Pub Date : 2024-09-11DOI: 10.1101/2024.09.11.612370
Matthias Felten, Luiz-Gustavo Teixeira Alves, Eleftheria Letsiou, Elvira Alfaro Arnedo, Sergio Pineiro-Hermida, Iciar P. Lopez, Kristina Dietert, Theresa C. Broemmel, Achim D. Gruber, Diana Fatykhova, Andreas C. Hocke, Tilmann Lingscheid, Jasmin Lienau, Gernot Rohde, Capnetz Study Group, Jose G. Pichel, Martin Witzenrath
Background:Streptococcus pneumoniae (S.pn) is the most prevalent causal bacterial pathogen in community-acquired pneumonia. Despite appropriate antimicrobial therapy, pneumococcal pneumonia can progress to acute respiratory distress syndrome where actual therapies are mainly supportive, and the discovery of new molecular targets is needed. Objective: To investigate the role of IGF1R (Insulin-like Growth Factor 1 Receptor) in pneumococcal pneumonia. Methods:Igf1r-deficient (UBC-CreERT2; Igf1rfl/fl) and control (Igf1rfl/fl) mice were infected with 5x106 S.pn (PN36) or PBS (sham infected). Mice were sacrificed 48 h after infection. Pulmonary permeability, local inflammatory response, and pulmonary and extra-pulmonary bacterial loads were analyzed. Further, IGF1R protein expression was determined in human lung tissue after S.pn infection and IGF1 and IGF1R levels were determined serum of pneumonia patients. Results: In patients and mice infected with S.pn, IGF1 signaling was significantly altered. Igf1r-deficient mice had significantly increased pulmonary permeability after infection with increased pulmonary inflammatory cytokine levels, while inflammatory cell recruitment was not altered compared to infected Igf1rfl/fl control animals. Pulmonary bacterial load was significantly higher in Igf1r-deficient mice, and histological analysis confirmed increased alveolar edema and necrosis compared to infected Igf1rfl/fl control and sham-infected mice. Ex vivo, S.pn caused a decrease in IGF1R protein expression in human lung tissue. Conclusion: Our results demonstrate a significant regulation of IGF1R in ex-vivo infected human lung tissue and in serum of S.pn pneumonia patients. Moreover, pneumonia severity was increased in Igf1r-deficient mice upon S.pn infection compared to Igf1rfl/fl control mice, suggesting that IGF1R plays a protective role in pneumococcal pneumonia.
{"title":"IGF1R is protective in pneumococcal pneumonia","authors":"Matthias Felten, Luiz-Gustavo Teixeira Alves, Eleftheria Letsiou, Elvira Alfaro Arnedo, Sergio Pineiro-Hermida, Iciar P. Lopez, Kristina Dietert, Theresa C. Broemmel, Achim D. Gruber, Diana Fatykhova, Andreas C. Hocke, Tilmann Lingscheid, Jasmin Lienau, Gernot Rohde, Capnetz Study Group, Jose G. Pichel, Martin Witzenrath","doi":"10.1101/2024.09.11.612370","DOIUrl":"https://doi.org/10.1101/2024.09.11.612370","url":null,"abstract":"<strong>Background:</strong> <em>Streptococcus pneumoniae (S.pn)</em> is the most prevalent causal bacterial pathogen in community-acquired pneumonia. Despite appropriate antimicrobial therapy, pneumococcal pneumonia can progress to acute respiratory distress syndrome where actual therapies are mainly supportive, and the discovery of new molecular targets is needed. <strong>Objective:</strong> To investigate the role of IGF1R (Insulin-like Growth Factor 1 Receptor) in pneumococcal pneumonia. <strong>Methods:</strong> <em>Igf1r</em>-deficient (<em>UBC-CreERT2; Igf1r<sup>fl/fl</sup></em>) and control (<em>Igf1r<sup>fl/fl</sup></em>) mice were infected with 5x106 <em>S.pn</em> (PN36) or PBS (sham infected). Mice were sacrificed 48 h after infection. Pulmonary permeability, local inflammatory response, and pulmonary and extra-pulmonary bacterial loads were analyzed. Further, IGF1R protein expression was determined in human lung tissue after <em>S.pn</em> infection and IGF1 and IGF1R levels were determined serum of pneumonia patients. <strong>Results: </strong>In patients and mice infected with <em>S.pn</em>, IGF1 signaling was significantly altered. <em>Igf1r</em>-deficient mice had significantly increased pulmonary permeability after infection with increased pulmonary inflammatory cytokine levels, while inflammatory cell recruitment was not altered compared to infected <em>Igf1r<sup>fl/fl</sup></em> control animals. Pulmonary