Pub Date : 2020-04-01DOI: 10.1096/fasebj.2020.34.s1.00476
Loretta Yang, Shengcheng Wu, Lu Meng, Christian Gerner-Smidt, R. Woods
As alternatives to established carbohydrate‐detection reagents (antibodies and lectins), Lectenz Bio is developing engineered proteins (known as Lectenz®), that target glycan sequences for with existing reagents either do not exist or are sub‐optimal. Lectenz® are engineered proteins derived from catalytically inactivated glycan‐processing enzymes that have been optimized for high affinity towards specific glycan sequences. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. Here we demonstrate the performance and specificity of two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme.
{"title":"Engineered High‐Specificity Affinity Reagents for the Detection of Glycan Sialylation","authors":"Loretta Yang, Shengcheng Wu, Lu Meng, Christian Gerner-Smidt, R. Woods","doi":"10.1096/fasebj.2020.34.s1.00476","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.00476","url":null,"abstract":"As alternatives to established carbohydrate‐detection reagents (antibodies and lectins), Lectenz Bio is developing engineered proteins (known as Lectenz®), that target glycan sequences for with existing reagents either do not exist or are sub‐optimal. Lectenz® are engineered proteins derived from catalytically inactivated glycan‐processing enzymes that have been optimized for high affinity towards specific glycan sequences. The conversion of such enzymes into affinity reagents is facilitated by computationally‐guided directed evolution. Lectenz® are being developed for a variety of glycan detection and enrichment applications including affinity chromatography, Western blot, and immunohistochemistry. Here we demonstrate the performance and specificity of two novel sialic acid recognizing Lectenz® engineered from a sialidase enzyme.","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":"141217766","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.s1.06067
Markus M. Rinschen, O. Palygin, Gary Siuzdak, A. Staruschenko
Hypertension is a persistent epidemic across the developed world that is closely associated with kidney disease. Here, we applied a metabolomics, phosphoproteomics and proteomics strategy to analyze the effect of hypertensive insults on kidneys. Our data established the metabolic aspects of hypertension‐induced glomerular sclerosis, including lipid breakdown at early disease stages and activation of anaplerotic pathways to regenerate energy equivalents to counter stress. For example, branched‐chain amino acids and proline, required for collagen synthesis, were depleted in glomeruli at early time points. Further, indicators of metabolic stress were reflected by low levels of ATP and NADH and increased abundance of oxidized lipids derived from lipid breakdown. These processes were specific to kidney glomeruli where metabolic signaling occurred through mTOR and AMPK signaling. Quantitative phosphoproteomics combined with computational modelling suggested that these processes controlled key molecules in glomeruli and specifically podocytes, including cytoskeletal components and GTP‐binding proteins, which would be expected to compete for decreasing amounts of GTP at early time points. As a result, glomeruli showed increased expression of metabolic enzymes of central carbon metabolism, amino acid degradation, and lipid oxidation, findings observed in previously published data from other disease models and patients with glomerular damage. Overall, these results suggest that metabolic interventions could be potentially useful in treating hypertension‐induced kidney disease.
