Pub Date : 2024-09-19DOI: 10.1101/2024.09.18.613678
Roxana Andreea Moldovan, Marta R Hidalgo, Helena Castane, Andrea Jimenez-Franco, Jorge Joven, Deborah J Burks, Amparo Galan, Francisco Garcia-Garcia
Obesity represents a significant risk factor in the development of type 2 diabetes (T2D), a chronic metabolic disorder characterized by elevated blood glucose levels. Significant sex differences have been identified in the prevalence, development, and pathophysiology of obesity and T2D; however, the underlying molecular mechanisms remain unclear. This study aims to identify sex-specific biomarkers in obesity and T2D and enhance our understanding of the underlying mechanisms associated with sex differences by integrating expression data. A systematic review, individual transcriptomic analysis, gene-level meta-analysis, and functional characterization were performed to achieve this aim. Eight studies and 236 subcutaneous adipose tissue samples were analyzed, identifying common and sex-specific biomarkers, many of which were previously associated with obesity or T2D. The obesity meta-analysis yielded nineteen differentially-expressed biomarkers from a sex-specific perspective (e.g., SPATA18, KREMEN1, NPY4R, and PRM3), while a comparison of the expression profiles between sexes in T2D prompted the identification and validation of specific transcriptomic signatures in males (SAMD9, NBPF3, LDHD, and EHD3) and females (RETN, HEY1, PLPP2, and PM20D2). At the functional level, we highlighted the fundamental role of the Wnt pathway in the development of obesity and T2D in females and the roles of more significant mitochondrial damage and free fatty acids in males. Overall, our sex-specific meta-analyses supported the detection of differentially expressed genes in males and females associated with the development of obesity and T2D, emphasizing the relevance of sex-based information in biomedical data and opening new avenues for research.
{"title":"Transcriptomic landscape of sex differences in obesity and type 2 diabetes in subcutaneous adipose tissue","authors":"Roxana Andreea Moldovan, Marta R Hidalgo, Helena Castane, Andrea Jimenez-Franco, Jorge Joven, Deborah J Burks, Amparo Galan, Francisco Garcia-Garcia","doi":"10.1101/2024.09.18.613678","DOIUrl":"https://doi.org/10.1101/2024.09.18.613678","url":null,"abstract":"Obesity represents a significant risk factor in the development of type 2 diabetes (T2D), a chronic metabolic disorder characterized by elevated blood glucose levels. Significant sex differences have been identified in the prevalence, development, and pathophysiology of obesity and T2D; however, the underlying molecular mechanisms remain unclear. This study aims to identify sex-specific biomarkers in obesity and T2D and enhance our understanding of the underlying mechanisms associated with sex differences by integrating expression data. A systematic review, individual transcriptomic analysis, gene-level meta-analysis, and functional characterization were performed to achieve this aim. Eight studies and 236 subcutaneous adipose tissue samples were analyzed, identifying common and sex-specific biomarkers, many of which were previously associated with obesity or T2D.\u0000The obesity meta-analysis yielded nineteen differentially-expressed biomarkers from a sex-specific perspective (e.g., SPATA18, KREMEN1, NPY4R, and PRM3), while a comparison of the expression profiles between sexes in T2D prompted the identification and validation of specific transcriptomic signatures in males (SAMD9, NBPF3, LDHD, and EHD3) and females (RETN, HEY1, PLPP2, and PM20D2). At the functional level, we highlighted the fundamental role of the Wnt pathway in the development of obesity and T2D in females and the roles of more significant mitochondrial damage and free fatty acids in males. Overall, our sex-specific meta-analyses supported the detection of differentially expressed genes in males and females associated with the development of obesity and T2D, emphasizing the relevance of sex-based information in biomedical data and opening new avenues for research.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"3 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253036","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 : 2024-09-18DOI: 10.1101/2024.09.16.613366
Nina Karamanova, Kaleb T Morrow, Alana Maerivoet, Jillian Madine, Ming Li, Raymond Q Migrino
Background: Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. Methods: VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Results: Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Conclusions: Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NF?B. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.
