为4±2天，而在F2中为700±100天。研究发现，在成年小鼠体内，昼夜节律调控基质蛋白的周转速度明显快于非昼夜节律调控基质蛋白，但这种区别在年长动物体内消失了。这项工作确定了蛋白质周转是肌腱基质昼夜节律/永久模型的基本机制，并表明昼夜节律调节的丧失是 ECM 老化的一个特征。
{"title":"Circadian and permanent pools of extracellular matrix co-exist in tendon tissue, but have distinct rates of turnover and differential responses to ageing","authors":"Anna Hoyle, Joan Chang, Marie FA Cutiongco, Ronan O'Cualain, Stacey Warwood, David Knight, Qing-Jun Meng, Karl E Kadler, Joe Swift","doi":"10.1101/2024.08.09.607297","DOIUrl":"https://doi.org/10.1101/2024.08.09.607297","url":null,"abstract":"Heavy carbon isotopes in the tendons of people who grew up in the age of nuclear bomb testing have shown that the extracellular matrix (ECM), assembled during development, stays with us for life. However, recent work suggests that type-I collagen in ECM-rich mouse tendon exists in two pools: a permanent matrix, and a more soluble, circadian-regulated matrix. Despite this, the underlying regulation of such distinct pools is not understood. Here, we demonstrate using stable isotope labelling coupled with mass spectrometry proteomics that circadian and permanent matrix pools have significantly different half-lives. Furthermore, the properties of the matrix pools are altered during development and ageing. Tail tendon tissue was harvested from mice fed on a heavy-lysine diet; protein was then extracted for analysis using a sequential two-step protocol. The first, soluble fraction (F1) was found to contain intracellular proteins, and a range of core and associated extracellular matrix proteins, including a pool of type-I collagen shown to be circadian-regulated. The remaining fraction (F2) contained primarily collagens, including type-I collagen which did not show rhythmicity. In adult mice, matrix proteins extracted in the F1 pool had significantly shorter half-lives than F2, including type-I collagen which had half-lives of 4 ± 2 days in F1, compared to 700 ± 100 days in F2. Circadian-regulated matrix proteins were found to have significantly faster turnover than non-circadian in adult mice, but this distinction was lost in older animals. This work identifies protein turnover as the underlying mechanism for the circadian/permanent model of tendon matrix, and suggests a loss of circadian regulation as a characteristic of ECM ageing.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941438","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 : 2024-08-10DOI: 10.1101/2024.08.09.607348
Courtney Clark, Amir Barzegar-Behrooz, Marco Cordani, Shahla Shojaei, Saeid Ghavami
Autophagy is a critical cellular process involved in the degradation and recycling of cytoplasmic components, playing a dual role in cancer by either promoting cell survival or facilitating cell death. In glioblastoma (GB), autophagy has been implicated in resistance to the chemotherapeutic agent Temozolomide (TMZ). This study presents a novel method to accurately measure autophagy flux in TMZ-resistant glioblastoma cells, combining advanced imaging techniques with biochemical assays. By quantifying key autophagy markers such as LC3-II and SQSTM1, our approach provides detailed insights into the dynamic processes of autophagosome formation and clearance under therapeutic stress. This method not only advances our understanding of autophagy in GB chemoresistance but also has significant implications for the development of autophagy-targeted therapies. The ability to monitor and manipulate autophagy flux in real-time offers a promising avenue for monitoring and understanding TMZ resistance and improving patient outcomes in glioblastoma treatment.
{"title":"Assessing Autophagy Flux in Glioblastoma Temozolomide Resistant Cells","authors":"Courtney Clark, Amir Barzegar-Behrooz, Marco Cordani, Shahla Shojaei, Saeid Ghavami","doi":"10.1101/2024.08.09.607348","DOIUrl":"https://doi.org/10.1101/2024.08.09.607348","url":null,"abstract":"Autophagy is a critical cellular process involved in the degradation and recycling of cytoplasmic components, playing a dual role in cancer by either promoting cell survival or facilitating cell death. In glioblastoma (GB), autophagy has been implicated in resistance to the chemotherapeutic agent Temozolomide (TMZ). This study presents a novel method to accurately measure autophagy flux in TMZ-resistant glioblastoma cells, combining advanced imaging techniques with biochemical assays. By quantifying key autophagy markers such as LC3-II and SQSTM1, our approach provides detailed insights into the dynamic processes of autophagosome formation and clearance under therapeutic stress. This method not only advances our understanding of autophagy in GB chemoresistance but also has significant implications for the development of autophagy-targeted therapies. The ability to monitor and manipulate autophagy flux in real-time offers a promising avenue for monitoring and understanding TMZ resistance and improving patient outcomes in glioblastoma treatment.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"44 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-08-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141941657","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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