bacterial load was significantly higher in <em>Igf1r</em>-deficient mice, and histological analysis confirmed increased alveolar edema and necrosis compared to infected <em>Igf1r<sup>fl/fl</sup></em> control and sham-infected mice. Ex vivo, <em>S.pn</em> caused a decrease in IGF1R protein expression in human lung tissue. <strong>Conclusion:</strong> Our results demonstrate a significant regulation of IGF1R in ex-vivo infected human lung tissue and in serum of <em>S.pn</em> pneumonia patients. Moreover, pneumonia severity was increased in <em>Igf1r</em>-deficient mice upon S.pn infection compared to <em>Igf1r<sup>fl/fl</sup></em> control mice, suggesting that IGF1R plays a protective role in pneumococcal pneumonia.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"59 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212783","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 : 2024-09-11DOI: 10.1101/2024.09.11.612516
Zhenyu Wang, Armaan Mehra, Quian-Chen Wang, Savita Gupta, Agatha Ribeiro da Silva, Thomas Juan, Stephan Gunther, Jan Detleffsen, Mario Looso, Didier Y.R. Stainier, Ruben Marin-Juez
VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1/FLT1, one can achieve more physiological VEGFA concentrations. We find that following cryoinjury, zebrafish flt1 mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in flt1 mutants abrogates these beneficial effects of flt1 deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene egr3. Using newly generated genetic tools, we observe egr3 upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via egr3 downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury.
{"title":"flt1 inactivation promotes zebrafish cardiac regeneration by enhancing endothelial activity and limiting the fibrotic response","authors":"Zhenyu Wang, Armaan Mehra, Quian-Chen Wang, Savita Gupta, Agatha Ribeiro da Silva, Thomas Juan, Stephan Gunther, Jan Detleffsen, Mario Looso, Didier Y.R. Stainier, Ruben Marin-Juez","doi":"10.1101/2024.09.11.612516","DOIUrl":"https://doi.org/10.1101/2024.09.11.612516","url":null,"abstract":"VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1/FLT1, one can achieve more physiological VEGFA concentrations. We find that following cryoinjury, zebrafish <em>flt1</em> mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in <em>flt1</em> mutants abrogates these beneficial effects of <em>flt1</em> deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene <em>egr3</em>. Using newly generated genetic tools, we observe <em>egr3</em> upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via <em>egr3</em> downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"32 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212746","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 : 2024-09-10DOI: 10.1101/2024.09.10.611870
Timothy Klouda, Savas T. Tsikis, Thomas I. Hirsch, Yunhye Kim, Tiffany Liu, Ingeborg Friehs, John Y.-J. Shyy, Gary Visner, Benjamin A Raby, Mark Puder, Ke Yuan
Patients with congenital heart disease (CHD) resulting in significant left-to-right shunting of blood are at risk for the development of pulmonary arterial hypertension (PAH). The underlying mechanism by which pulmonary overcirculation and shear stress lead to vascular remodeling remains unclear. Our study established a new two-hit murine model of severe pulmonary hypertension (PH) by combining left pneumonectomy and exposure to hypoxia (LP/Hx). Utilizing transgenic reporter lines, immunofluorescence staining, and advanced microscopy, we conducted cell-lineage tracing experiments for endothelial cells (ECs), smooth muscle cells (SMCs), and pericytes. We identified that SMCs is a primary contributor to distal arteriolar remodeling after LP/Hx. Subsequent qPCR analysis on isolated cells demonstrated that Cxcl12 was upregulated in both ECs and SMCs from LP/Hx animals. Likewise, CXCL12 was overexpressed in the SMC layer of arterioles in patients with acyanotic PAH-CHD. These findings provide novel insights into the contribution of SMCs and Cxcl12 to pulmonary flow-induced vascular remodeling. This newly established murine model of PH will serve as a new tool for research and targeted therapeutics for patients with PAH.