{"title":"Medin Induces Pro-Inflammatory Activation of Human Brain Vascular Smooth Muscle Cells","authors":"Nina Karamanova, Kaleb T Morrow, Alana Maerivoet, Jillian Madine, Ming Li, Raymond Q Migrino","doi":"10.1101/2024.09.16.613366","DOIUrl":"https://doi.org/10.1101/2024.09.16.613366","url":null,"abstract":"Background: Medin is one of the most common amyloidogenic proteins and accumulates in the vasculature with aging. Vascular medin accumulation is associated with Alzheimer's disease, vascular dementia and aortic aneurysms. Medin impairs smooth muscle-dependent vasodilation in isolated human brain cerebral arteries. The role of medin in vascular smooth muscle (VSMC) activation is unknown. We aim to evaluate the effects of medin on human brain VSMC activation. Methods: VSMCs were exposed to physiologic doses of medin (0.5, 1 and 5 µM) without or with small molecule nuclear factor-κB (NFκB) inhibitor RO106-9920 (10 µM) for 20 hours. Polymerase chain reaction, Western blot/enzyme-linked immunosorbent assay were used to quantify gene and protein expressions/secretions, respectively, of pro-inflammatory factors (interleukin (IL)-6, IL-8 and monocyte chemoattractant protein (MCP)-1) and structural and enzyme proteins associated with VSMC phenotypic transformation (smooth muscle actin alpha 2 (ACTA2), myosin heavy chain 11 (MYH11) and NADPH oxidase 4 (NOX4)). Results: Medin exposure increased VSMC gene expression and protein secretion of IL-6, IL-8 and MCP-1 (protein secretion 46.0±12.8x, 20.2±4.1x and 8.7±3.1x, respectively, medin 5 µM versus vehicle, all p<0.05). There was no change in gene or protein expressions of ACTA2, MYH11 and NOX4. Co-treatment with RO106-9920 reduced medin-induced increases in IL-6 and IL-8 and a trend towards reduced MCP-1 secretion. Conclusions: Medin induced pro-inflammatory activation of human brain VSMCs that is mediated, at least in part, by NF?B. Acute medin treatment did not alter structural proteins involved in VSMC phenotypic transformation. The findings support medin as a potential novel mediator of and therapeutic target for vascular aging pathology.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"21 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142268925","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 : 2024-09-18DOI: 10.1101/2024.09.18.613780
Gloria R Garcia, Murali Palangat, Josquin Moraly, Benjamin T. Donovan, Bixuan Wang, Ira Phadke, David Sturgill, Jiji Chen, Brittney Short, Gustavo A. Rivero, David M. Swoboda, Naomi Taylor, Kathy McGraw, Daniel R Larson
The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA binding proteins U2AF1/2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-transcribed mitochondrial mRNAs and proteins, inhibits translation, localizes to the outer mitochondrial membrane, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point-mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, and OXPHOS-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial targeting of nuclear-encoded mRNAs.
{"title":"U2AF regulates the translation and localization of nuclear-encoded mitochondrial mRNAs","authors":"Gloria R Garcia, Murali Palangat, Josquin Moraly, Benjamin T. Donovan, Bixuan Wang, Ira Phadke, David Sturgill, Jiji Chen, Brittney Short, Gustavo A. Rivero, David M. Swoboda, Naomi Taylor, Kathy McGraw, Daniel R Larson","doi":"10.1101/2024.09.18.613780","DOIUrl":"https://doi.org/10.1101/2024.09.18.613780","url":null,"abstract":"The mechanisms underlying molecular targeting to mitochondria remain enigmatic, yet this process is crucial for normal cellular function. The RNA binding proteins U2AF1/2 form a heterodimer (U2AF) that shuttles between the nucleus and cytoplasm, regulating splicing in the nucleus and translation in the cytoplasm. Our study identifies an unexpected role for U2AF in mitochondrial function. We demonstrate that U2AF interacts with nuclear-transcribed mitochondrial mRNAs and proteins, inhibits translation, localizes to the outer mitochondrial membrane, and regulates mRNA localization to mitochondria. Moreover, an oncogenic point-mutation in U2AF1(S34F) disrupts this regulation, leading to altered mitochondrial structure, increased translation, and OXPHOS-dependent metabolic rewiring, recapitulating changes observed in bone marrow progenitors from patients with myelodysplastic syndromes. These findings reveal a non-canonical role for U2AF, where it modulates multiple aspects of mitochondrial function by regulating the translation and mitochondrial targeting of nuclear-encoded mRNAs.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253037","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 : 2024-09-18DOI: 10.1101/2024.09.17.613571
Sophie Bonnal, Simon Bajew, Rosa Martinez Corral, Manuel Irimia
Microexons are essential for proper functioning of neurons and pancreatic endocrine cells, where their inclusion depends on the splicing factors SRRM3/4. However, in pancreatic cells, lower expression of these regulators limits inclusion to only the most sensitive subset among all neuronal microexons. Although various cis-acting elements can contribute to microexon regulation, how they determine this differential dose response and high or low sensitivity to SRRM3/4 remains unknown. Here, Massively Parallel Splicing Assays probing 28,535 variants show that sensitivity to SRRM4 is conserved across vertebrates and support a regulatory model whereby high or low microexon sensitivity is largely determined by an interplay between core splicing architecture and length constraints. This conclusion is further supported by distinct spliceosome activities in the absence of SRRM3/4 and by a mathematical model that assumes that the two types of microexons differ only in their efficiency to recruit early spliceosomal components.
