Browsing by Subject "Lipids"
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Item A positive feedback loop mediates crosstalk between calcium, cyclic nucleotide and lipid signalling in calcium-induced Toxoplasma gondii egress(Public Library of Science, 2022-10-20) Nofal, Stephanie D.; Dominicus, Caia; Broncel, Malgorzata; Katris, Nicholas J.; Flynn, Helen R.; Arrizabalaga, Gustavo; Botté, Cyrille Y.; Invergo, Brandon M.; Treeck, Moritz; Pharmacology and Toxicology, School of MedicineFundamental processes that govern the lytic cycle of the intracellular parasite Toxoplasma gondii are regulated by several signalling pathways. However, how these pathways are connected remains largely unknown. Here, we compare the phospho-signalling networks during Toxoplasma egress from its host cell by artificially raising cGMP or calcium levels. We show that both egress inducers trigger indistinguishable signalling responses and provide evidence for a positive feedback loop linking calcium and cyclic nucleotide signalling. Using WT and conditional knockout parasites of the non-essential calcium-dependent protein kinase 3 (CDPK3), which display a delay in calcium inonophore-mediated egress, we explore changes in phosphorylation and lipid signalling in sub-minute timecourses after inducing Ca2+ release. These studies indicate that cAMP and lipid metabolism are central to the feedback loop, which is partly dependent on CDPK3 and allows the parasite to respond faster to inducers of egress. Biochemical analysis of 4 phosphodiesterases (PDEs) identified in our phosphoproteomes establishes PDE2 as a cAMP-specific PDE which regulates Ca2+ induced egress in a CDPK3-independent manner. The other PDEs display dual hydrolytic activity and play no role in Ca2+ induced egress. In summary, we uncover a positive feedback loop that enhances signalling during egress, thereby linking several signalling pathways.Item Adaptive enzyme formation during hyperlipogenesis(1966) Hicks, Sonja ElaineItem Advancements in APOE and dementia research: Highlights from the 2023 AAIC Advancements: APOE conference(Wiley, 2024) Kloske, Courtney M.; Belloy, Michael E.; Blue, Elizabeth E.; Bowman, Gregory R.; Carrillo, Maria C.; Chen, Xiaoying; Chiba-Falek, Ornit; Davis, Albert A.; Di Paolo, Gilbert; Garretti, Francesca; Gate, David; Golden, Lesley R.; Heinecke, Jay W.; Herz, Joachim; Huang, Yadong; Iadecola, Costantino; Johnson, Lance A.; Kanekiyo, Takahisa; Karch, Celeste M.; Khvorova, Anastasia; Koppes-den Hertog, Sascha J.; Lamb, Bruce T.; Lawler, Paige E.; Le Guen, Yann; Litvinchuk, Alexandra; Liu, Chia-Chen; Mahinrad, Simin; Marcora, Edoardo; Marino, Claudia; Michaelson, Danny M.; Miller, Justin J.; Morganti, Josh M.; Narayan, Priyanka S.; Naslavsky, Michel S.; Oosthoek, Marlies; Ramachandran, Kapil V.; Ramakrishnan, Abhirami; Raulin, Ana-Caroline; Robert, Aiko; Saleh, Rasha N. M.; Sexton, Claire; Shah, Nilomi; Shue, Francis; Sible, Isabel J.; Soranno, Andrea; Strickland, Michael R.; Tcw, Julia; Thierry, Manon; Tsai, Li-Huei; Tuckey, Ryan A.; Ulrich, Jason D.; van der Kant, Rik; Wang, Na; Wellington, Cheryl L.; Weninger, Stacie C.; Yassine, Hussein N.; Zhao, Na; Bu, Guojun; Goate, Alison M.; Holtzman, David M.; Neurology, School of MedicineIntroduction: The apolipoprotein E gene (APOE) is an established central player in the pathogenesis of Alzheimer's disease (AD), with distinct apoE isoforms exerting diverse effects. apoE influences not only amyloid-beta and tau pathologies but also lipid and energy metabolism, neuroinflammation, cerebral vascular health, and sex-dependent disease manifestations. Furthermore, ancestral background may significantly impact the link between APOE and AD, underscoring the need for more inclusive research. Methods: In 2023, the Alzheimer's Association convened multidisciplinary researchers at the "AAIC Advancements: APOE" conference to discuss various topics, including apoE isoforms and their roles in AD pathogenesis, progress in apoE-targeted therapeutic strategies, updates on disease models and interventions that modulate apoE expression and function. Results: This manuscript presents highlights from the conference and provides an overview of opportunities for further research in the field. Discussion: Understanding apoE's multifaceted roles in AD pathogenesis will help develop targeted interventions for AD and advance the field of AD precision medicine. Highlights: APOE is a central player in the pathogenesis