{"title":"Smooth muscle Cxcl12 activation is associated with vascular remodeling in flow-induced pulmonary hypertension","authors":"Timothy Klouda, Savas T. Tsikis, Thomas I. Hirsch, Yunhye Kim, Tiffany Liu, Ingeborg Friehs, John Y.-J. Shyy, Gary Visner, Benjamin A Raby, Mark Puder, Ke Yuan","doi":"10.1101/2024.09.10.611870","DOIUrl":"https://doi.org/10.1101/2024.09.10.611870","url":null,"abstract":"Patients with congenital heart disease (CHD) resulting in significant left-to-right shunting of blood are at risk for the development of pulmonary arterial hypertension (PAH). The underlying mechanism by which pulmonary overcirculation and shear stress lead to vascular remodeling remains unclear. Our study established a new two-hit murine model of severe pulmonary hypertension (PH) by combining left pneumonectomy and exposure to hypoxia (LP/Hx). Utilizing transgenic reporter lines, immunofluorescence staining, and advanced microscopy, we conducted cell-lineage tracing experiments for endothelial cells (ECs), smooth muscle cells (SMCs), and pericytes. We identified that SMCs is a primary contributor to distal arteriolar remodeling after LP/Hx. Subsequent qPCR analysis on isolated cells demonstrated that Cxcl12 was upregulated in both ECs and SMCs from LP/Hx animals. Likewise, CXCL12 was overexpressed in the SMC layer of arterioles in patients with acyanotic PAH-CHD. These findings provide novel insights into the contribution of SMCs and Cxcl12 to pulmonary flow-induced vascular remodeling. This newly established murine model of PH will serve as a new tool for research and targeted therapeutics for patients with PAH.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"273 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212791","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 : 2024-09-10DOI: 10.1101/2024.09.09.612157
Mingqing Chen, Jianke Gong
There are several causes of mating-induced physiological changes in hermaphrodites exposed to males, such as functional male sperms causing shrinking and the male pheromone mediating shortened lifespan, which utilize different molecular pathways and are shared across species. However, it is unclear whether the male seminal fluid protein contributes to this post-mating regulation. Here, we investigated the transmit way and the impacts of the Caenorhabditis male seminal fluid protein SFP-1 in mated hermaphrodite tissues. We find that SFP-1 is the key component in seminal fluid to induce post-mating physiological changes in mated hermaphrodites. It acts as a cargo packing into exophers which require the phospholipid scramblase ANOH-1 and ANOH-2 to develop in male seminal vesicle. Exophers carrying SFP-1 cross over the somatic gonad uterus and eventually the protein is uptaken by the intestinal cells via endocytosis. Within the intestine, the NTF2-like domain of SFP-1 assists the association and interaction with the transcription factors SKN-1 and DAF-16 to induce post-mating somatic fat depletion and a shortened lifespan. Our study reveals the elaborate strategies of the male seminal fluid protein on triggering mating-induced physiological changes elicited by sexual interactions that could exist in other species.
{"title":"A male seminal fluid protein SFP-1 regulates hermaphrodite post-mating longevity and fat metabolism in Caenorhabditis elegans","authors":"Mingqing Chen, Jianke Gong","doi":"10.1101/2024.09.09.612157","DOIUrl":"https://doi.org/10.1101/2024.09.09.612157","url":null,"abstract":"There are several causes of mating-induced physiological changes in hermaphrodites exposed to males, such as functional male sperms causing shrinking and the male pheromone mediating shortened lifespan, which utilize different molecular pathways and are shared across species. However, it is unclear whether the male seminal fluid protein contributes to this post-mating regulation. Here, we investigated the transmit way and the impacts of the Caenorhabditis male seminal fluid protein SFP-1 in mated hermaphrodite tissues. We find that SFP-1 is the key component in seminal fluid to induce post-mating physiological changes in mated hermaphrodites. It acts as a cargo packing into exophers which require the phospholipid scramblase ANOH-1 and ANOH-2 to develop in male seminal vesicle. Exophers carrying SFP-1 cross over the somatic gonad uterus and eventually the protein is uptaken by the intestinal cells via endocytosis. Within the intestine, the NTF2-like domain of SFP-1 assists the association and interaction with the transcription factors SKN-1 and DAF-16 to induce post-mating somatic fat depletion and a shortened lifespan. Our study reveals the elaborate strategies of the male seminal fluid protein on triggering mating-induced physiological changes elicited by sexual interactions that could exist in other species.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"22 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212806","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 : 2024-09-10DOI: 10.1101/2024.09.09.612167
Ruijie Yu, Prabhjeet Phalora, Nan Li, Till Boecking, David A Jacques
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development.
