Pub Date : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.02101
Vashendriya V. V. Hira, Barbara Breznik, Annique Loncq de Jong, M. Khurshed, Remco J. Molenaar, Tamara Lah, Cornelis van Noorden
Glioblastomais the most aggressive primary brain tumor. Slowly dividing and therapy‐resistant glioblastoma stem cells (GSCs) reside in protective peri‐arteriolar niches and are held responsible for GSC maintenance and glioblastoma recurrence. Recently, we showed similarities between GSC niches and hematopoietic stem cell (HSC) niches in bone marrow. Acute myeloid leukemia(AML) cells hijack HSC niches and are transformed into slowly‐dividing and therapy‐resistant leukemic stem cells (LSCs). Current clinical trials are focussed on removal of LSCs out of HSC niches to differentiate and to become sensitized to chemotherapy. In the present study, we elaborated further on these similarities by immunohistochemical analyses and fluorescence microscopy of17 biomarkers in paraffin sections of human glioblastoma and human healthy bone marrow. We found all 17 biomarkers to be expressed both in hypoxic peri‐arteriolar HSC niches in bone marrow and hypoxic peri‐arteriolar GSC niches in glioblastoma. Our findings implicate that GSC niches are being formed in glioblastoma as a copy of HSC niches in bone marrow. These similarities between HSC niches and GSC niches provide a theoretic basis for the development of novel strategies to force GSCs out of their niches, in a similar manner as in AML, to induce GSC differentiation and proliferation to render them more sensitive to anti‐glioblastoma therapies.
{"title":"Similarities Between Stem Cell Niches in Glioblastoma and Bone Marrow: Rays of Hope for Novel Treatment Strategies","authors":"Vashendriya V. V. Hira, Barbara Breznik, Annique Loncq de Jong, M. Khurshed, Remco J. Molenaar, Tamara Lah, Cornelis van Noorden","doi":"10.1096/fasebj.2020.34.s1.02101","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.02101","url":null,"abstract":"Glioblastomais the most aggressive primary brain tumor. Slowly dividing and therapy‐resistant glioblastoma stem cells (GSCs) reside in protective peri‐arteriolar niches and are held responsible for GSC maintenance and glioblastoma recurrence. Recently, we showed similarities between GSC niches and hematopoietic stem cell (HSC) niches in bone marrow. Acute myeloid leukemia(AML) cells hijack HSC niches and are transformed into slowly‐dividing and therapy‐resistant leukemic stem cells (LSCs). Current clinical trials are focussed on removal of LSCs out of HSC niches to differentiate and to become sensitized to chemotherapy. In the present study, we elaborated further on these similarities by immunohistochemical analyses and fluorescence microscopy of17 biomarkers in paraffin sections of human glioblastoma and human healthy bone marrow. We found all 17 biomarkers to be expressed both in hypoxic peri‐arteriolar HSC niches in bone marrow and hypoxic peri‐arteriolar GSC niches in glioblastoma. Our findings implicate that GSC niches are being formed in glioblastoma as a copy of HSC niches in bone marrow. These similarities between HSC niches and GSC niches provide a theoretic basis for the development of novel strategies to force GSCs out of their niches, in a similar manner as in AML, to induce GSC differentiation and proliferation to render them more sensitive to anti‐glioblastoma therapies.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141217925","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.04350
Laura C Miller Conrad
Pseudomonas aeruginosa is an emerging public health threat. A common Gram‐negative source of secondary infections, it has few effective treatment options, which are currently being undermined by the rise of resistance. New therapies are urgently needed. We had previously pursued an antivirulence strategy to block the production of pyocyanin, a redox‐active virulence factor. A photoaffinity analog of an antipyocyanin compound was developed to interrogate the inhibitor’s molecular mechanism of action. In the process, antibiotic adjuvant activity was suggested by the proteomics results. Using susceptibility assays, we found that these compounds amplify the bactericidal activity of colistin, a well‐characterized antibiotic, suggesting they may represent a first‐in‐class antibiotic adjuvant therapy. Analogs have the potential to not only widen the therapeutic index of cationic antibiotic peptides like colistin, but to be effective against colistin‐resistant strains. Multidrug‐resistant infections with P. aeruginosa are currently treated with colistin and the related polymyxin b, however, resistance to these drugs is also increasingly encountered. The adjuvants have the potential to preserve the life‐saving therapy of colistin when used in a combination therapy.