{"title":"Core splicing architecture and early spliceosomal recognition determine microexon sensitivity to SRRM3/4","authors":"Sophie Bonnal, Simon Bajew, Rosa Martinez Corral, Manuel Irimia","doi":"10.1101/2024.09.17.613571","DOIUrl":"https://doi.org/10.1101/2024.09.17.613571","url":null,"abstract":"Microexons are essential for proper functioning of neurons and pancreatic endocrine cells, where their inclusion depends on the splicing factors SRRM3/4. However, in pancreatic cells, lower expression of these regulators limits inclusion to only the most sensitive subset among all neuronal microexons. Although various cis-acting elements can contribute to microexon regulation, how they determine this differential dose response and high or low sensitivity to SRRM3/4 remains unknown. Here, Massively Parallel Splicing Assays probing 28,535 variants show that sensitivity to SRRM4 is conserved across vertebrates and support a regulatory model whereby high or low microexon sensitivity is largely determined by an interplay between core splicing architecture and length constraints. This conclusion is further supported by distinct spliceosome activities in the absence of SRRM3/4 and by a mathematical model that assumes that the two types of microexons differ only in their efficiency to recruit early spliceosomal components.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"206 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253038","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 : 2024-09-18DOI: 10.1101/2024.09.17.613470
Jillis Grubben, Gerard Bijsterbosch, Burak Aktürk, Richard G. F. Visser, Henk Schouten
Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks (DSBs) for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the Tomato Mosaic Virus (ToMV) resistance locus in Solanum lycopersicum. We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing DSBs in both alleles of F1 tomato seedlings to promote non-homologous end joining (NHEJ) and homology-directed repair (HDR); second, targeting a single allele in a heterozygous background to induce HDR in seedlings. CRISPR/Cas9 activity was confirmed in F1 seedlings by detecting NHEJ-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing single nucleotide polymorphisms (SNPs) between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F1 progeny, no increase in recombination frequency was observed compared to wild-type plants. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F1 plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs are insufficient to break the genetic linkage at the ToMV locus on chromosome 9 in recombination cold spots within somatic cells.