{"title":"Metabolic rewiring of the hypertensive kidney","authors":"Markus M. Rinschen, O. Palygin, Gary Siuzdak, A. Staruschenko","doi":"10.1096/fasebj.2020.34.s1.06067","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.06067","url":null,"abstract":"Hypertension is a persistent epidemic across the developed world that is closely associated with kidney disease. Here, we applied a metabolomics, phosphoproteomics and proteomics strategy to analyze the effect of hypertensive insults on kidneys. Our data established the metabolic aspects of hypertension‐induced glomerular sclerosis, including lipid breakdown at early disease stages and activation of anaplerotic pathways to regenerate energy equivalents to counter stress. For example, branched‐chain amino acids and proline, required for collagen synthesis, were depleted in glomeruli at early time points. Further, indicators of metabolic stress were reflected by low levels of ATP and NADH and increased abundance of oxidized lipids derived from lipid breakdown. These processes were specific to kidney glomeruli where metabolic signaling occurred through mTOR and AMPK signaling. Quantitative phosphoproteomics combined with computational modelling suggested that these processes controlled key molecules in glomeruli and specifically podocytes, including cytoskeletal components and GTP‐binding proteins, which would be expected to compete for decreasing amounts of GTP at early time points. As a result, glomeruli showed increased expression of metabolic enzymes of central carbon metabolism, amino acid degradation, and lipid oxidation, findings observed in previously published data from other disease models and patients with glomerular damage. Overall, these results suggest that metabolic interventions could be potentially useful in treating hypertension‐induced kidney disease.","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":"141216979","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.s1.04661
G. Fulop, C. Ahire, Tamás Csípő, S. Tarantini, T. Kiss, Priya Balasubramanian, A. Yabluchanskiy, E. Farkas, Attila Toth, Ádám Nyúl-Tóth, Peter Toth, A. Csiszar, Z. Ungvari
Cognitive impairment is one of the most common co‐occurring chronic conditions among elderly heart failure patients (incidence: up to ~ 80%); however, the underlying mechanisms are not completely understood. It is hypothesized that in addition to decreased cardiac output, increases in central‐and consequentially, cerebral‐venous pressure (backward failure) also contribute significantly to the genesis of cognitive impairment. To test this hypothesis and elucidate the specific pathogenic role of venous congestion in the brain, we have established a novel model of increased cerebral venous pressure: mice with jugular vein ligation (JVL). To test the hypothesis that increased venous pressure in the brain contributes to the development of cognitive deficits by causing blood‐brain barrier disruption, dysregulation of blood flow, and/or promoting neuroinflammation, in C57BL/6 mice, the internal and external jugular veins were ligated. Cognitive function (radial arm water maze), gait function (CatWalk), and motor coordination (rotarod) were tested post‐JVL. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow in the whisker barrel cortex in response to contralateral whisker stimulation by laser speckle contrast imaging through a closed cranial window. Blood‐brain barrier integrity (IgG extravasation) and microglia activation (Iba1 staining) were assessed in brain slices by immunohistochemistry. Neuroinflammation‐related gene expression profile was assessed by a targeted qPCR array. After jugular vein ligation, mice exhibited impaired spatial learning and memory, altered motor coordination, and impaired gait function, mimicking important aspects of altered brain function observed in human heart failure patients. JVL did not alter neurovascular coupling responses. In the brains of mice with JVL, significant extravasation of IgG was detected, indicating blood‐brain barrier disruption, which was associated with histological markers of neuroinflammation (increased presence of activated microglia) and a proinflammatory shift in gene expression profile. Thus, cerebral venous congestion per se can cause blood‐brain barrier disruption and neuroinflammation, which likely contribute to the genesis of cognitive impairment. These findings have relevance to the pathogenesis of cognitive decline associated with heart failure as well as increased cerebal venous pressure due to increased jugular venous reflux in elderly human patients.
{"title":"Cerebral venous congestion promotes blood‐brain barrier disruption and neuroinflammation, impairing cognitive function in mice.","authors":"G. Fulop, C. Ahire, Tamás Csípő, S. Tarantini, T. Kiss, Priya Balasubramanian, A. Yabluchanskiy, E. Farkas, Attila Toth, Ádám Nyúl-Tóth, Peter Toth, A. Csiszar, Z. Ungvari","doi":"10.1096/fasebj.2020.34.s1.04661","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.04661","url":null,"abstract":"Cognitive impairment is one of the most common co‐occurring chronic conditions among elderly heart failure patients (incidence: up to ~ 80%); however, the underlying mechanisms are not completely understood. It is hypothesized that in addition to decreased cardiac output, increases in central‐and consequentially, cerebral‐venous pressure (backward failure) also contribute significantly to the genesis of cognitive impairment. To test this hypothesis and elucidate the specific pathogenic role of venous congestion in the brain, we have established a novel model of increased cerebral venous pressure: mice with jugular vein ligation (JVL). To test the hypothesis that increased venous pressure