Pub Date : 2024-09-19DOI: 10.1101/2024.09.17.613393
Oluwamuyiwa T. Amusan, Shuqi Wang, Chaoran Yin, Heather S. Koehler, Yixun Li, Tencho Tenev, Rebecca Wilson, Benjamin Bellenie, Ting Zhang, Jian Wang, chang liu, kim seong, Seyedeh L. Poorbaghi, Joseph Yates, Yuchen Shen, Jason W. Upton, Pascal Meier, Siddharth Balachandra, Hongyan Guo
Necroptosis initiated by the host sensor Z-NA Binding Protein-1 (ZBP1) is essential for host defense against a growing number of viruses, including Herpes Simplex Virus-1 (HSV-1). Studies with HSV-1 and other necroptogenic stimuli in murine settings have suggested that ZBP1 triggers necroptosis by directly complexing with the kinase RIPK3. Whether this is also the case in human cells, or whether additional co-factors are needed for ZBP1-mediated necroptosis, is unclear. Here, we show that ZBP1-induced necroptosis in human cells requires RIPK1. We have found that RIPK1 is essential for forming a stable and functional ZBP1-RIPK3 complex in human cells, but is dispensable for the formation of the equivalent murine complex. The RIP Homology Interaction Motif (RHIM) in RIPK3 is responsible for this difference between the two species, because replacing the RHIM in human RIPK3 with the RHIM from murine RIPK3 is sufficient to overcome the requirement for RIPK1 in human cells. These observations describe a critical mechanistic difference between mice and humans in how ZBP1 engages in necroptosis, with important implications for treating human diseases.
{"title":"RIPK1 is essential for Herpes Simplex Virus-triggered ZBP1-dependent necroptosis in human cells","authors":"Oluwamuyiwa T. Amusan, Shuqi Wang, Chaoran Yin, Heather S. Koehler, Yixun Li, Tencho Tenev, Rebecca Wilson, Benjamin Bellenie, Ting Zhang, Jian Wang, chang liu, kim seong, Seyedeh L. Poorbaghi, Joseph Yates, Yuchen Shen, Jason W. Upton, Pascal Meier, Siddharth Balachandra, Hongyan Guo","doi":"10.1101/2024.09.17.613393","DOIUrl":"https://doi.org/10.1101/2024.09.17.613393","url":null,"abstract":"Necroptosis initiated by the host sensor Z-NA Binding Protein-1 (ZBP1) is essential for host defense against a growing number of viruses, including Herpes Simplex Virus-1 (HSV-1). Studies with HSV-1 and other necroptogenic stimuli in murine settings have suggested that ZBP1 triggers necroptosis by directly complexing with the kinase RIPK3. Whether this is also the case in human cells, or whether additional co-factors are needed for ZBP1-mediated necroptosis, is unclear. Here, we show that ZBP1-induced necroptosis in human cells requires RIPK1. We have found that RIPK1 is essential for forming a stable and functional ZBP1-RIPK3 complex in human cells, but is dispensable for the formation of the equivalent murine complex. The RIP Homology Interaction Motif (RHIM) in RIPK3 is responsible for this difference between the two species, because replacing the RHIM in human RIPK3 with the RHIM from murine RIPK3 is sufficient to overcome the requirement for RIPK1 in human cells. These observations describe a critical mechanistic difference between mice and humans in how ZBP1 engages in necroptosis, with important implications for treating human diseases.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265194","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}
Giardia lamblia does not encode Rpn12 and Sem1, two proteins crucial for assembling the proteasome lid. To understand how the interactions between the giardial proteasome lid subunits may have changed to compensate for their absence, we used the yeast two-hybrid assay to generate a binary protein interaction map of the Giardia lid subunits. Most interactions within the Giardia proteasome lid are stronger than those within the Saccharomyces cerevisiae lid. These may compensate for the absence of Rpn12 and Sem1. A notable exception was the weaker interaction between GlRpn11 and GlRpn8, compared to the strong interaction between Rpn11-Rpn8 of yeast. The Rpn11-Rpn8 dimer provides a platform for lid assembly and their interaction involves the insertion of a methionine residue of Rpn11 into a hydrophobic pocket of Rpn8. Molecular modeling indicates that GlRpn8s pocket is wider, reconciling the experimental observation of its weak interaction with GlRpn11. This weaker interaction may have evolved to support extra proteasomal functions of GlRpn11, which localizes to multiple subcellular regions where other proteasome subunits have not been detected. One such location is the mitosome. Functional complementation in yeast shows that GlRpn11 can influence mitochondrial function and distribution. This, together with its mitosomal localization, indicates that GlRpn11 functions at the mitosome. Thus, this parasites proteasome lid has a simpler subunit architecture and structural attributes that may support dual functionalities for GlRpn11. Such parasite-specific proteasome features could provide new avenues for controlling the transmission of Giardia.