of Alzheimer's disease. APOE exerts a numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The AAIC Advancements: APOE conference encouraged discussions and collaborations on understanding the role of APOE.Item Analysis of human gliomas by swab touch spray-mass spectrometry: applications to intraoperative assessment of surgical margins and presence of oncometabolites(Royal Society of Chemistry, 2017-10-23) Pirro, Valentina; Llor, Raquel Sero; Jarmusch, Alan K.; Alfaro, Clint M.; Cohen-Gadol, Aaron A.; Hattab, Eyas M.; Cooks, R. Graham; Neurological Surgery, School of MedicineTouch spray mass spectrometry using medical swabs is an ambient ionization technique (ionization of unprocessed sample in the open air) that has potential intraoperative application in quickly identifying the disease state of tissue and in better characterizing the resection margin. To explore this potential, we studied 29 human brain tumor specimens and obtained evidence that this technique can provide diagnostic molecular information that is relevant to brain cancer. Touch spray using medical swabs involves the physical sampling of tissue using a medical swab on a spatial scale of a few mm2 with subsequent ionization occurring directly from the swab tip upon addition of solvent and application of a high voltage. Using a tertiary mixture of acetonitrile, N,N-dimethylformamide, and ethanol, membrane-derived phospholipids and oncometabolites are extracted from the tissue, incorporated into the sprayed microdroplets, vacuumed into the mass spectrometer, and characterized in the resulting mass spectra. The tumor cell load was assessed from the complex phospholipid pattern in the mass spectra and also separately by measurement of N-acetylaspartate. Mutation status of the isocitrate dehydrogenase gene was determined via detection of the oncometabolite 2-hydroxyglutarate. The lack of sample pretreatment makes touch spray mass spectrometry using medical swabs a feasible intraoperative strategy for rapid surgical assessment.Item Benefits of Icosapent Ethyl Across the Range of Kidney Function in Patients With Established Cardiovascular Disease or Diabetes: REDUCE-IT RENAL(Wolters Kluwer, 2021-11-30) Majithia, Arjun; Bhatt, Deepak L.; Friedman, Allon N.; Miller, Michael; Steg, Ph. Gabriel; Brinton, Eliot A.; Jacobson, Terry A.; Ketchum, Steven B.; Juliano, Rebecca A.; Jiao, Lixia; Doyle, Ralph T., Jr.; Granowitz, Craig; Budoff, Matthew; Mason, R. Preston; Tardif, Jean-Claude; Boden, William E.; Ballantyne, Christie M.; Medicine, School of MedicineBackground: Chronic kidney disease is associated with adverse outcomes among patients with established cardiovascular disease (CVD) or diabetes. Commonly used medications to treat CVD are less effective among patients with reduced kidney function. Methods: REDUCE-IT (Reduction of Cardiovascular Events with Icosapent Ethyl-Intervention Trial) was a multicenter, double-blind, placebo-controlled trial that randomly assigned statin-treated patients with elevated triglycerides (135-499 mg/dL) who had CVD or diabetes and 1 additional risk factor to treatment with icosapent ethyl (4 g daily) or placebo. Patients from REDUCE-IT were categorized by prespecified estimated glomerular filtration rate (eGFR) categories to analyze the effect of icosapent ethyl on the primary end point (composite of cardiovascular death, nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or unstable angina) and key secondary end point (a composite of cardiovascular death, nonfatal myocardial infarction, or nonfatal stroke). Results: Among the 8179 REDUCE-IT patients, median baseline eGFR was 75 mL·min-1·1.73 m-2 (range, 17-123 mL·min-1·1.73 m-2). There were no meaningful changes in median eGFR for icosapent ethyl versus placebo across study visits. Treatment with icosapent ethyl led to consistent reduction in both the primary and key secondary composite end points across baseline eGFR categories. Patients with eGFR <60 mL·min-1·1.73 m-2 treated with icosapent ethyl had the largest absolute and similar relative risk reduction for the primary composite end point (icosapent ethyl versus placebo, 21.8% versus 28.9%; hazard ratio [HR], 0.71 [95% CI, 0.59-0.85]; P=0.0002) and key secondary composite end point (16.8% versus 22.5%; HR 0.71 [95% CI, 0.57-0.88]; P=0.001). The numeric reduction in cardiovascular death was greatest in the eGFR <60 mL·min-1·1.73 m-2 group (icosapent ethyl: 7.6%; placebo: 10.6%; HR, 0.70 [95% CI, 