人类 T 细胞白血病病毒 1 型（HTLV-1）是一种无法治疗的逆转录病毒，可导致致命的恶性肿瘤和退行性炎症。由于我们对基本病毒学的认识存在巨大差距，特别是与密切相关的艾滋病毒相比，有效的治疗方法迟迟未能问世。最近开发的一种非常有效的抗艾滋病病毒策略是针对病毒使用药物，干扰病毒的外壳完整性以及与宿主的相互作用。重要的是，首个获批的抗病毒囊药物来那卡巴韦可提供长效的暴露前预防。这种特性为预防 HTLV-1 的传播提供了一种手段，但其噬菌体以前从未被视为药物靶点。在这里，我们描述了 HTLV-1 外壳蛋白的首个高分辨率晶体结构，定义了重要的晶格界面，并确定了一个以前未知的配体结合口袋。我们发现这个口袋对病毒的感染性至关重要，为未来抗病毒药物的开发提供了一个潜在靶点。
{"title":"The Human T-cell Leukemia Virus capsid protein is a potential drug target","authors":"Ruijie Yu, Prabhjeet Phalora, Nan Li, Till Boecking, David A Jacques","doi":"10.1101/2024.09.09.612167","DOIUrl":"https://doi.org/10.1101/2024.09.09.612167","url":null,"abstract":"Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"52 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212789","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 : 2024-09-10DOI: 10.1101/2024.09.10.612038
Shardul Kulkarni, Alexis Morrissey, Aswathy Sebastian, Cheryl A Keller, Belinda Giardine, Courtney Smith, Oluwasegun T Akinniyi, Alexei Arnaoutov, Istvan Albert, Shaun Mahony, Joseph C. Reese
CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation, and translation. It has been well studied in budding yeast; however, relatively less is known about its regulation and functions in mammals. To characterize the functions of the human CCR4-NOT complex, we developed a rapid auxin-induced degron system to deplete CNOT1 (the scaffold of the complex) and CNOT4 (E3 ubiquitin ligase) in cell culture. Transcriptome-wide measurements of gene-expression revealed that depleting CNOT1 changed several thousand transcripts, wherein most mRNAs were increased and resulted in a global decrease in mRNA decay rates. In contrast to what was observed in CNOT1-depleted cells, CNOT4 depletion only modestly changed RNA steady-state levels and, surprisingly, led to a global acceleration in mRNA decay. To further investigate the role of CCR4-NOT in transcription, we used transient transcriptome sequencing (TT-seq) to measure ongoing RNA synthesis. Depletion of either subunit resulted in increased RNA synthesis of several thousand genes. In contrast to most of the genome, a rapid reduction in the synthesis of KRAB-Zinc-Finger-proteins (KZNFs) genes, especially those clustered on chromosome 19, was observed. KZNFs are transcriptional repressors of retro-transposable elements (rTEs), and consistent with the decreased KZNFs expression, we observed a significant and rapid activation of rTEs, mainly Long interspersed Nuclear Elements (LINEs). Our data reveal that CCR4-NOT regulates gene expression and silences retrotransposons across the genome by maintaining KZNF expression. These data establish CCR4-NOT as a global regulator of gene expression, and we have identified a novel mammalian-specific function of the complex, the suppression of rTEs.