{"title":"The binary protein interactome mapping of the Giardia lamblia proteasome lid reveals extra proteasomal functions of GlRpn11","authors":"Ankita Das, Atrayee Ray, Nibedita Ray Chaudhuri, Soumyajit Mukherjee, Shubhra Ghosh Dastidar, Alok Ghosh, Sandipan Ganguly, Kuladip Jana, Srimonti Sarkar","doi":"10.1101/2024.09.18.613619","DOIUrl":"https://doi.org/10.1101/2024.09.18.613619","url":null,"abstract":"Giardia lamblia does not encode Rpn12 and Sem1, two proteins crucial for assembling the proteasome lid. To understand how the interactions between the giardial proteasome lid subunits may have changed to compensate for their absence, we used the yeast two-hybrid assay to generate a binary protein interaction map of the Giardia lid subunits. Most interactions within the Giardia proteasome lid are stronger than those within the Saccharomyces cerevisiae lid. These may compensate for the absence of Rpn12 and Sem1. A notable exception was the weaker interaction between GlRpn11 and GlRpn8, compared to the strong interaction between Rpn11-Rpn8 of yeast. The Rpn11-Rpn8 dimer provides a platform for lid assembly and their interaction involves the insertion of a methionine residue of Rpn11 into a hydrophobic pocket of Rpn8. Molecular modeling indicates that GlRpn8s pocket is wider, reconciling the experimental observation of its weak interaction with GlRpn11. This weaker interaction may have evolved to support extra proteasomal functions of GlRpn11, which localizes to multiple subcellular regions where other proteasome subunits have not been detected. One such location is the mitosome. Functional complementation in yeast shows that GlRpn11 can influence mitochondrial function and distribution. This, together with its mitosomal localization, indicates that GlRpn11 functions at the mitosome. Thus, this parasites proteasome lid has a simpler subunit architecture and structural attributes that may support dual functionalities for GlRpn11. Such parasite-specific proteasome features could provide new avenues for controlling the transmission of Giardia.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265195","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 : 2024-09-19DOI: 10.1101/2024.09.16.608435
Annika Meid
Cell migration is a fundamental process in both normal and cancerous tissues, playing a crucial role in development, immune responses, and, in the case of cancer cells, metastasis- a leading cause of cancer-related mortality. Understanding the differences between healthy and cancerous cell migration is essential for uncovering potential therapeutic targets. This study aims to elucidate these differences by comparing the migratory behaviors of healthy cells (nHDF cells) and cancer cells (MDA-MB-231 cells). Our findings reveal that cancer cells significantly reduce their stiffness during migration through narrow channels, a phenomenon not observed in healthy cells. Additionally, DNA and membrane repair mechanisms are more active in healthy cells during migration compared to tumor cells. Notably, the use of MDA-MB-231 FUCCI cells demonstrates that the cell cycle profoundly influences cell migration under confined conditions. These insights provide a deeper understanding of the cellular mechanisms driving migration in both healthy and cancerous cells.