尽管 CRISPR/Cas9 在诱导 DNA 双链断裂(DSB)以进行基因组编辑方面取得了成功,但在体细胞中实现靶向重组仍具有挑战性,尤其是在重组冷点,如番茄花叶病毒(ToMV)在番茄中的抗性基因座。我们研究了 CRISPR/Cas9 诱导的体细胞定向重组克服 ToMV 基因座周围连锁阻力的潜力。我们采用了两种策略:第一,在 F1 番茄幼苗的两个等位基因中诱导 DSB,以促进非同源末端连接(NHEJ)和同源定向修复(HDR);第二,在杂合背景中靶向单个等位基因,以诱导幼苗中的 HDR。通过检测 ToMV 目标位点上 NHEJ 介导的突变,在 F1 幼苗中证实了 CRISPR/Cas9 的活性。我们开发了一个生物信息学管道,通过分析亲本单倍型之间的单核苷酸多态性(SNP)来识别目标重组子,从而精确追踪 SNP 变异。该研究采用了一种二维汇集策略,以区分真正的重组事件和 PCR 伪影。尽管取得了这些进展,F1 后代中的 CRISPR/Cas9 系统也很活跃,但与野生型植物相比,重组频率并没有增加。我们将研究扩展到原生质体,以评估 CRISPR/Cas9 能否在不同细胞条件下诱导同一基因座的定向重组。与 F1 植物的研究结果一致,我们在原生质体中观察到的重组模式与野生型对照相比没有增加。我们的研究结果表明,CRISPR/Cas9诱导的DSB不足以打破体细胞内重组冷点中9号染色体上ToMV基因座的遗传连接。
{"title":"CRISPR/Cas9-induced breaks are insufficient to break linkage drag surrounding the ToMV locus of Solanum lycopersicum","authors":"Jillis Grubben, Gerard Bijsterbosch, Burak Aktürk, Richard G. F. Visser, Henk Schouten","doi":"10.1101/2024.09.17.613470","DOIUrl":"https://doi.org/10.1101/2024.09.17.613470","url":null,"abstract":"Despite the success of CRISPR/Cas9 in inducing DNA double-strand breaks (DSBs) for genome editing, achieving targeted recombination in somatic cells remains challenging, particularly at recombination cold spots like the Tomato Mosaic Virus (ToMV) resistance locus in <em>Solanum lycopersicum</em>. We investigated the potential of CRISPR/Cas9-induced targeted recombination in somatic cells to overcome linkage drag surrounding the ToMV locus. We employed two strategies: first, inducing DSBs in both alleles of F<sub>1</sub> tomato seedlings to promote non-homologous end joining (NHEJ) and homology-directed repair (HDR); second, targeting a single allele in a heterozygous background to induce HDR in seedlings. CRISPR/Cas9 activity was confirmed in F<sub>1</sub> seedlings by detecting NHEJ-mediated mutations at the target sites in ToMV. We developed a bioinformatics pipeline to identify targeted recombinants by analyzing single nucleotide polymorphisms (SNPs) between parental haplotypes, allowing precise tracking of SNP variations. A two-dimensional pooling strategy was employed to distinguish genuine recombination events from PCR artifacts. Despite these advances and the active CRISPR/Cas9 system in F<sub>1</sub> progeny, no increase in recombination frequency was observed compared to wild-type plants. We extended our research to protoplasts to assess whether CRISPR/Cas9 could induce targeted recombination under different cellular conditions at the same locus. Consistent with our findings in F<sub>1</sub> plants, we observed no increase in recombinant patterns compared to wild-type controls in protoplasts. Our findings suggest that CRISPR/Cas9-induced DSBs are insufficient to break the genetic linkage at the ToMV locus on chromosome 9 in recombination cold spots within somatic cells.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"16 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253039","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 : 2024-09-17DOI: 10.1101/2024.09.17.613413
Ana S Falcao, Mafalda Lopes da Silva, Pedro Antas, Cristina Escrevente, Margarida Pedro, Rita Coelho, Ines S Ferreira, Ines P Santos, Thomas C. Ciossek, Paul Nicklin, Sandra Tenreiro, Miguel C. Seabra
Age-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be in the retinal pigment epithelium (RPE). RPE is responsible for the daily digestion of photoreceptor outer segments (POS), which imposes a heavy continuous burden on the lysosomal network. POS feeding assay in vitro suggested that the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo, derives from lysosomal dysfunction. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether induction of the mTOR/TFEB axis could improve digestion of POS and hence reduce AFG load. Treatment of POS-fed cells with rapamycin, an mTORC1 inhibitor after the appearance of AFG results in 30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the mTOR/TFEB axis in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.