in the brain contributes to the development of cognitive deficits by causing blood‐brain barrier disruption, dysregulation of blood flow, and/or promoting neuroinflammation, in C57BL/6 mice, the internal and external jugular veins were ligated. Cognitive function (radial arm water maze), gait function (CatWalk), and motor coordination (rotarod) were tested post‐JVL. Neurovascular coupling responses were assessed by measuring changes in cerebral blood flow in the whisker barrel cortex in response to contralateral whisker stimulation by laser speckle contrast imaging through a closed cranial window. Blood‐brain barrier integrity (IgG extravasation) and microglia activation (Iba1 staining) were assessed in brain slices by immunohistochemistry. Neuroinflammation‐related gene expression profile was assessed by a targeted qPCR array. After jugular vein ligation, mice exhibited impaired spatial learning and memory, altered motor coordination, and impaired gait function, mimicking important aspects of altered brain function observed in human heart failure patients. JVL did not alter neurovascular coupling responses. In the brains of mice with JVL, significant extravasation of IgG was detected, indicating blood‐brain barrier disruption, which was associated with histological markers of neuroinflammation (increased presence of activated microglia) and a proinflammatory shift in gene expression profile. Thus, cerebral venous congestion per se can cause blood‐brain barrier disruption and neuroinflammation, which likely contribute to the genesis of cognitive impairment. These findings have relevance to the pathogenesis of cognitive decline associated with heart failure as well as increased cerebal venous pressure due to increased jugular venous reflux in elderly human patients.","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":"141217361","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.s1.05287
C. M. Lawrence, Ross Hartman, B. Eilers, Mark J. Young
Sulfolobus turreted icosahedral virus (STIV) is a model archaeal virus with a pseudo‐T=31 icosahedral virion with a mass of ~64 mega‐Daltons. Although STIV employs pyramidal lysis structures to exit the host, knowledge of the viral entry process is lacking. We therefore initiated crystallographic and cryo‐electron tomographic (CET) studies on STIV attachment and entry. Cryoelectron micrographs showed virion attachment to pili‐like structures emanating from the Sulfolobus host. Tomographic reconstruction and sub‐tomogram averaging revealed pili recognition by the STIV C381 turret protein. Specifically, the triple jelly roll structure of C381 determined by X‐ray crystallography shows that pilus recognition is mediated by conserved surface residues in the second and third domains. In addition, the STIV petal protein (C557), when present, occludes the pili binding site, suggesting that it functions as a maturation protein. Combined, these results demonstrate a role for the namesake STIV turrets in initial cellular attachment and provide the first molecular model for viral attachment in the archaeal domain of life. The work also nicely illustrates the synergistic power of hybrid structural studies utilizing cryo‐electron tomography, single particle analysis and crystallography to model cellular structures at the molecular level.
{"title":"The Molecular Mechanism of Cellular Attachment for an Archaeal Virus","authors":"C. M. Lawrence, Ross Hartman, B. Eilers, Mark J. Young","doi":"10.1096/fasebj.2020.34.s1.05287","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.05287","url":null,"abstract":"Sulfolobus turreted icosahedral virus (STIV) is a model archaeal virus with a pseudo‐T=31 icosahedral virion with a mass of ~64 mega‐Daltons. Although STIV employs pyramidal lysis structures to exit the host, knowledge of the viral entry process is lacking. We therefore initiated crystallographic and cryo‐electron tomographic (CET) studies on STIV attachment and entry. Cryoelectron micrographs showed virion attachment to pili‐like structures emanating from the Sulfolobus host. Tomographic reconstruction and sub‐tomogram averaging revealed pili recognition by the STIV C381 turret protein. Specifically, the triple jelly roll structure of C381 determined by X‐ray crystallography shows that pilus recognition is mediated by conserved surface residues in the second and third domains. In addition, the STIV petal protein (C557), when present, occludes the pili binding site, suggesting that it functions as a maturation protein. Combined, these results demonstrate a role for the namesake STIV turrets in initial cellular attachment and provide the first molecular model for viral attachment in the archaeal domain of life. The work also nicely illustrates the synergistic power of hybrid structural studies utilizing cryo‐electron tomography, single particle analysis and crystallography to model cellular structures at the molecular level.","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":"141217815","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.s1.05501
A. Kawashima, Cassandra J. Wong, C. King, A. Gingras, A. Newton
PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. A number of other PHLPP1 targets have been identified, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. During mitotic exit, PHLPP1 protein levels decrease, suggesting that PHLPP1 is degraded during mitotic exit. This correlates with an increase in Akt Ser473 phosphorylation, a validated cellular target of PHLPP1. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows proper passage through mitosis. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, plays a role in controlling proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.