铜绿假单胞菌是一种新出现的公共卫生威胁。铜绿假单胞菌是一种常见的革兰氏阴性菌，可引起继发性感染，但它的有效治疗方案却很少，目前抗药性的增加又削弱了它的治疗效果。我们迫切需要新的疗法。此前，我们一直在寻求一种抗病毒策略，以阻断具有氧化还原作用的毒力因子--焦花青素的产生。我们开发了一种抗焦花青素化合物的光亲和类似物，以探究抑制剂的分子作用机制。在此过程中，蛋白质组学结果表明了抗生素的辅助活性。通过药敏试验，我们发现这些化合物放大了可乐定（一种特性良好的抗生素）的杀菌活性，这表明它们可能代表了第一类抗生素辅助疗法。类似物不仅有可能扩大阳离子抗生素肽（如秋水仙碱）的治疗指数，还能有效抑制耐秋水仙碱菌株。铜绿假单胞菌对多种药物产生耐药性，目前主要使用秋水仙素和相关的多粘菌素 b 进行治疗，但对这些药物产生耐药性的情况也越来越多。辅助剂在联合疗法中使用时，有可能保留可乐定的救命疗法。
{"title":"Photoaffinity Approach Reveals Antibiotic Adjuvant Activity toward Pseudomonas aeruginosa","authors":"Laura C Miller Conrad","doi":"10.1096/fasebj.2020.34.s1.04350","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.04350","url":null,"abstract":"Pseudomonas aeruginosa is an emerging public health threat. A common Gram‐negative source of secondary infections, it has few effective treatment options, which are currently being undermined by the rise of resistance. New therapies are urgently needed. We had previously pursued an antivirulence strategy to block the production of pyocyanin, a redox‐active virulence factor. A photoaffinity analog of an antipyocyanin compound was developed to interrogate the inhibitor’s molecular mechanism of action. In the process, antibiotic adjuvant activity was suggested by the proteomics results. Using susceptibility assays, we found that these compounds amplify the bactericidal activity of colistin, a well‐characterized antibiotic, suggesting they may represent a first‐in‐class antibiotic adjuvant therapy. Analogs have the potential to not only widen the therapeutic index of cationic antibiotic peptides like colistin, but to be effective against colistin‐resistant strains. Multidrug‐resistant infections with P. aeruginosa are currently treated with colistin and the related polymyxin b, however, resistance to these drugs is also increasingly encountered. The adjuvants have the potential to preserve the life‐saving therapy of colistin when used in a combination therapy.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141217053","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.00150
Kris A. DeMali, Alicia M. Salvi, Jennifer L. Bays
External forces are sensed by cell surface adhesion receptors, such as E‐cadherin, and trigger robust actin cytoskeletal rearrangements that allow the cell to withstand the force. These actin rearrangements are energetically costly and require approximately half of the ATP in a cell. Previous work in our laboratory demonstrated that in response to force, E‐cadherin signals for enhanced energy production by stimulating AMP‐activated protein kinase (AMPK). AMPK triggers glucose uptake and its oxidation to ATP thereby providing the energy necessary for actin cytoskeletal remodeling. How mechanical force stimulates glucose uptake remains completely unexplored. In this study, we identify glucose transporter‐1 (GLUT1) as a force‐sensitive protein that is highly enriched in the plasma membrane in cells under tension. GLUT1 is necessary for the uptake of glucose and the reinforcement of the actin cytoskeleton that occurs when E‐cadherin experiences force. Intriguingly, GLUT1 is recruited to the cell‐cell junctions and forms a complex with E‐cadherin via a novel linkage mediated by the spectrin adaptor protein, Ankyrin G. Loss of Ankyrin G binding to E‐cadherin inhibits GLUT1 retention to the membrane, glucose uptake, and reinforcement of the actin cytoskeleton in response to force. This study provides the first mechanism for how cell mechanics stimulates the uptake of glucose.