细胞迁移是正常组织和癌变组织的基本过程，在发育、免疫反应以及癌细胞转移（癌症相关死亡的主要原因）中发挥着至关重要的作用。了解健康细胞和癌细胞迁移之间的差异对于发现潜在的治疗靶点至关重要。本研究旨在通过比较健康细胞（nHDF 细胞）和癌细胞（MDA-MB-231 细胞）的迁移行为来阐明这些差异。我们的研究结果表明，癌细胞在通过狭窄通道迁移的过程中会明显降低其硬度，而这是在健康细胞中观察不到的现象。此外，与肿瘤细胞相比，健康细胞在迁移过程中的 DNA 和膜修复机制更为活跃。值得注意的是，MDA-MB-231 FUCCI 细胞的使用表明，在封闭条件下，细胞周期对细胞迁移有深刻影响。这些见解加深了人们对驱动健康细胞和癌细胞迁移的细胞机制的理解。
{"title":"The role of disease state in confined migration","authors":"Annika Meid","doi":"10.1101/2024.09.16.608435","DOIUrl":"https://doi.org/10.1101/2024.09.16.608435","url":null,"abstract":"Cell migration is a fundamental process in both normal and cancerous tissues, playing a crucial role in development, immune responses, and, in the case of cancer cells, metastasis- a leading cause of cancer-related mortality. Understanding the differences between healthy and cancerous cell migration is essential for uncovering potential therapeutic targets. This study aims to elucidate these differences by comparing the migratory behaviors of healthy cells (nHDF cells) and cancer cells (MDA-MB-231 cells). Our findings reveal that cancer cells significantly reduce their stiffness during migration through narrow channels, a phenomenon not observed in healthy cells. Additionally, DNA and membrane repair mechanisms are more active in healthy cells during migration compared to tumor cells. Notably, the use of MDA-MB-231 FUCCI cells demonstrates that the cell cycle profoundly influences cell migration under confined conditions. These insights provide a deeper understanding of the cellular mechanisms driving migration in both healthy and cancerous cells.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265192","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 : 2024-09-19DOI: 10.1101/2024.09.17.613249
Clara Douadi, Ilias Theodorou, Quentin Lamy-Besnier, Yanis Sbardella, Loic Brot, Paolo Emidio Costantini, Roberto Saporetti, Alberto Danielli, Matteo Calvaresi, Marianne De Paepe, Harry Sokol, Diego Garcia-Weber, Veronique Carriere, Sophie Thenet, Luisa De Sordi
Impaired intestinal barrier function is a major feature of Crohn's disease (CD), leading to exacerbated inflammation in response to the microbiota. In this context, the translocation of intestinal bacteriophages (phages) and their effects on the host have been little investigated. We used phage fluorescence imaging coupled with ex-vivo and in-vitro models that mimic physiological and inflammatory conditions and found that phages can translocate across the intestinal barrier without disrupting its integrity. Although the translocation rate across the intestinal epithelium depended on phage morphology and the condition of the barrier, these factors did not influence the crossing of phage across the vascular endothelium. Virome analysis confirmed that viral sequences shared between blood and stool samples are more abundant in CD patients than healthy subjects, indicating that a barrier defect facilitates phage translocation from the gut to the bloodstream.