{"title":"Human CCR4-NOT is a global regulator of gene expression and is a novel silencer of retrotransposon activation","authors":"Shardul Kulkarni, Alexis Morrissey, Aswathy Sebastian, Cheryl A Keller, Belinda Giardine, Courtney Smith, Oluwasegun T Akinniyi, Alexei Arnaoutov, Istvan Albert, Shaun Mahony, Joseph C. Reese","doi":"10.1101/2024.09.10.612038","DOIUrl":"https://doi.org/10.1101/2024.09.10.612038","url":null,"abstract":"CCR4-NOT regulates multiple steps in gene regulation, including transcription, mRNA decay, protein ubiquitylation, and translation. It has been well studied in budding yeast; however, relatively less is known about its regulation and functions in mammals. To characterize the functions of the human CCR4-NOT complex, we developed a rapid auxin-induced degron system to deplete CNOT1 (the scaffold of the complex) and CNOT4 (E3 ubiquitin ligase) in cell culture. Transcriptome-wide measurements of gene-expression revealed that depleting CNOT1 changed several thousand transcripts, wherein most mRNAs were increased and resulted in a global decrease in mRNA decay rates. In contrast to what was observed in CNOT1-depleted cells, CNOT4 depletion only modestly changed RNA steady-state levels and, surprisingly, led to a global acceleration in mRNA decay. To further investigate the role of CCR4-NOT in transcription, we used transient transcriptome sequencing (TT-seq) to measure ongoing RNA synthesis. Depletion of either subunit resulted in increased RNA synthesis of several thousand genes. In contrast to most of the genome, a rapid reduction in the synthesis of KRAB-Zinc-Finger-proteins (KZNFs) genes, especially those clustered on chromosome 19, was observed. KZNFs are transcriptional repressors of retro-transposable elements (rTEs), and consistent with the decreased KZNFs expression, we observed a significant and rapid activation of rTEs, mainly Long interspersed Nuclear Elements (LINEs). Our data reveal that CCR4-NOT regulates gene expression and silences retrotransposons across the genome by maintaining KZNF expression. These data establish CCR4-NOT as a global regulator of gene expression, and we have identified a novel mammalian-specific function of the complex, the suppression of rTEs.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212784","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 : 2024-09-10DOI: 10.1101/2024.09.10.612331
Brittany Cain, Zhenyu Yuan, Evelyn Thoman, Rhett Kovall, Brian Gebelein
How homeodomain proteins gain sufficient DNA binding specificity to regulate diverse processes has been a long-standing question. Here, we determine how the ALX4 Paired-like protein achieves DNA binding specificity for a TAATNNNATTA dimer site. We first show that ALX4 binds this motif independently of its co-factor, TWIST1, in cranial neural crest cells. Structural analysis identified seven ALX4 residues that participate in dimer binding, many of which are conserved across the Paired-like family, but not other homeodomain proteins. Unexpectedly, the two ALX4 proteins within the dimer use distinct residues to form asymmetric protein-protein and protein-DNA interactions to mediate cooperativity. Moreover, we found that ALX4 cooperativity is required for transcriptional activation and that ALX4 disease variants cause distinct molecular defects that include loss of cooperativity. These findings provide new insights into how Paired-like factors gain DNA specificity and show how disease variants can be stratified based on their molecular defects.
{"title":"Structural and biochemical characterization of the ALX4 dimer reveals novel insights into how disease alleles impact ALX4 function","authors":"Brittany Cain, Zhenyu Yuan, Evelyn Thoman, Rhett Kovall, Brian Gebelein","doi":"10.1101/2024.09.10.612331","DOIUrl":"https://doi.org/10.1101/2024.09.10.612331","url":null,"abstract":"How homeodomain proteins gain sufficient DNA binding specificity to regulate diverse processes has been a long-standing question. Here, we determine how the ALX4 Paired-like protein achieves DNA binding specificity for a TAATNNNATTA dimer site. We first show that ALX4 binds this motif independently of its co-factor, TWIST1, in cranial neural crest cells. Structural analysis identified seven ALX4 residues that participate in dimer binding, many of which are conserved across the Paired-like family, but not other homeodomain proteins. Unexpectedly, the two ALX4 proteins within the dimer use distinct residues to form asymmetric protein-protein and protein-DNA interactions to mediate cooperativity. Moreover, we found that ALX4 cooperativity is required for transcriptional activation and that ALX4 disease variants cause distinct molecular defects that include loss of cooperativity. These findings provide new insights into how Paired-like factors gain DNA specificity and show how disease variants can be stratified based on their molecular defects.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212788","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 : 2024-09-10DOI: 10.1101/2024.09.10.612279
Christopher J. Walkey, Kathy J. Snow, Jote Bulcha, Aaron R. Cox, Alexa E. Martinez, M. Cecilia Ljungberg, Denise G. Lanza, Marco De Giorgi, Marcel A. Chuecos, Michele Alves-Bezerra, Carlos Flores Suarez, Sean M. Hartig, Susan G. Hilsenbeck, Chih-Wei Hsu, Ethan Saville, Yaned Gaitan, Jeff Duryea, Seth Hannigan, Mary E. Dickinson, Oleg Mirochnitchenko, Dan Wang, Cathleen M. Lutz, Jason D. Heaney, Guangping Gao, Steve A. Murray, William R. Lagor
Gene therapy with Adeno-Associated Viral (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of ten naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 and AAVrh74) following systemic delivery into male and female mice. A transgene expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.