{"title":"Enhancing lysosome function via mTOR/TFEB activation reduces lipofuscin-like granules in early Age-related Macular Degeneration","authors":"Ana S Falcao, Mafalda Lopes da Silva, Pedro Antas, Cristina Escrevente, Margarida Pedro, Rita Coelho, Ines S Ferreira, Ines P Santos, Thomas C. Ciossek, Paul Nicklin, Sandra Tenreiro, Miguel C. Seabra","doi":"10.1101/2024.09.17.613413","DOIUrl":"https://doi.org/10.1101/2024.09.17.613413","url":null,"abstract":"Age-related macular degeneration (AMD) is the most common blinding disease in the western world and is currently incurable. Although the exact causes of AMD are not clear, the primary origin of pathology appears to be in the retinal pigment epithelium (RPE). RPE is responsible for the daily digestion of photoreceptor outer segments (POS), which imposes a heavy continuous burden on the lysosomal network. POS feeding assay in vitro suggested that the accumulation of autofluorescence granules (AFG), similar to lipofuscin in vivo, derives from lysosomal dysfunction. Here we show that synchronous phagocytosis of POS leads to early transient mTOR activation followed by inhibition in late phagosome maturation. One of its substrates, the transcription factor EB (TFEB) increases during phagosome maturation albeit mostly in its inactive phosphorylated form. We questioned whether induction of the mTOR/TFEB axis could improve digestion of POS and hence reduce AFG load. Treatment of POS-fed cells with rapamycin, an mTORC1 inhibitor after the appearance of AFG results in 30% reduction of AFG load. This effect is dependent on active lysosomal enzymes and induction of active dephosphorylated TFEB with consequent activation of GADD34 and lysosomal biogenesis. As a proof of concept, we show that overexpressing a constitutively active form of unphosphorylated TFEB dramatically reduces POS-dependent AFG accumulation. Overall, this study suggests that viral or pharmacological approaches activating the mTOR/TFEB axis in the RPE could be beneficial as cell-protective treatment of early/intermediate cases of AMD, acting to delay progression of the disease.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253045","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 : 2024-09-17DOI: 10.1101/2024.09.17.613497
Marten L. Chaillet, Sander Roet, Remco C. Veltkamp, Friedrich Förster
Template matching (TM) in cryo-electron tomography (cryo-ET) enables in situ detection and localization of known macromolecules. However, TM faces challenges such as interfering features with a high signal-to-noise ratio and the need for manual curation of results. To address these challenges, we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.
{"title":"pytom-match-pick: a tophat-transform constraint for automated classification in template matching","authors":"Marten L. Chaillet, Sander Roet, Remco C. Veltkamp, Friedrich Förster","doi":"10.1101/2024.09.17.613497","DOIUrl":"https://doi.org/10.1101/2024.09.17.613497","url":null,"abstract":"Template matching (TM) in cryo-electron tomography (cryo-ET) enables in situ detection and localization of known macromolecules. However, TM faces challenges such as interfering features with a high signal-to-noise ratio and the need for manual curation of results. To address these challenges, we introduce pytom-match-pick, a GPU-accelerated, open-source command line interface for enhanced TM in cryo-ET. Using pytom-match-pick, we first quantify the effects of point spread function (PSF) weighting and show that a tilt-weighted PSF outperforms a binary wedge with a single defocus estimate. We also assess previously introduced background normalization methods for classification performance. This indicates that phase randomization is more effective than spectrum whitening in reducing false positives. Furthermore, a novel application of the tophat transform on score maps, combined with a dual-constraint thresholding strategy, reduces false positives and improves precision. We benchmarked pytom-match-pick on public datasets, demonstrating improved classification and localization of macromolecules like ribosomal subunits and proteasomes that led to fewer artifacts in subtomogram averages. This tool promises to advance visual proteomics by improving the efficiency and accuracy of macromolecule detection in cellular contexts.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"23 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253044","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}
The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPARg is mediated by Transportin-1, but PPARg lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPARg in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPARg, indicating that PPARg recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPARg.