{"title":"Mechanisms Linking Mechanotransduction and Cell Metabolism","authors":"Kris A. DeMali, Alicia M. Salvi, Jennifer L. Bays","doi":"10.1096/fasebj.2020.34.s1.00150","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.00150","url":null,"abstract":"External forces are sensed by cell surface adhesion receptors, such as E‐cadherin, and trigger robust actin cytoskeletal rearrangements that allow the cell to withstand the force. These actin rearrangements are energetically costly and require approximately half of the ATP in a cell. Previous work in our laboratory demonstrated that in response to force, E‐cadherin signals for enhanced energy production by stimulating AMP‐activated protein kinase (AMPK). AMPK triggers glucose uptake and its oxidation to ATP thereby providing the energy necessary for actin cytoskeletal remodeling. How mechanical force stimulates glucose uptake remains completely unexplored. In this study, we identify glucose transporter‐1 (GLUT1) as a force‐sensitive protein that is highly enriched in the plasma membrane in cells under tension. GLUT1 is necessary for the uptake of glucose and the reinforcement of the actin cytoskeleton that occurs when E‐cadherin experiences force. Intriguingly, GLUT1 is recruited to the cell‐cell junctions and forms a complex with E‐cadherin via a novel linkage mediated by the spectrin adaptor protein, Ankyrin G. Loss of Ankyrin G binding to E‐cadherin inhibits GLUT1 retention to the membrane, glucose uptake, and reinforcement of the actin cytoskeleton in response to force. This study provides the first mechanism for how cell mechanics stimulates the uptake of glucose.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141218097","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.03469
Geoffroy Noel, Esther S.H. Kang, Marija Popovic
Anatomy teaching to Medical Physics (MP) trainees is recognized by the Commission on Accreditation of Medical Physics Education Programs, and yet anatomy laboratory sessions have not been integrated in MP training curricula. This study outlines the development of anatomy laboratory sessions for MP trainees and serves as the initial step to developing an anatomical curriculum guideline for MP. The two objectives of this qualitative study are: (a) to explore the educational potential of integrated anatomy laboratory sessions in the MP curriculum and (b) to evaluate the benefits of interprofessional education activities between MP trainees and Radiation Oncology residents.
{"title":"Integration of Gross Anatomy Laboratory sessions into Medical Physics Curriculum using Segmentation and Augmented‐Reality","authors":"Geoffroy Noel, Esther S.H. Kang, Marija Popovic","doi":"10.1096/fasebj.2020.34.s1.03469","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.03469","url":null,"abstract":"Anatomy teaching to Medical Physics (MP) trainees is recognized by the Commission on Accreditation of Medical Physics Education Programs, and yet anatomy laboratory sessions have not been integrated in MP training curricula. This study outlines the development of anatomy laboratory sessions for MP trainees and serves as the initial step to developing an anatomical curriculum guideline for MP. The two objectives of this qualitative study are: (a) to explore the educational potential of integrated anatomy laboratory sessions in the MP curriculum and (b) to evaluate the benefits of interprofessional education activities between MP trainees and Radiation Oncology residents.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141218748","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.06096
Amanda Daniels, Carolina Briseno, N. Nikolaidis
HspA1A, a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cell survival. In addition to its functions in protein homeostasis, HspA1A also functions at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although the interactions between HspA1A and lipids have important biological consequences, their mechanistic details remain elusive and unexplored because the amino acids responsible for the lipid binding remain largely uncharacterized and the relationship between the chaperone and lipid‐binding functions remains unknown. To clarify the mechanism of the HspA1A‐lipid interactions, we first characterized the properties of a mutation from Tryptophan to Phenylalanine (W90F), known to affect binding of HspA1A to BMP and a Lysine to Alanine (K71A) mutation, which results in complete loss of the chaperone function. Experiments using the lipid vesicle sedimentation (LVS) method and Surface Plasmon Resonance revealed that although the W90F‐HspA1A binding to BMP was different from the WT, this mutation did not affect the binding to PS. Furthermore, our results revealed that the K71A mutation did not significantly change the binding of HspA1A to lipids under any of the conditions tested. These results allowed us to make two predictions: first, the lipid‐binding sites for PS and BMP are lipid‐specific, and second, the chaperone and lipid‐binding functions are distinct. To support the first prediction, we used a combination of structural superimpositions, sequence alignments, and literature observations and predicted three putative lipid‐binding regions on the HspA1A molecule. We tested these predictions using the LVS assay for 20 single or double mutations spanning two of these regions. Our results revealed that only two of these mutations affected the binding to PS, while a third one altered the binding to BMP. To test the second prediction, we performed a series of experiments to determine the effect of lipid binding to the secondary structure and chaperone functions of HspA1A. We used Circular dichroism spectrometry, measured the release of inorganic phosphate, and determined the rate of refolding of chemically denatured beta‐galactosidase in the presence or absence of particular lipid. These experiments revealed that lipid binding did not alter the secondary structure of the protein and affected neither the rate of ATP hydrolysis nor the rate of protein refolding. Together our findings provide further evidence that HspA1A binds to PS and BMP using different amino acid sites and support the notion that the chaperone and lipid‐binding functions of HspA1A do not overlap. These findings provide the basis for future experiments to test the effects of these mutations on the membrane‐localized functions of HspA1A in cancer and stressed cells.