{"title":"CDK1‐dependent Phosphorylation of the Tumor Suppressor Phosphatase, PHLPP1, Regulates the Mitotic PHLPP1 Interactome","authors":"A. Kawashima, Cassandra J. Wong, C. King, A. Gingras, A. Newton","doi":"10.1096/fasebj.2020.34.s1.05501","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.05501","url":null,"abstract":"PH domain Leucine Rich Repeat Protein Phosphatase 1 (PHLPP1) is a tumor suppressor originally discovered for its ability to directly dephosphorylate and inactivate the pro‐survival kinase Akt, a key transducer of growth factor signaling. A number of other PHLPP1 targets have been identified, but still little is known about the molecular mechanisms governing the function and regulation of PHLPP1 itself. Here we report that PHLPP1 is hyperphosphorylated during mitosis in a CDK1‐dependent manner, and that this hyperphosphorylation regulates its interaction with mitotic proteins. Specifically, we show that PHLPP1 undergoes an electrophoretic mobility shift in mitotic cells that is lost with lambda phosphatase treatment, and is prevented by CDK1 inhibition. This mobility shift can be recreated in vitro using recombinant CDK1‐Cyclin B. Mass spectrometry and biochemical analysis reveals that these phosphorylations modify the N‐terminus of PHLPP1, a functionally uncharacterized region. A proximity dependent biotin identification (BioID) interaction screen revealed that mitotic PHLPP1 interacts with components of the mitotic spindle apparatus and the kinetochore. Additionally, the data suggest that the N‐terminus is required for the dissociation of PHLPP1 from interphase scaffolds, such as Scribble, during mitosis. During mitotic exit, PHLPP1 protein levels decrease, suggesting that PHLPP1 is degraded during mitotic exit. This correlates with an increase in Akt Ser473 phosphorylation, a validated cellular target of PHLPP1. Our data are consistent with a model in which phosphorylation of PHLPP1 during mitosis regulates binding to its mitotic partners and allows proper passage through mitosis. Reversible protein phosphorylation, orchestrated by kinases and phosphatases, plays a role in controlling proper progression through mitosis, which is essential as errors in mitosis can result in aneuploidy, a hallmark of cancer. The finding that PHLPP1 binds mitotic proteins in a cell cycle and phosphorylation‐dependent manner may have relevance to its tumor suppressive function.","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":"141219190","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.s1.06948
Soyeon Park, Asrafun Nahar
The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as ‘Rpt ring’, assembles through six ATPase subunits (Rpt1‐Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2 and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early‐stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism—degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully‐formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone‐mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation.
{"title":"‐ Two Alternative Mechanisms Regulate the Onset of Chaperone‐mediated Assembly of the Proteasomal ATPases","authors":"Soyeon Park, Asrafun Nahar","doi":"10.1096/fasebj.2020.34.s1.06948","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.06948","url":null,"abstract":"The proteasome holoenzyme is a molecular machine that degrades most proteins in eukaryotes. In the holoenzyme, its heterohexameric ATPase injects protein substrates into the proteolytic core particle, where degradation occurs. The heterohexameric ATPase, referred to as ‘Rpt ring’, assembles through six ATPase subunits (Rpt1‐Rpt6) individually binding to specific chaperones (Rpn14, Nas6, Nas2 and Hsm3). Here, our findings suggest that the onset of Rpt ring assembly can be regulated by two alternative mechanisms. Excess Rpt subunits relative to their chaperones are sequestered into multiple puncta specifically during early‐stage Rpt ring assembly. Sequestration occurs during stressed conditions, for example heat, which transcriptionally induce Rpt subunits. When the free Rpt pool is limited experimentally, Rpt subunits are competent for proteasome assembly even without their cognate chaperones. These data suggest that sequestration may regulate amounts of individual Rpt subunits relative to their chaperones, allowing for proper onset of Rpt ring assembly. Indeed, Rpt subunits in the puncta can later resume their assembly into the proteasome. Intriguingly, when proteasome assembly resumes in stressed cells or is ongoing in unstressed cells, excess Rpt subunits are recognized by an alternative mechanism—degradation by the proteasome holoenzyme itself. Rpt subunits undergo proteasome assembly until the holoenzyme complex is generated at a sufficient level. The fully‐formed holoenzyme can then degrade any remaining excess Rpt subunits, thereby regulating its own Rpt ring assembly. These two alternative mechanisms, degradation and sequestration of Rpt subunits, may help control the onset of chaperone‐mediated Rpt ring assembly, thereby promoting proper proteasome holoenzyme formation.","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":"141217342","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.s1.02093