{"title":"A Comprehensive Atlas of AAV Tropism in the Mouse","authors":"Christopher J. Walkey, Kathy J. Snow, Jote Bulcha, Aaron R. Cox, Alexa E. Martinez, M. Cecilia Ljungberg, Denise G. Lanza, Marco De Giorgi, Marcel A. Chuecos, Michele Alves-Bezerra, Carlos Flores Suarez, Sean M. Hartig, Susan G. Hilsenbeck, Chih-Wei Hsu, Ethan Saville, Yaned Gaitan, Jeff Duryea, Seth Hannigan, Mary E. Dickinson, Oleg Mirochnitchenko, Dan Wang, Cathleen M. Lutz, Jason D. Heaney, Guangping Gao, Steve A. Murray, William R. Lagor","doi":"10.1101/2024.09.10.612279","DOIUrl":"https://doi.org/10.1101/2024.09.10.612279","url":null,"abstract":"Gene therapy with Adeno-Associated Viral (AAV) vectors requires knowledge of their tropism within the body. Here we analyze the tropism of ten naturally occurring AAV serotypes (AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, AAVrh10 and AAVrh74) following systemic delivery into male and female mice. A transgene expressing ZsGreen and Cre recombinase was used to identify transduction in a cell-dependent manner based on fluorescence. Cre-driven activation of tdTomato fluorescence offered superior sensitivity for transduced cells. All serotypes except AAV3B and AAV4 had high liver tropism. Fluorescence activation revealed transduction of unexpected tissues, including adrenals, testes and ovaries. Rare transduced cells within tissues were also readily visualized. Biodistribution of AAV genomes correlated with fluorescence, except in immune tissues. AAV4 was found to have a pan-endothelial tropism while also targeting pancreatic beta cells. This public resource enables selection of the best AAV serotypes for basic science and preclinical applications in mice.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"2 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212790","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 : 2024-09-10DOI: 10.1101/2024.09.10.612082
Ekaterina Shuvalova, Alexey Shuvalov, Walaa Al Sheikh, Alexander V. Ivanov, Nikita Biziaev, Tatiana V. Egorova, Sergey E. Dmitriev, Ilya M. Terenin, Elena Alkalaeva
Eukaryotic translation initiation factor eIF4F, comprising subunits eIF4G, eIF4E, and eIF4A, plays a pivotal role in the 48S preinitiation complex assembly and ribosomal scanning. Additionally, eIF4B enhances the helicase activity of eIF4A. eIF4F also interacts with PABP bound to the poly(A) tail of mRNA, thereby forming a closed-loop structure. PABP, in turn, interacts with eRF3, stimulating translation termination. Here, we employed a reconstituted mammalian system to directly demonstrate that eIF4F potently enhances translation termination. Specifically, eIF4A and eIF4B promote the loading of eRF1 into the A site of the ribosome, while eIF4G1 stimulates the GTPase activity of eRF3 and facilitates the dissociation of release factors following peptide release. We also identified MIF4G as the minimal domain required for this activity and show that eIF4G2/DAP5 can also promote termination. Our findings provide compelling evidence that the closed-loop mRNA structure facilitates translation termination, with PABP and eIF4F directly involved in this process.