{"title":"HspA1A, a 70‐kDa heat‐shock protein, contains several distinct lipid‐binding sites","authors":"Amanda Daniels, Carolina Briseno, N. Nikolaidis","doi":"10.1096/fasebj.2020.34.s1.06096","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.06096","url":null,"abstract":"HspA1A, a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cell survival. In addition to its functions in protein homeostasis, HspA1A also functions at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although the interactions between HspA1A and lipids have important biological consequences, their mechanistic details remain elusive and unexplored because the amino acids responsible for the lipid binding remain largely uncharacterized and the relationship between the chaperone and lipid‐binding functions remains unknown. To clarify the mechanism of the HspA1A‐lipid interactions, we first characterized the properties of a mutation from Tryptophan to Phenylalanine (W90F), known to affect binding of HspA1A to BMP and a Lysine to Alanine (K71A) mutation, which results in complete loss of the chaperone function. Experiments using the lipid vesicle sedimentation (LVS) method and Surface Plasmon Resonance revealed that although the W90F‐HspA1A binding to BMP was different from the WT, this mutation did not affect the binding to PS. Furthermore, our results revealed that the K71A mutation did not significantly change the binding of HspA1A to lipids under any of the conditions tested. These results allowed us to make two predictions: first, the lipid‐binding sites for PS and BMP are lipid‐specific, and second, the chaperone and lipid‐binding functions are distinct. To support the first prediction, we used a combination of structural superimpositions, sequence alignments, and literature observations and predicted three putative lipid‐binding regions on the HspA1A molecule. We tested these predictions using the LVS assay for 20 single or double mutations spanning two of these regions. Our results revealed that only two of these mutations affected the binding to PS, while a third one altered the binding to BMP. To test the second prediction, we performed a series of experiments to determine the effect of lipid binding to the secondary structure and chaperone functions of HspA1A. We used Circular dichroism spectrometry, measured the release of inorganic phosphate, and determined the rate of refolding of chemically denatured beta‐galactosidase in the presence or absence of particular lipid. These experiments revealed that lipid binding did not alter the secondary structure of the protein and affected neither the rate of ATP hydrolysis nor the rate of protein refolding. Together our findings provide further evidence that HspA1A binds to PS and BMP using different amino acid sites and support the notion that the chaperone and lipid‐binding functions of HspA1A do not overlap. These findings provide the basis for future experiments to test the effects of these mutations on the membrane‐localized functions of HspA1A in cancer and stressed cells.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141219092","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.09661
R. Calvani
Dopaminergic nigrostriatal denervation and widespread intracellular α‐synuclein accumulation are neuropathologic hallmarks of Parkinson’s disease (PD). A constellation of peripheral processes, including metabolic and inflammatory changes, are thought to contribute to neurodegeneration. In the present study, we sought to obtain insight into the multifaceted pathophysiology of PD through the application of a multi‐marker discovery approach. Fifty older adults aged 70+, 20 with PD and 30 age‐matched controls were enrolled as part of the EXosomes in PArkiNson Disease (EXPAND) study. A panel of 68 circulating mediators of inflammation, neurogenesis and neural plasticity, and amino acid metabolism was assayed. Biomarker selection was accomplished through sequential and orthogonalized covariance selection (SO‐CovSel), a multi‐platform regression method developed to handle highly correlated variables organized in multi‐block datasets. The SO‐CovSel model with the best prediction ability using the smallest number of variables was built with seven biomolecules. The model allowed correct classification of 94.2 ± 3.1% participants with PD and 100% controls. The biomarker profile of older adults with PD was defined by higher circulating levels of interleukin (IL) 8, macrophage inflammatory protein (MIP)‐1β, phosphoethanolamine, and proline, and by lower concentrations of citrulline, IL9, and MIP‐1α. Our innovative approach allowed identifying and evaluating the classification performance of a set of potential biomarkers for PD in older adults. Future studies are warranted to establish whether these biomolecules could serve as valuable biomarkers for PD as well as unveil new targets for interventions.