Lee-Young Chau, Yaw-Wen Hsu, Ming-Tsai Chiang, Fu-Fei Hsu, Takashi Angata, Paul R. Crocker
The accumulation of lipid‐laden macrophages, foam cells, within sub‐endothelial intima is a key feature of early atherosclerosis. Siglec‐E is a member of sialic acid binding lectin predominantly expressed on myeloid cells to transduce inhibitory signal upon interacting with its ligands. Whether Siglec‐E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. To this end, both apoE‐deficient and apoE/Siglec‐E‐double deficient mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were then assessed. The results showed that Siglec‐E deficiency accelerated atherosclerosis without affecting lipid profile in apoE deficient mice. In vitro experiments demonstrated that Siglec‐E deletion facilitated the uptake of acetylated or oxidized low density lipoprotein (LDL) and augmented foam cell formation in macrophages. By performing proximity labeling and proteomic analysis, we identified CD36 as a cell surface protein interacting with Siglec‐E. Notably, the interaction between Siglec‐E and CD36 was not affected by the sialylation status of CD36. Further experiments demonstrated that oxidized LDL induced transient Siglec‐E phosphorylation and recruitment of SHP‐1 in macrophages. VAV, a downstream effector implicated in CD36‐mediated oxidized LDL uptake, was shown to interact with SHP‐1 following oxidized LDL treatment. Moreover, Siglec‐E deficiency enhanced VAV phosphorylation induced by oxidized LDL. Collectively, these data demonstrate that Siglec‐E attenuates atherosclerosis in apoE‐deficient mice through suppressing CD36‐mediated signaling responsible for modified LDL uptake and foam cell formation in macrophages.
{"title":"Siglec‐E Retards Atherosclerosis by Inhibiting CD36‐Mediated Foam Cell Formation","authors":"Lee-Young Chau, Yaw-Wen Hsu, Ming-Tsai Chiang, Fu-Fei Hsu, Takashi Angata, Paul R. Crocker","doi":"10.1096/fasebj.2020.34.s1.02093","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.02093","url":null,"abstract":"The accumulation of lipid‐laden macrophages, foam cells, within sub‐endothelial intima is a key feature of early atherosclerosis. Siglec‐E is a member of sialic acid binding lectin predominantly expressed on myeloid cells to transduce inhibitory signal upon interacting with its ligands. Whether Siglec‐E expression on macrophages impacts foam cell formation and atherosclerosis remains to be established. To this end, both apoE‐deficient and apoE/Siglec‐E‐double deficient mice were placed on high fat diet for 3 months and their lipid profiles and severities of atherosclerosis were then assessed. The results showed that Siglec‐E deficiency accelerated atherosclerosis without affecting lipid profile in apoE deficient mice. In vitro experiments demonstrated that Siglec‐E deletion facilitated the uptake of acetylated or oxidized low density lipoprotein (LDL) and augmented foam cell formation in macrophages. By performing proximity labeling and proteomic analysis, we identified CD36 as a cell surface protein interacting with Siglec‐E. Notably, the interaction between Siglec‐E and CD36 was not affected by the sialylation status of CD36. Further experiments demonstrated that oxidized LDL induced transient Siglec‐E phosphorylation and recruitment of SHP‐1 in macrophages. VAV, a downstream effector implicated in CD36‐mediated oxidized LDL uptake, was shown to interact with SHP‐1 following oxidized LDL treatment. Moreover, Siglec‐E deficiency enhanced VAV phosphorylation induced by oxidized LDL. Collectively, these data demonstrate that Siglec‐E attenuates atherosclerosis in apoE‐deficient mice through suppressing CD36‐mediated signaling responsible for modified LDL uptake and foam cell formation in macrophages.","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":"141217810","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.s1.02010
J. Galligan, Dominique O. Gaffney, E. Jennings, Colin C Anderson, John O. Marentette, Taoda Shi, Anne-Mette Schou Oxvig, Matthew D. Streeter, Mogens Johannsen, David A. Spiegel, Eli Chapman, James R. Roede
Post‐translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feed‐forward mechanisms of regulation. We have identified a novel PTM that is derived from the glycolytic by‐product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D‐lactate. We have identified the non‐enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a ‘LactoylLys’ modification on proteins. GLO2 knockout cells have elevated LGSH and consequently, a marked increase in LactoylLys. Using an alkyne‐tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg‐like conditions.