0.51-0.95]; P=0.02). Although patients with eGFR <60 mL·min-1·1.73 m-2 treated with icosapent ethyl had the highest numeric rates of atrial fibrillation/flutter (icosapent ethyl: 4.2%; placebo 3.0%; HR 1.42 [95% CI, 0.86-2.32]; P=0.17) and serious bleeding (icosapent ethyl: 5.4%; placebo 3.6%; HR, 1.40 [95% CI, 0.90-2.18]; P=0.13), HRs for atrial fibrillation/flutter and serious bleeding were similar across eGFR categories (P-interaction for atrial fibrillation/flutter=0.92; P-interaction for serious bleeding=0.76). Conclusions: In REDUCE-IT, icosapent ethyl reduced fatal and nonfatal ischemic events across the broad range of baseline eGFR categories.Item Comparative distribution of carbohydrates and lipid droplets in the Golgi apparatus of intestinal absorptive cells(Rockefeller University Press, 1971) Sage, Jean A.; Jersild, Ralph A., Jr.; Anatomy, Cell Biology and Physiology, School of MedicineItem Conformational Changes in Two Inter-Helical Loops of Mhp1 Membrane Transporter(PLOS, 2015-07-17) Song, Hyun Deok; Zhu, Fangqiang; Department of Physics, School of ScienceMhp1 is a bacterial secondary transporter with high-resolution crystal structures available for both the outward- and inward-facing conformations. Through molecular dynamics simulations of the ligand-free Mhp1 as well as analysis of its crystal structures, here we show that two inter-helical loops, respectively located at the extra- and intracellular ends of the “hash motif” in the protein, play important roles in the conformational transition. In the outward- and inward-facing states of the protein, the loops adopt different secondary structures, either wrapped to the end of an alpha-helix, or unwrapped to extended conformations. In equilibrium simulations of 100 ns with Mhp1 in explicit lipids and water, the loop conformations remain largely stable. In targeted molecular dynamics simulations with the protein structure driven from one state to the other, the loops exhibit resistance and only undergo abrupt changes when other parts of the protein already approach the target conformation. Free energy calculations on the isolated loops further confirm that the wrapping/unwrapping transitions are associated with substantial energetic barriers, and consist of multiple sequential steps involving the rotation of certain backbone torsion angles. Furthermore, in simulations with the loops driven from one state to the other, a large part of the protein follows the loops to the target conformation. Taken together, our simulations suggest that changes of the loop secondary structures would be among the slow degrees of freedom in the conformational transition of the entire protein. Incorporation of detailed loop structures into the reaction coordinate, therefore, should improve the convergence and relevance of the resulting conformational free energy.Item Crystal structure of RahU, an aegerolysin protein from the human pathogen Pseudomonas aeruginosa, and its interaction with membrane ceramide phosphorylethanolamine(Springer Nature, 2021-03-22) Kočar, Eva; Lenarčič, Tea; Hodnik, Vesna; Panevska, Anastasija; Huang, Yunjie; Bajc, Gregor; Kostanjšek, Rok; Naren, Anjaparavanda P.; Maček, Peter; Anderluh, Gregor; Sepčić, Kristina; Podobnik, Marjetka; Butala, Matej; Pediatrics, School of MedicineAegerolysins are proteins produced by bacteria, fungi, plants and protozoa. The most studied fungal aegerolysins share a common property of interacting with membranes enriched with cholesterol in combination with either sphingomyelin or ceramide phosphorylethanolamine (CPE), major sphingolipids in the cell membranes of vertebrates and invertebrates, respectively. However, genome analyses show a particularly high frequency of aegerolysin genes in bacteria, including the pathogenic genera Pseudomonas and Vibrio; these are human pathogens of high clinical relevance and can thrive in a variety of other species. The knowledge on bacterial aegerolysin-lipid interactions is scarce. We show that Pseudomonas aeruginosa aegerolysin RahU interacts with CPE, but not with sphingomyelin-enriched artificial membranes, and that RahU interacts with the insect cell line producing CPE. We report crystal structures of RahU alone and in complex with tris(hydroxymethyl)aminomethane (Tris), which, like the phosphorylethanolamine head group of CPE, contains a primary amine. The RahU structures reveal that the two loops proximal to the amino terminus form a cavity that accommodates