{"title":"A Novel Multi‐marker Discovery Approach Identifies New Biomarkers for Parkinson’s Disease in Older People: an EXosomes in PArkiNson Disease (EXPAND) Ancillary Study","authors":"R. Calvani","doi":"10.1096/fasebj.2020.34.s1.09661","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.09661","url":null,"abstract":"Dopaminergic nigrostriatal denervation and widespread intracellular α‐synuclein accumulation are neuropathologic hallmarks of Parkinson’s disease (PD). A constellation of peripheral processes, including metabolic and inflammatory changes, are thought to contribute to neurodegeneration. In the present study, we sought to obtain insight into the multifaceted pathophysiology of PD through the application of a multi‐marker discovery approach. Fifty older adults aged 70+, 20 with PD and 30 age‐matched controls were enrolled as part of the EXosomes in PArkiNson Disease (EXPAND) study. A panel of 68 circulating mediators of inflammation, neurogenesis and neural plasticity, and amino acid metabolism was assayed. Biomarker selection was accomplished through sequential and orthogonalized covariance selection (SO‐CovSel), a multi‐platform regression method developed to handle highly correlated variables organized in multi‐block datasets. The SO‐CovSel model with the best prediction ability using the smallest number of variables was built with seven biomolecules. The model allowed correct classification of 94.2 ± 3.1% participants with PD and 100% controls. The biomarker profile of older adults with PD was defined by higher circulating levels of interleukin (IL) 8, macrophage inflammatory protein (MIP)‐1β, phosphoethanolamine, and proline, and by lower concentrations of citrulline, IL9, and MIP‐1α. Our innovative approach allowed identifying and evaluating the classification performance of a set of potential biomarkers for PD in older adults. Future studies are warranted to establish whether these biomolecules could serve as valuable biomarkers for PD as well as unveil new targets for interventions.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141217063","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.02954
L. Litzau
The wheat dwarf virus Rep domain is an HUH‐endonuclease and is involved in rolling‐circle replication. HUH‐endonucleases, or HUH‐tags, form covalent protein‐ssDNA adducts by coordinating a divalent metal ion to cleave a specific ssDNA sequence and form a phosphotyrosine linkage. This protein‐ssDNA fusion is useful for various biotechnology applications such as cellular imaging, cellular barcoding, DNA‐guided protein localization, and single molecule manipulation of DNA‐tethered proteins. Solving the structure of the Rep domain in complex with DNA could present necessary information regarding HUH‐tag sequence specificity and allow for rational engineering of protein‐DNA interactions. Here, the structure of WDV Rep domain in the apo state was solved with a crystal diffracting to 1.24 Å. While ssDNA soaks were attempted, they proved ineffective. However, the solved structure represents a step towards solving protein‐ssDNA complex.