{"title":"Non‐Enzymatic Lysine Lactoylation of Glycolytic Enzymes","authors":"J. Galligan, Dominique O. Gaffney, E. Jennings, Colin C Anderson, John O. Marentette, Taoda Shi, Anne-Mette Schou Oxvig, Matthew D. Streeter, Mogens Johannsen, David A. Spiegel, Eli Chapman, James R. Roede","doi":"10.1096/fasebj.2020.34.s1.02010","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.02010","url":null,"abstract":"Post‐translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feed‐forward mechanisms of regulation. We have identified a novel PTM that is derived from the glycolytic by‐product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D‐lactate. We have identified the non‐enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a ‘LactoylLys’ modification on proteins. GLO2 knockout cells have elevated LGSH and consequently, a marked increase in LactoylLys. Using an alkyne‐tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg‐like conditions.","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":"141217020","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.s1.00112
Mary P. Nivison, Allegra VanderWilde, Pravita Balijepalli, Kathryn E. Meier
Dietary and nutritional factors are considered critical modulating factors in prostate cancer development. Epidemiological data have suggested to the idea that dietary consumption of lycopene, found in tomatoes, may prevent prostate cancer. This study examined the mechanism by which lycopene interacts with prostate cancer cells, with the goal of discovering less toxic treatment and prevention options. The hypothesis that was addressed was that lycopene interferes with growth factor‐mediated signal transduction in prostate cancer cells. The effects of lycopene on PC‐3, a human prostate cancer cell line, were analyzed using proliferation assays, immunoblot analysis, and confocal microscopy. The growth factors used were lysophosphatidic acid (LPA) and epidermal growth factor (EGF). Lycopene (10 μM) inhibited LPA and EGF‐induced proliferation of PC‐3 cells, confirming previous unpublished results. Lycopene also inhibited activation of Akt in response to LPA and EGF as assessed by immunoblotting. Confocal immunofluorescence microscopy showed that the decrease in activated Akt was most prominent in the cell nucleus. These results confirm the ability of lycopene to inhibit growth factor response in human prostate cancer cells, and suggest new directions for future studies.
{"title":"Effects of Lycopene on Growth Factor Response in Prostate Cancer Cells","authors":"Mary P. Nivison, Allegra VanderWilde, Pravita Balijepalli, Kathryn E. Meier","doi":"10.1096/fasebj.2020.34.s1.00112","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.00112","url":null,"abstract":"Dietary and nutritional factors are considered critical modulating factors in prostate cancer development. Epidemiological data have suggested to the idea that dietary consumption of lycopene, found in tomatoes, may prevent prostate cancer. This study examined the mechanism by which lycopene interacts with prostate cancer cells, with the goal of discovering less toxic treatment and prevention options. The hypothesis that was addressed was that lycopene interferes with growth factor‐mediated signal transduction in prostate cancer cells. The effects of lycopene on PC‐3, a human prostate cancer cell line, were analyzed using proliferation assays, immunoblot analysis, and confocal microscopy. The growth factors used were lysophosphatidic acid (LPA) and epidermal growth factor (EGF). Lycopene (10 μM) inhibited LPA and EGF‐induced proliferation of PC‐3 cells, confirming previous unpublished results. Lycopene also inhibited activation of Akt in response to LPA and EGF as assessed by immunoblotting. Confocal immunofluorescence microscopy showed that the decrease in activated Akt was most prominent in the cell nucleus. These results confirm the ability of lycopene to inhibit growth factor response in human prostate cancer cells, and suggest new directions for future studies.","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":"89112038","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.s1.00244
Cayce E. Dorrier, Dvir Aran, Ezekiel Haenelt, C. Lizama, K. Cautivo, Ryan N. Sheehy, Geoffrey A. Weiner, Thomas Arnold, R. Daneman
Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) in which the body’s immune system attacks the myelin sheath that surrounds and insulates axons. In many cases this myelin is not repaired by myelinating oligodendrocytes, which decreases the efficiency of action potential conduction and leads to neural dysfunction. We hypothesized that a barrier preventing oligodendrocyte lineage cells from repairing damaged myelin is a fibrotic scar. Following CNS injury, a scar consisting of an outer glial scar made up of reactive astrocytes and an inner fibrotic scar made of proteins such as collagen I forms around the site of trauma. In MS the glial scar has also been characterized, but the presence of a fibrotic scar has not been investigated. I have shown that following