{"title":"Structural insight into the nuclear transportation mechanism of PPARg by Transportin-1","authors":"Sachiko TOMA-FUKAI, Yutaro Nakamura, Akihiro Kawamoto, Hikaru Shimizu, Koki Hayama, Ruri Kojima, Kanami Yoshimura, Masaki Ishii, Mika Hirose, Toshiaki Teratani, Shinya Ohata, Takayuki Kato, Hironari Kamikubo, Toshimasa Itoh, Kengo Tomita, Toshiyuki Shimizu","doi":"10.1101/2024.09.17.612794","DOIUrl":"https://doi.org/10.1101/2024.09.17.612794","url":null,"abstract":"The spatial and temporal control of protein is essential for normal cellular function. Proteins working in the nucleus have nuclear localization signal (NLS) sequences and are escorted into the nucleus by cognate nuclear transport receptors. A wealth of experimental data about NLS has been accumulated, and nuclear transportation mechanisms are established at the biochemical and structural levels. The peroxisome proliferator-activated receptors (PPARs) are ligand-dependent transcription factors that control various biological responses. We recently reported that the transportation of PPARg is mediated by Transportin-1, but PPARg lacks a typical NLS sequence recognized by Transportin-1. Moreover, the recognition mechanism remains largely unknown. In this study, we determined the Cryo-EM structure of PPARg in complex with Transportin-1 and revealed that Transportin-1 gripped the folded DNA binding domain and the Hinge region of PPARg, indicating that PPARg recognizes a folded domain with an extended region as a nuclear localization signal, not a canonical unstructured signal sequence, confirmed by the mutation analyses in vitro and in cultured cells. Our study is the first snapshot structure working in nuclear transportation, not in transcription, of PPARg.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"5 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253042","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 : 2024-09-17DOI: 10.1101/2024.09.17.613402
Chenyang Gu, Shoji Takada, Giovani B. Brandani, Tsuyoshi Terakawa
The SMC complex cohesin mediates interphase chromatin structural formation in eukaryotic cells through DNA loop extrusion. Here, we sought to investigate its mechanism using molecular dynamics simulations. To achieve this, we first constructed the amino-acid-residue-resolution structural models of the cohesin subunits, SMC1, SMC3, STAG1, and NIPBL. By simulating these subunits with double-stranded DNA molecules, we predicted DNA binding patches on each subunit and quantified the affinities of these patches to DNA using their dissociation rate constants as a proxy. Then, we constructed the structural model of the whole cohesin complex and mapped the predicted high-affinity DNA binding patches on the structure. From the spatial relations of the predicted patches, we identified that multiple patches on the SMC1, SMC3, STAG1, and NIPBL subunits form a DNA clamping patch group. The simulations of the whole complex with double-stranded DNA molecules suggest that this patch group facilitates DNA bending and helps capture a DNA segment in the cohesin ring formed by the SMC1 and SMC3 subunits. In previous studies, these have been identified as critical steps in DNA loop extrusion. Therefore, this study provides experimentally testable predictions of DNA binding sites implicated in previously proposed DNA loop extrusion mechanisms and highlights the essential roles of the accessory subunits STAG1 and NIPBL in the mechanism.
在真核细胞中,SMC 复合物凝聚素通过 DNA 环挤压介导染色质间期结构的形成。在此,我们试图利用分子动力学模拟研究其机制。为此,我们首先构建了SMC1、SMC3、STAG1和NIPBL等凝聚素亚基的氨基酸分辨率结构模型。通过用双链DNA分子模拟这些亚基,我们预测了每个亚基上的DNA结合斑块,并以这些斑块的解离率常数为代表,量化了这些斑块与DNA的亲和力。然后,我们构建了整个凝聚蛋白复合物的结构模型,并在该结构上绘制了预测的高亲和性 DNA 结合斑块。从预测的补丁的空间关系中,我们发现SMC1、SMC3、STAG1和NIPBL亚基上的多个补丁组成了一个DNA夹持补丁组。用双链 DNA 分子模拟整个复合体的结果表明,这个贴片组有利于 DNA 弯曲,并有助于在 SMC1 和 SMC3 亚基形成的凝聚素环中捕获 DNA 片段。在以前的研究中,这些已被确定为 DNA 环挤出的关键步骤。因此,本研究对之前提出的 DNA 环挤出机制中涉及的 DNA 结合位点进行了可实验检验的预测,并强调了附属亚基 STAG1 和 NIPBL 在该机制中的重要作用。