Tris, and that the flexibility of these two loops is important for this interaction. We show that Tris interferes with CPE-enriched membranes for binding to RahU, implying on the importance of the ligand cavity between the loops and its proximity in RahU membrane interaction. We further support this by studying the interaction of single amino acid substitution mutants of RahU with the CPE-enriched membranes. Our results thus represent a starting point for a better understanding of the role of P. aeruginosa RahU, and possibly other bacterial aegerolysins, in bacterial interactions with other organisms.Item Differential Lipid Profiles of Normal Human Brain Matter and Gliomas by Positive and Negative Mode Desorption Electrospray Ionization – Mass Spectrometry Imaging.(PLOS, 2016) Jarmusch, Alan K.; Alfaro, Clint M.; Pirro, Valentina; Hattab, Eyas M.; Cohen-Gadol, Aaron A.; Cooks, R. Graham; Department of Neurological Surgery, IU School of MedicineDesorption electrospray ionization—mass spectrometry (DESI-MS) imaging was used to analyze unmodified human brain tissue sections from 39 subjects sequentially in the positive and negative ionization modes. Acquisition of both MS polarities allowed more complete analysis of the human brain tumor lipidome as some phospholipids ionize preferentially in the positive and others in the negative ion mode. Normal brain parenchyma, comprised of grey matter and white matter, was differentiated from glioma using positive and negative ion mode DESI-MS lipid profiles with the aid of principal component analysis along with linear discriminant analysis. Principal component–linear discriminant analyses of the positive mode lipid profiles was able to distinguish grey matter, white matter, and glioma with an average sensitivity of 93.2% and specificity of 96.6%, while the negative mode lipid profiles had an average sensitivity of 94.1% and specificity of 97.4%. The positive and negative mode lipid profiles provided complementary information. Principal component–linear discriminant analysis of the combined positive and negative mode lipid profiles, via data fusion, resulted in approximately the same average sensitivity (94.7%) and specificity (97.6%) of the positive and negative modes when used individually. However, they complemented each other by improving the sensitivity and specificity of all classes (grey matter, white matter, and glioma) beyond 90% when used in combination. Further principal component analysis using the fused data resulted in the subgrouping of glioma into two groups associated with grey and white matter, respectively, a separation not apparent in the principal component analysis scores plots of the separate positive and negative mode data. The interrelationship of tumor cell percentage and the lipid profiles is discussed, and how such a measure could be used to measure residual tumor at surgical margins.Item Direct Detection of Isolevuglandins in Tissues Using a D11 scFv-Alkaline Phosphatase Fusion Protein and Immunofluorescence(MyJove Corporation, 2021-07-05) Warden, Cassandra; Simmons, Alan J.; Pasic, Lejla; Pitzer, Ashley; Davies, Sean S.; Layer, Justin H.; Mernaugh, Raymond L.; Kirabo, Annet; Medicine, School of MedicineIsolevuglandins (IsoLGs) are highly reactive gamma ketoaldehydes formed from H2-isoprostanes through lipid peroxidation and crosslink proteins leading to inflammation and various diseases including hypertension. Detection of IsoLG accumulation in tissues is crucial in shedding light on their involvement in the disease processes. However, measurement of IsoLGs in tissues is extremely difficult, and currently available tools, including mass spectrometry analysis, are laborious and extremely expensive. Here we describe a novel method for in situ detection of IsoLGs in tissues using alkaline phosphatase-conjugated D11 ScFv and a recombinant phage-display antibody produced in E. coli by immunofluorescent microscopy. Four controls were used for validating the staining: (1) staining with and without D11, (2) staining with bacterial periplasmic extract with the alkaline phosphatase linker, (3) irrelevant scFV antibody staining, and (4) competitive control with IsoLG prior to the staining. We demonstrate the effectiveness of the alkaline phosphatase-conjugated D11 in both human and mouse tissues with or without hypertension. This method will likely serve as an important tool to study the role of IsoLGs in a wide variety of disease processes.