小麦矮小病毒 Rep 结构域是一种 HUH 内切酶，参与滚圆复制。HUH 内切酶或 HUH 标签通过二价金属离子配位形成共价蛋白质-ssDNA 加合物，从而裂解特定的 ssDNA 序列并形成磷酪氨酸连接。这种蛋白质-ssDNA 融合可用于各种生物技术应用，如细胞成像、细胞条形码、DNA 引导的蛋白质定位以及 DNA 系链蛋白质的单分子操作。解决 Rep 结构域与 DNA 复合物的结构问题可以提供有关 HUH-tag 序列特异性的必要信息，并允许对蛋白质-DNA 相互作用进行合理的工程设计。在这里，我们用衍射到 1.24 Å 的晶体解出了 WDV Rep 结构域的 apo 状态结构。不过，所解决的结构代表着向解决蛋白质-ssDNA复合物迈出了一步。
{"title":"Crystal structure of the wheat dwarf virus Rep domain","authors":"L. Litzau","doi":"10.1096/fasebj.2020.34.s1.02954","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.02954","url":null,"abstract":"The wheat dwarf virus Rep domain is an HUH‐endonuclease and is involved in rolling‐circle replication. HUH‐endonucleases, or HUH‐tags, form covalent protein‐ssDNA adducts by coordinating a divalent metal ion to cleave a specific ssDNA sequence and form a phosphotyrosine linkage. This protein‐ssDNA fusion is useful for various biotechnology applications such as cellular imaging, cellular barcoding, DNA‐guided protein localization, and single molecule manipulation of DNA‐tethered proteins. Solving the structure of the Rep domain in complex with DNA could present necessary information regarding HUH‐tag sequence specificity and allow for rational engineering of protein‐DNA interactions. Here, the structure of WDV Rep domain in the apo state was solved with a crystal diffracting to 1.24 Å. While ssDNA soaks were attempted, they proved ineffective. However, the solved structure represents a step towards solving protein‐ssDNA complex.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141218455","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.03748
Clinita E. Randolph, P. Wedegaertner
Constitutively activating mutations in Gαq and Gα11 (Gαq/11) have been reported in up to 93% of uveal melanomas. Although constitutively active Gαq/11 promote uveal melanoma tumorigenesis through activation of multiple downstream pathways, no therapies inhibit constitutively active Gαq/11. Studies suggest that increased palmitate turnover and increased cytoplasmic localization occur upon activation of some Gα subunits. The purpose of our studies is to understand the role of palmitoylation in trafficking and signaling of constitutively active Gαq/11 in uveal melanoma. Using live cell imaging and cellular fractionation of HEK 293 cells and uveal melanoma cells, constitutively active GαqQ209L and GαqQ209P show decreased localization at membranes compared to wild type Gαq, suggesting increased turnover of attached palmitate in constitutively active mutants. Moreover, immunofluorescence microscopy revealed that a palmitoylation‐deficient constitutively active mutant displays a complete loss of plasma membrane localization and an inability to signal as measured by Rho‐ and Rac‐dependent YAP translocation into the nucleus, TEAD‐dependent luciferase activity, and ERK phosphorylation. These studies demonstrate that palmitoylation of mutationally activated Gαq/11 is required for its signaling functions. Although live cell imaging and cellular fractionation experiments reveal decreased membrane localization of constitutively active Gαq compared to wild‐type Gαq, our studies suggest that complete loss of membrane localization abolishes constitutively active Gαq‐dependent signaling. Disruption of constitutively active Gαq/11 palmitoylation and localization may be an effective strategy for inhibiting constitutively active Gαq/11 oncogenic signaling in uveal melanoma.
{"title":"Understanding Gαq/11 Localization and Trafficking in Uveal Melanoma","authors":"Clinita E. Randolph, P. Wedegaertner","doi":"10.1096/fasebj.2020.34.s1.03748","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.03748","url":null,"abstract":"Constitutively activating mutations in Gαq and Gα11 (Gαq/11) have been reported in up to 93% of uveal melanomas. Although constitutively active Gαq/11 promote uveal melanoma tumorigenesis through activation of multiple downstream pathways, no therapies inhibit constitutively active Gαq/11. Studies suggest that increased palmitate turnover and increased cytoplasmic localization occur upon activation of some Gα subunits. The purpose of our studies is to understand the role of palmitoylation in trafficking and signaling of constitutively active Gαq/11 in uveal melanoma. Using live cell imaging and cellular fractionation of HEK 293 cells and uveal melanoma cells, constitutively active GαqQ209L and GαqQ209P show decreased localization at membranes compared to wild type Gαq, suggesting increased turnover of attached palmitate in constitutively active mutants. Moreover, immunofluorescence microscopy revealed that a palmitoylation‐deficient constitutively active mutant displays a complete loss of plasma membrane localization and an inability to signal as measured by Rho‐ and Rac‐dependent YAP translocation into the nucleus, TEAD‐dependent luciferase activity, and ERK phosphorylation. These studies demonstrate that palmitoylation of mutationally activated Gαq/11 is required for its signaling functions. Although live cell imaging and cellular fractionation experiments reveal decreased membrane localization of constitutively active Gαq compared to wild‐type Gαq, our studies suggest that complete loss of membrane localization abolishes constitutively active Gαq‐dependent signaling. Disruption of constitutively active Gαq/11 palmitoylation and localization may be an effective strategy for inhibiting constitutively active Gαq/11 oncogenic signaling in uveal melanoma.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141217922","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.08854
A. Domashevskiy
Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A‐chain (RTA) and a viral genome‐linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N‐terminal truncated variant, VPg1‐110, bind to RTA and abolish ricin’s catalytic depurination of 28S rRNA in vitro and in a cell‐free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten‐fold as temperature elevates (5 °C to 37 °C). RTA‐VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA‐VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.