{"title":"Differential translocation of bacteriophages across the intestinal barrier in health and Crohn's disease","authors":"Clara Douadi, Ilias Theodorou, Quentin Lamy-Besnier, Yanis Sbardella, Loic Brot, Paolo Emidio Costantini, Roberto Saporetti, Alberto Danielli, Matteo Calvaresi, Marianne De Paepe, Harry Sokol, Diego Garcia-Weber, Veronique Carriere, Sophie Thenet, Luisa De Sordi","doi":"10.1101/2024.09.17.613249","DOIUrl":"https://doi.org/10.1101/2024.09.17.613249","url":null,"abstract":"Impaired intestinal barrier function is a major feature of Crohn's disease (CD), leading to exacerbated inflammation in response to the microbiota. In this context, the translocation of intestinal bacteriophages (phages) and their effects on the host have been little investigated. We used phage fluorescence imaging coupled with ex-vivo and in-vitro models that mimic physiological and inflammatory conditions and found that phages can translocate across the intestinal barrier without disrupting its integrity. Although the translocation rate across the intestinal epithelium depended on phage morphology and the condition of the barrier, these factors did not influence the crossing of phage across the vascular endothelium. Virome analysis confirmed that viral sequences shared between blood and stool samples are more abundant in CD patients than healthy subjects, indicating that a barrier defect facilitates phage translocation from the gut to the bloodstream.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265189","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 : 2024-09-19DOI: 10.1101/2024.09.18.613713
Michelle M Conti, Jillian P Bail, Rui Li, Lihua Julie Zhu, Jennifer A Benanti
Cell survival depends upon the ability to adapt to changing environments. Environmental stressors trigger an acute stress response program that rewires cell physiology, downregulates proliferation genes and pauses the cell cycle until the cell adapts. Here, we show that dynamic phosphorylation of the yeast cell cycle-regulatory transcription factor Hcm1 is required to maintain fitness in chronic stress. Hcm1 is activated by cyclin dependent kinase (CDK) and inactivated by the phosphatase calcineurin (CN) in response to stressors that signal through increases in cytosolic Ca2+. Expression of a constitutively active, phosphomimetic Hcm1 mutant reduces fitness in stress, suggesting Hcm1 inactivation is required. However, a comprehensive analysis of Hcm1 phosphomutants revealed that Hcm1 activity is also important to survive stress, demonstrating that Hcm1 activity must be toggled on and off to promote gene expression and fitness. These results suggest that dynamic control of cell cycle regulators is critical for survival in stressful environments.
{"title":"Dynamic phosphorylation of Hcm1 promotes fitness in chronic stress","authors":"Michelle M Conti, Jillian P Bail, Rui Li, Lihua Julie Zhu, Jennifer A Benanti","doi":"10.1101/2024.09.18.613713","DOIUrl":"https://doi.org/10.1101/2024.09.18.613713","url":null,"abstract":"Cell survival depends upon the ability to adapt to changing environments. Environmental stressors trigger an acute stress response program that rewires cell physiology, downregulates proliferation genes and pauses the cell cycle until the cell adapts. Here, we show that dynamic phosphorylation of the yeast cell cycle-regulatory transcription factor Hcm1 is required to maintain fitness in chronic stress. Hcm1 is activated by cyclin dependent kinase (CDK) and inactivated by the phosphatase calcineurin (CN) in response to stressors that signal through increases in cytosolic Ca<sup>2+</sup>. Expression of a constitutively active, phosphomimetic Hcm1 mutant reduces fitness in stress, suggesting Hcm1 inactivation is required. However, a comprehensive analysis of Hcm1 phosphomutants revealed that Hcm1 activity is also important to survive stress, demonstrating that Hcm1 activity must be toggled on and off to promote gene expression and fitness. These results suggest that dynamic control of cell cycle regulators is critical for survival in stressful environments.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265190","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 : 2024-09-19DOI: 10.1101/2024.09.18.613519
Anoop Kumar Yadav, Abhijeet S. Jadhav, Pawel Szczepanik, Paolo Fagherazzi, Ivo Kabelka, Robert Vacha, Jakub Svenda, Hana Polasek-Sedlackova
HaloTag technology represents a versatile tool for studying proteins. Fluorescent HaloTag ligands employed in sequential labeling led to the discovery of distinct protein variants for histones, cohesins, and MCM complexes. Nonetheless, an efficient biochemical approach to separate the distinct protein variants to study their biological functions is missing. Principally being a gap in technology, the HaloTag toolbox lacks affinity ligands displaying good cell permeability and efficient affinity capture. Here, we describe the design, synthesis, and validation of a new cell-permeable Biotin-HaloTag ligand, which allows rapid labeling of Halo-tagged proteins in live cells and their efficient separation using streptavidin pull-down. Our work outlines how to use the herein-developed affinity ligand in sequential labeling to biochemically separate distinct protein variants and study their biological properties. The approach holds immense potential for addressing fundamental questions concerning essential cellular processes, including genome duplication and chromatin maintenance.