{"title":"Molecular dynamics simulations of human cohesin subunits identify DNA binding sites and their potential roles in DNA loop extrusion","authors":"Chenyang Gu, Shoji Takada, Giovani B. Brandani, Tsuyoshi Terakawa","doi":"10.1101/2024.09.17.613402","DOIUrl":"https://doi.org/10.1101/2024.09.17.613402","url":null,"abstract":"The SMC complex cohesin mediates interphase chromatin structural formation in eukaryotic cells through DNA loop extrusion. Here, we sought to investigate its mechanism using molecular dynamics simulations. To achieve this, we first constructed the amino-acid-residue-resolution structural models of the cohesin subunits, SMC1, SMC3, STAG1, and NIPBL. By simulating these subunits with double-stranded DNA molecules, we predicted DNA binding patches on each subunit and quantified the affinities of these patches to DNA using their dissociation rate constants as a proxy. Then, we constructed the structural model of the whole cohesin complex and mapped the predicted high-affinity DNA binding patches on the structure. From the spatial relations of the predicted patches, we identified that multiple patches on the SMC1, SMC3, STAG1, and NIPBL subunits form a DNA clamping patch group. The simulations of the whole complex with double-stranded DNA molecules suggest that this patch group facilitates DNA bending and helps capture a DNA segment in the cohesin ring formed by the SMC1 and SMC3 subunits. In previous studies, these have been identified as critical steps in DNA loop extrusion. Therefore, this study provides experimentally testable predictions of DNA binding sites implicated in previously proposed DNA loop extrusion mechanisms and highlights the essential roles of the accessory subunits STAG1 and NIPBL in the mechanism.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"9 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253040","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 : 2024-09-17DOI: 10.1101/2024.09.17.613539
Sri Karthika Shanmugam, Scott A Kanner, Xinle M Zou, Enoch Amarh, Papiya Choudhury, Rajesh SONI, Robert S Kass, Henry M Colecraft
Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the ubiquitin code that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.
{"title":"Decoding polyubiquitin regulation of KV7. 1 functional expression with engineered linkage-selective deubiquitinases","authors":"Sri Karthika Shanmugam, Scott A Kanner, Xinle M Zou, Enoch Amarh, Papiya Choudhury, Rajesh SONI, Robert S Kass, Henry M Colecraft","doi":"10.1101/2024.09.17.613539","DOIUrl":"https://doi.org/10.1101/2024.09.17.613539","url":null,"abstract":"Protein posttranslational modification with distinct polyubiquitin linkage chains is a critical component of the ubiquitin code that universally regulates protein expression and function to control biology. Functional consequences of diverse polyubiquitin linkages on proteins are mostly unknown, with progress hindered by a lack of methods to specifically tune polyubiquitin linkages on individual proteins in live cells. Here, we bridge this gap by exploiting deubiquitinases (DUBs) with preferences for hydrolyzing different polyubiquitin linkages: OTUD1 - K63; OTUD4 - K48; Cezanne - K11; TRABID - K29/K33; and USP21 - non-specific. We developed a suite of engineered deubiquitinases (enDUBs) comprised of DUB catalytic domains fused to a GFP-targeted nanobody and used them to investigate polyubiquitin linkage regulation of an ion channel, YFP-KCNQ1. Mass spectrometry of YFP-KCNQ1 expressed in HEK293 cells indicated channel polyubiquitination with K48 (72%) and K63 (24%) linkages being dominant. NEDD4-2 and ITCH both decreased KCNQ1 functional expression but with distinctive polyubiquitination signatures. All enDUBs reduced KCNQ1 ubiquitination but yielded unique effects on channel expression, surface density, ionic currents, and subcellular localization. The pattern of outcomes indicates K11, K29/K33, and K63 chains mediate net KCNQ1-YFP intracellular retention, but achieved in different ways: K11 promotes ER retention/degradation, enhances endocytosis, and reduces recycling; K29/K33 promotes ER retention/degradation; K63 enhances endocytosis and reduces recycling. The pattern of enDUB effects on KCNQ1-YFP differed in cardiomyocytes, emphasizing ubiquitin code mutability. Surprisingly, enDUB-O4 decreased KCNQ1-YFP surface density suggesting a role for K48 in forward trafficking. Lastly, linkage-selective enDUBs displayed varying capabilities to rescue distinct trafficking-deficient long QT syndrome type 1 mutations. The results reveal distinct polyubiquitin chains control different aspects of KCNQ1 functional expression, demonstrate ubiquitin code plasticity, and introduce linkage-selective enDUBs as a potent tool to help demystify the polyubiquitin code.","PeriodicalId":501108,"journal":{"name":"bioRxiv - Molecular Biology","volume":"27 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142253041","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}