{"title":"Eukaryotic initiation factors eIF4F and eIF4B promote translation termination upon closed-loop formation","authors":"Ekaterina Shuvalova, Alexey Shuvalov, Walaa Al Sheikh, Alexander V. Ivanov, Nikita Biziaev, Tatiana V. Egorova, Sergey E. Dmitriev, Ilya M. Terenin, Elena Alkalaeva","doi":"10.1101/2024.09.10.612082","DOIUrl":"https://doi.org/10.1101/2024.09.10.612082","url":null,"abstract":"Eukaryotic translation initiation factor eIF4F, comprising subunits eIF4G, eIF4E, and eIF4A, plays a pivotal role in the 48S preinitiation complex assembly and ribosomal scanning. Additionally, eIF4B enhances the helicase activity of eIF4A. eIF4F also interacts with PABP bound to the poly(A) tail of mRNA, thereby forming a closed-loop structure. PABP, in turn, interacts with eRF3, stimulating translation termination. Here, we employed a reconstituted mammalian system to directly demonstrate that eIF4F potently enhances translation termination. Specifically, eIF4A and eIF4B promote the loading of eRF1 into the A site of the ribosome, while eIF4G1 stimulates the GTPase activity of eRF3 and facilitates the dissociation of release factors following peptide release. We also identified MIF4G as the minimal domain required for this activity and show that eIF4G2/DAP5 can also promote termination. Our findings provide compelling evidence that the closed-loop mRNA structure facilitates translation termination, with PABP and eIF4F directly involved in this process.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"7 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212792","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 : 2024-09-10DOI: 10.1101/2024.09.10.612337
Lihu Gong, Xiuli Liu, Xin Yang, Ze Yu, Siming Chen, Chao Xing, Xin Liu
Polycomb repressive complex 2 (PRC2) mediates developmental gene repression as two classes of holocomplexes, PRC2.1 and PRC2.2. EPOP is an accessory subunit specific to PRC2.1, which also contains PCL proteins. Unlike other accessory subunits that collectively facilitate PRC2 targeting, EPOP was implicated in an enigmatic inhibitory role, together with its interactor Elongin BC. We report an unusual molecular mechanism whereby EPOP regulates PRC2.1 by directly modulating its oligomerization state. EPOP disrupts the PRC2.1 dimer and weakens its chromatin association, likely by disabling the avidity effect conferred by the dimeric complex. Congruently, an EPOP mutant specifically defective in PRC2 binding enhances genome-wide enrichments of MTF2 and H3K27me3 in mouse epiblast-like cells. Elongin BC is largely dispensable for the EPOP-mediated inhibition of PRC2.1. EPOP defines a distinct subclass of PRC2.1, which uniquely maintains an epigenetic program by preventing the over-repression of key gene regulators along the continuum of early differentiation.
{"title":"EPOP Restricts PRC2.1 Targeting to Chromatin by Directly Modulating Enzyme Complex Dimerization","authors":"Lihu Gong, Xiuli Liu, Xin Yang, Ze Yu, Siming Chen, Chao Xing, Xin Liu","doi":"10.1101/2024.09.10.612337","DOIUrl":"https://doi.org/10.1101/2024.09.10.612337","url":null,"abstract":"Polycomb repressive complex 2 (PRC2) mediates developmental gene repression as two classes of holocomplexes, PRC2.1 and PRC2.2. EPOP is an accessory subunit specific to PRC2.1, which also contains PCL proteins. Unlike other accessory subunits that collectively facilitate PRC2 targeting, EPOP was implicated in an enigmatic inhibitory role, together with its interactor Elongin BC. We report an unusual molecular mechanism whereby EPOP regulates PRC2.1 by directly modulating its oligomerization state. EPOP disrupts the PRC2.1 dimer and weakens its chromatin association, likely by disabling the avidity effect conferred by the dimeric complex. Congruently, an EPOP mutant specifically defective in PRC2 binding enhances genome-wide enrichments of MTF2 and H3K27me3 in mouse epiblast-like cells. Elongin BC is largely dispensable for the EPOP-mediated inhibition of PRC2.1. EPOP defines a distinct subclass of PRC2.1, which uniquely maintains an epigenetic program by preventing the over-repression of key gene regulators along the continuum of early differentiation.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"33 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142212785","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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