{"title":"Development of a cell-permeable Biotin-HaloTag ligand to explore functional differences between protein variants across cellular generations","authors":"Anoop Kumar Yadav, Abhijeet S. Jadhav, Pawel Szczepanik, Paolo Fagherazzi, Ivo Kabelka, Robert Vacha, Jakub Svenda, Hana Polasek-Sedlackova","doi":"10.1101/2024.09.18.613519","DOIUrl":"https://doi.org/10.1101/2024.09.18.613519","url":null,"abstract":"HaloTag technology represents a versatile tool for studying proteins. Fluorescent HaloTag ligands employed in sequential labeling led to the discovery of distinct protein variants for histones, cohesins, and MCM complexes. Nonetheless, an efficient biochemical approach to separate the distinct protein variants to study their biological functions is missing. Principally being a gap in technology, the HaloTag toolbox lacks affinity ligands displaying good cell permeability and efficient affinity capture. Here, we describe the design, synthesis, and validation of a new cell-permeable Biotin-HaloTag ligand, which allows rapid labeling of Halo-tagged proteins in live cells and their efficient separation using streptavidin pull-down. Our work outlines how to use the herein-developed affinity ligand in sequential labeling to biochemically separate distinct protein variants and study their biological properties. The approach holds immense potential for addressing fundamental questions concerning essential cellular processes, including genome duplication and chromatin maintenance.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265191","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 : 2024-09-19DOI: 10.1101/2024.09.18.613649
Chaitra Rao, Daniel T Cater, Saptarshi Roy, Jerry Xu, Andre De G Olivera, Carmella Evans-Molina, Decio L Eizirik, Jon D Piganelli, Raghavendra Mirmira, Emily Kristen Sims
Aims/hypothesis: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death. Methods: Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls. Results: PD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN-α or IFN-ɣ treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretation: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.
目的/假设：1型糖尿病患者中存活的β细胞会通过上调程序性死亡配体1（PD-L1）来应对炎症，从而与免疫细胞程序性死亡-1（PD-1）结合，限制自我反应免疫细胞的破坏。细胞外囊泡 (EV) 及其载体可作为β细胞健康的生物标志物，并有助于胰岛细胞间的交流。我们假设，1 型糖尿病的炎症环境会增加贝塔细胞 EV 货物中的 PD-L1，而 EV PD-L1 可保护贝塔细胞免受免疫介导的细胞死亡：方法：用促炎细胞因子处理β细胞系和人类胰岛，以模拟促炎的1型糖尿病微环境。使用超速离心法或尺寸排阻色谱法分离 EV，并通过免疫印迹、流式细胞术和 ELISA 进行分析。使用竞争性结合试验和体外功能试验评估了 EV PD-L1：PD-1 的结合情况，测试 EV PD-L1 抑制 NOD CD8 T 细胞的能力。对胰岛自身抗体阳性（Ab+）或近期发病的1型糖尿病患者血浆中的EV和可溶性PD-L1进行了检测，并与非糖尿病对照组进行了比较：结果：PD-L1蛋白在细胞内与四泛素相关蛋白共定位，并在β细胞EV表面被检测到。24小时IFN-α或IFN-ɣ处理诱导的EV PD-L1货物增加了两倍，但EV数量没有相应增加。IFN暴露主要增加了β细胞EV表面的PD-L1表达，β细胞EV PD-L1与PD-1的结合能力呈剂量依赖性。功能实验证明，β细胞 EV PD-L1 有抑制小鼠 CD8 T 细胞增殖和细胞毒性的特异性作用。此外，在胰岛Ab+或1型糖尿病患者（而非对照组）中，血浆EV PD-L1水平与循环C肽呈正相关，这表明较高的EV-PD-L1水平可保护残留的β细胞功能。结论/解释：IFN暴露会增加β细胞EV表面的PD-L1。Beta 细胞 EV PD-L1 与 PD1 结合，抑制 CD8 T 细胞增殖和细胞毒性。与对照组相比，胰岛自身抗体阳性患者的循环EV PD-L1更高。在1型糖尿病进展的不同阶段，循环中的EV PD-L1水平与残留的C肽相关。这些研究结果表明，EV PD-L1 可导致 1 型糖尿病进展和残留β细胞功能的异质性，并提出了利用 EV PD-L1 抑制免疫介导的β细胞死亡的可能性。
{"title":"Beta cell extracellular vesicle PD-L1 as a novel regulator of CD8+ T cell activity and biomarker during the evolution of Type 1 Diabetes","authors":"Chaitra Rao, Daniel T Cater, Saptarshi Roy, Jerry Xu, Andre De G Olivera, Carmella Evans-Molina, Decio L Eizirik, Jon D Piganelli, Raghavendra Mirmira, Emily Kristen Sims","doi":"10.1101/2024.09.18.613649","DOIUrl":"https://doi.org/10.1101/2024.09.18.613649","url":null,"abstract":"Aims/hypothesis: Surviving beta cells in type 1 diabetes respond to inflammation by upregulating programmed death-ligand 1 (PD-L1) to engage immune cell programmed death-1 (PD-1) and limit destruction by self-reactive immune cells. Extracellular vesicles (EVs) and their cargo can serve as biomarkers of beta cell health and contribute to islet intercellular communication. We hypothesized that the inflammatory milieu of type 1 diabetes increases PD-L1 in beta cell EV cargo and that EV PD-L1 may protect beta cells against immune-mediated cell death.