{"title":"Inhibition of ricin A‐chain (RTA) catalytic activity by a viral genome‐linked protein (VPg).","authors":"A. Domashevskiy","doi":"10.1096/fasebj.2020.34.s1.08854","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.08854","url":null,"abstract":"Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A‐chain (RTA) and a viral genome‐linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N‐terminal truncated variant, VPg1‐110, bind to RTA and abolish ricin’s catalytic depurination of 28S rRNA in vitro and in a cell‐free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten‐fold as temperature elevates (5 °C to 37 °C). RTA‐VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA‐VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141218723","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 : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.03324
Cynthia J. Metz, Michael J. Metz
Many professors are interested in providing science outreach to local K‐12 students, but may not have the time or resources needed to create an engaging presentation. The American Physiological Society supports outreach initiatives through programs such as the Physiology Understanding (PhUn) Week. Building upon these activities, it was desired to create and assess an immersive and engaging outreach program in a disadvantaged K‐12 student population. Three distinct modules were created on cardiovascular physiology, respiratory physiology, and oral health. All resources for these modules underwent peer‐review and publication, allowing other professors to easily execute their own outreach programming. The outreach modules were presented to 288 students in 3rd, 5th, 6th, and 7th grade classes. Implementation of the modules resulted in significant increases in student content knowledge, ranging from 32–57% improvement (P<0.001, dependent t‐test) with an average increase of 46%. K‐12 science teachers reported that the program was at an appropriate educational level, increased students’ enthusiasm for science, and increased students’ exposure to science careers. Additionally, the presenters of the outreach program were perceived to be enthusiastic, knowledgeable, and proficient at interacting with the students. On open‐response survey items, the science teachers indicated a high level of satisfaction with the program and an enthusiasm for continued collaborations. These results indicate the importance of organized and interactive science activities for the success of a new outreach program.
{"title":"The In’s and Out’s of Science Outreach: Assessment of an Engaging New Program","authors":"Cynthia J. Metz, Michael J. Metz","doi":"10.1096/fasebj.2020.34.s1.03324","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.03324","url":null,"abstract":"Many professors are interested in providing science outreach to local K‐12 students, but may not have the time or resources needed to create an engaging presentation. The American Physiological Society supports outreach initiatives through programs such as the Physiology Understanding (PhUn) Week. Building upon these activities, it was desired to create and assess an immersive and engaging outreach program in a disadvantaged K‐12 student population. Three distinct modules were created on cardiovascular physiology, respiratory physiology, and oral health. All resources for these modules underwent peer‐review and publication, allowing other professors to easily execute their own outreach programming. The outreach modules were presented to 288 students in 3rd, 5th, 6th, and 7th grade classes. Implementation of the modules resulted in significant increases in student content knowledge, ranging from 32–57% improvement (P<0.001, dependent t‐test) with an average increase of 46%. K‐12 science teachers reported that the program was at an appropriate educational level, increased students’ enthusiasm for science, and increased students’ exposure to science careers. Additionally, the presenters of the outreach program were perceived to be enthusiastic, knowledgeable, and proficient at interacting with the students. On open‐response survey items, the science teachers indicated a high level of satisfaction with the program and an enthusiasm for continued collaborations. These results indicate the importance of organized and interactive science activities for the success of a new outreach program.","PeriodicalId":22447,"journal":{"name":"The FASEB Journal","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2020-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141218340","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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