induction of experimental autoimmune encephalomyelitis (EAE) in mice, which is used as a model of MS, an extensive fibrotic scar forms in the lesioned tissue. Scar‐forming cells were visualized in this tissue using a Col1a1GFP mouse model. The number of these cells increased rapidly in the lesion site following symptom onset and remained high throughout the course of the disease. Lineage tracing and single cell RNA sequencing determined that these cells arise from the proliferation of fibroblasts and not other cells such as pericytes turning on the production of collagen. The objective of this project is to determine the role of the fibrotic scar in tissue repair and to determine mechanisms of scar formation in the CNS. To determine the role of the fibrotic scar following EAE I used the herpes thymidine kinase system to ablate proliferating fibroblasts. Using this paradigm I was able to reduce scar formation by 60% in EAE and found that this reduction resulted in a decrease in motor symptoms in the later stages of disease concurrent with an increase in oligodendrocyte lineage cells in the inflammatory lesions. To understand the signaling pathways that play a role in CNS scar formation, I used FACS to purify Col1a1GFP+ fibroblasts from spinal cords of healthy mice and mice with EAE and analyzed their transcriptome by RNA sequencing. I found that these cells upregulate inflammatory signaling pathways in disease such as the interferon gamma pathway. Deleting the interferon gamma receptor in CNS fibroblasts resulted in a decrease in scar formation following EAE and may be a potential therapeutic target for CNS disorders with fibrotic scarring. In conclusion I identified a fibrotic scar forms following neuroinflammation that arises from the proliferation of CNS fibroblasts, plays a role in disease recovery and forms in part through interferon gamma signaling.
{"title":"The Role of the Fibrotic Scar in Repair Following Neuroinflammation","authors":"Cayce E. Dorrier, Dvir Aran, Ezekiel Haenelt, C. Lizama, K. Cautivo, Ryan N. Sheehy, Geoffrey A. Weiner, Thomas Arnold, R. Daneman","doi":"10.1096/fasebj.2020.34.s1.00244","DOIUrl":"https://doi.org/10.1096/fasebj.2020.34.s1.00244","url":null,"abstract":"Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) in which the body’s immune system attacks the myelin sheath that surrounds and insulates axons. In many cases this myelin is not repaired by myelinating oligodendrocytes, which decreases the efficiency of action potential conduction and leads to neural dysfunction. We hypothesized that a barrier preventing oligodendrocyte lineage cells from repairing damaged myelin is a fibrotic scar. Following CNS injury, a scar consisting of an outer glial scar made up of reactive astrocytes and an inner fibrotic scar made of proteins such as collagen I forms around the site of trauma. In MS the glial scar has also been characterized, but the presence of a fibrotic scar has not been investigated. I have shown that following induction of experimental autoimmune encephalomyelitis (EAE) in mice, which is used as a model of MS, an extensive fibrotic scar forms in the lesioned tissue. Scar‐forming cells were visualized in this tissue using a Col1a1GFP mouse model. The number of these cells increased rapidly in the lesion site following symptom onset and remained high throughout the course of the disease. Lineage tracing and single cell RNA sequencing determined that these cells arise from the proliferation of fibroblasts and not other cells such as pericytes turning on the production of collagen. The objective of this project is to determine the role of the fibrotic scar in tissue repair and to determine mechanisms of scar formation in the CNS. To determine the role of the fibrotic scar following EAE I used the herpes thymidine kinase system to ablate proliferating fibroblasts. Using this paradigm I was able to reduce scar formation by 60% in EAE and found that this reduction resulted in a decrease in motor symptoms in the later stages of disease concurrent with an increase in oligodendrocyte lineage cells in the inflammatory lesions. To understand the signaling pathways that play a role in CNS scar formation, I used FACS to purify Col1a1GFP+ fibroblasts from spinal cords of healthy mice and mice with EAE and analyzed their transcriptome by RNA sequencing. I found that these cells upregulate inflammatory signaling pathways in disease such as the interferon gamma pathway. Deleting the interferon gamma receptor in CNS fibroblasts resulted in a decrease in scar formation following EAE and may be a potential therapeutic target for CNS disorders with fibrotic scarring. In conclusion I identified a fibrotic scar forms following neuroinflammation that arises from the proliferation of CNS fibroblasts, plays a role in disease recovery and forms in part through interferon gamma signaling.","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":"141218789","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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