\u0000Methods: Beta cell lines and human islets were treated with proinflammatory cytokines to model the proinflammatory type 1 diabetes microenvironment. EVs were isolated using ultracentrifugation or size exclusion chromatography and analysed via immunoblot, flow cytometry, and ELISA. EV PD-L1: PD-1 binding was assessed using a competitive binding assay and in vitro functional assays testing the ability of EV PD-L1 to inhibit NOD CD8 T cells. Plasma EV and soluble PD-L1 were assayed in plasma of individuals with islet autoantibody positivity (Ab+) or recent-onset type 1 diabetes and compared to non-diabetic controls.\u0000Results: PD-L1 protein colocalized with tetraspanin-associated proteins intracellularly and was detected on the surface of beta cell EVs. 24-h IFN-α or IFN-ɣ treatment induced a two-fold increase in EV PD-L1 cargo without a corresponding increase in number of EVs. IFN exposure predominantly increased PD-L1 expression on the surface of beta cell EVs and beta cell EV PD-L1 showed a dose-dependent capacity to bind PD-1. Functional experiments demonstrated specific effects of beta cell EV PD-L1 to suppress proliferation and cytotoxicity of murine CD8 T cells. Plasma EV PD-L1 levels were increased in islet Ab+ individuals, particularly in those with single Ab+, Additionally, in from individuals with either Ab+ or type 1 diabetes, but not in controls, plasma EV PD-L1 positively correlated with circulating C-peptide, suggesting that higher EV-PD-L1 could be protective for residual beta cell function. Conclusions/interpretation: IFN exposure increases PD-L1 on the beta cell EV surface. Beta cell EV PD-L1 binds PD1 and inhibits CD8 T cell proliferation and cytotoxicity. Circulating EV PD-L1 is higher in islet autoantibody positive patients compared to controls. Circulating EV PD-L1 levels correlate with residual C-peptide at different stages in type 1 diabetes progression. These findings suggest that EV PD-L1 could contribute to heterogeneity in type 1 diabetes progression and residual beta cell function and raise the possibility that EV PD-L1 could be exploited as a means to inhibit immune-mediated beta cell death.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265188","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 : 2024-09-19DOI: 10.1101/2024.09.18.613687
Samuel J. Atkinson, William Vincent Ritchie, Kyle Thompson, Dawn Thompson, James N. Hislop, Riko Hatakeyama
The mammalian/mechanistic Target of Rapamycin Complex 1 (mTORC1) kinase controls cell growth in response to various external stimuli. mTORC1 has a myriad of substrates, and the activation of distinct downstream pathways has important physiological outcomes. Emerging evidence suggests that mTORC1 can respond to upstream signals in a nuanced manner, differentially regulating individual substrates and downstream biological processes. The nature of signals that determine the signaling selectivity of mTORC1 is not fully understood. Here, we studied mTORC1 regulation by G protein-coupled receptors (GPCRs). We found that muscarinic acetylcholine receptor M5, formyl peptide receptor 2, and beta-2 adrenergic receptor, which are coupled to distinct effector G proteins, all trigger substrate-specific activation of mTORC1. Remarkably, phosphorylation of the TFEB transcription factor, a non-canonical mTORC1 substrate that controls lysosome biogenesis, responded to GPCRs differently, sometimes even oppositely, compared to canonical mTORC1 substrates such as S6K1 and 4EBP1. This study highlights the need to re-evaluate the effects of GPCRs on mTORC1 by parallelly monitoring individual substrates, an important consideration to be made when assessing GPCR ligands as therapeutic tools to manipulate the mTORC1 pathway.
{"title":"Substrate-specific regulation of mTORC1 activity by G protein-coupled receptors","authors":"Samuel J. Atkinson, William Vincent Ritchie, Kyle Thompson, Dawn Thompson, James N. Hislop, Riko Hatakeyama","doi":"10.1101/2024.09.18.613687","DOIUrl":"https://doi.org/10.1101/2024.09.18.613687","url":null,"abstract":"The mammalian/mechanistic Target of Rapamycin Complex 1 (mTORC1) kinase controls cell growth in response to various external stimuli. mTORC1 has a myriad of substrates, and the activation of distinct downstream pathways has important physiological outcomes. Emerging evidence suggests that mTORC1 can respond to upstream signals in a nuanced manner, differentially regulating individual substrates and downstream biological processes. The nature of signals that determine the signaling selectivity of mTORC1 is not fully understood. Here, we studied mTORC1 regulation by G protein-coupled receptors (GPCRs). We found that muscarinic acetylcholine receptor M5, formyl peptide receptor 2, and beta-2 adrenergic receptor, which are coupled to distinct effector G proteins, all trigger substrate-specific activation of mTORC1. Remarkably, phosphorylation of the TFEB transcription factor, a non-canonical mTORC1 substrate that controls lysosome biogenesis, responded to GPCRs differently, sometimes even oppositely, compared to canonical mTORC1 substrates such as S6K1 and 4EBP1. This study highlights the need to re-evaluate the effects of GPCRs on mTORC1 by parallelly monitoring individual substrates, an important consideration to be made when assessing GPCR ligands as therapeutic tools to manipulate the mTORC1 pathway.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265193","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 : 2024-09-19DOI: 10.1101/2024.09.18.613772
Ethan G Stoddard, Linglan Fang, Yuhao Zhong, Zachary E Potter, Daniel S Brush, Jessica J Simon, Martin Golkowski, Dustin J Maly
Raf kinases are crucial effectors in the Ras-Raf-Mek-Erk signaling pathway, making them important targets for the development of cancer therapeutics. This study investigates the variable potency of DFG-out-stabilizing Raf inhibitors in mutant KRas-expressing cell lines. We demonstrate that inhibitor potency correlates with basal Raf activity, with more active Raf being more susceptible to inhibition. We further show that DFG-out-stabilizing inhibitors disrupt high-affinity Raf-Mek interactions, promoting the formation of inhibited Raf dimers. Furthermore, we identify cobimetinib as a Mek inhibitor that uniquely sensitizes Raf to DFG-out inhibitors by disrupting autoinhibited Raf-Mek complexes. Building on this insight, we developed cobimetinib analogs with enhanced sensitization properties. Our findings provide a mechanistic framework for understanding the cellular determinants of DFG-out-stabilizing inhibitor sensitivity and offer strategies for optimizing synergistic Raf-Mek inhibitor combinations.
{"title":"Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies","authors":"Ethan G Stoddard, Linglan Fang, Yuhao Zhong, Zachary E Potter, Daniel S Brush, Jessica J Simon, Martin Golkowski, Dustin J Maly","doi":"10.1101/2024.09.18.613772","DOIUrl":"https://doi.org/10.1101/2024.09.18.613772","url":null,"abstract":"Raf kinases are crucial effectors in the Ras-Raf-Mek-Erk signaling pathway, making them important targets for the development of cancer therapeutics. This study investigates the variable potency of DFG-out-stabilizing Raf inhibitors in mutant KRas-expressing cell lines. We demonstrate that inhibitor potency correlates with basal Raf activity, with more active Raf being more susceptible to inhibition. We further show that DFG-out-stabilizing inhibitors disrupt high-affinity Raf-Mek interactions, promoting the formation of inhibited Raf dimers. Furthermore, we identify cobimetinib as a Mek inhibitor that uniquely sensitizes Raf to DFG-out inhibitors by disrupting autoinhibited Raf-Mek complexes. Building on this insight, we developed cobimetinib analogs with enhanced sensitization properties. Our findings provide a mechanistic framework for understanding the cellular determinants of DFG-out-stabilizing inhibitor sensitivity and offer strategies for optimizing synergistic Raf-Mek inhibitor combinations.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265196","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: 10.1101/2024.09.17.613449
Eva Wiesner, Julia Binz-Lotter, Simon E. Troeder, David Unnersjoe-Jess, Nelli Rutkowski, Branko Zevnik, Thomas Benzing, Roland Wedlich-Soldner, Matthias J. Hackl
F-actin is a major component of the cellular cytoskeleton, responsible for maintaining cell shape, enabling movement and facilitating intracellular transport. In the kidney, glomerular podocytes are highly dependent on their actin cytoskeleton shaping their unique foot processes. Hereditary mutations in actin-binding proteins cause focal segmental glomerulosclerosis, while other organs remain largely unaffected. So far, actin visualization in podocytes has been limited to electron microscopy or indirect immunofluorescent labeling of actin-binding proteins. However, the short F-actin-binding peptide Lifeact enables researchers to study actin dynamics in vitro and in vivo with minimal interference with actin metabolism. Here we introduce a new mouse model with conditional expression of a Lifeact.mScarlet I fusion protein providing red labeling of actin. Cre recombinase-mediated activity allows cell-specific and mosaic expression in podocytes, enabling selective labeling of individual cells to contrast with non-expressing neighboring cells. Transgenic mice are born healthy and young animals display no kidney-related phenotype. By intravital imaging and super-resolution microscopy, we show subcellular localization of actin to the foot processes in a resolution previously only obtainable by electron microscopy. Our novel mouse line provides the opportunity to study the actin cytoskeleton in podocytes and other cell types by intravital imaging and other conventional light microscopy techniques.
{"title":"A Red Fluorescent Lifeact Marker to Study Actin Morphology in Podocytes","authors":"Eva Wiesner, Julia Binz-Lotter, Simon E. Troeder, David Unnersjoe-Jess, Nelli Rutkowski, Branko Zevnik, Thomas Benzing, Roland Wedlich-Soldner, Matthias J. Hackl","doi":"10.1101/2024.09.17.613449","DOIUrl":"https://doi.org/10.1101/2024.09.17.613449","url":null,"abstract":"F-actin is a major component of the cellular cytoskeleton, responsible for maintaining cell shape, enabling movement and facilitating intracellular transport. In the kidney, glomerular podocytes are highly dependent on their actin cytoskeleton shaping their unique foot processes. Hereditary mutations in actin-binding proteins cause focal segmental glomerulosclerosis, while other organs remain largely unaffected.\u0000So far, actin visualization in podocytes has been limited to electron microscopy or indirect immunofluorescent labeling of actin-binding proteins. However, the short F-actin-binding peptide Lifeact enables researchers to study actin dynamics in vitro and in vivo with minimal interference with actin metabolism.\u0000Here we introduce a new mouse model with conditional expression of a Lifeact.mScarlet I fusion protein providing red labeling of actin. Cre recombinase-mediated activity allows cell-specific and mosaic expression in podocytes, enabling selective labeling of individual cells to contrast with non-expressing neighboring cells. Transgenic mice are born healthy and young animals display no kidney-related phenotype. By intravital imaging and super-resolution microscopy, we show subcellular localization of actin to the foot processes in a resolution previously only obtainable by electron microscopy. Our novel mouse line provides the opportunity to study the actin cytoskeleton in podocytes and other cell types by intravital imaging and other conventional light microscopy techniques.","PeriodicalId":501590,"journal":{"name":"bioRxiv - Cell Biology","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142265200","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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