ABSTRACT
The cholesterol-sensing protein Scap induces cholesterol synthesis by transporting membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum (ER) to the Golgi apparatus for proteolytic activation. Transport requires interaction between Scap's two ER luminal loops (L1 and L7), which flank an intramembrane sterol-sensing domain (SSD). Cholesterol inhibits Scap transport by binding to L1, which triggers Scap's binding to Insig, an ER retention protein. Here we used cryoelectron microscopy (cryo-EM) to elucidate two structures of full-length chicken Scap: (1) a wild-type free of Insigs and (2) mutant Scap bound to chicken Insig without cholesterol. Strikingly, L1 and L7 intertwine tightly to form a globular domain that acts as a luminal platform connecting the SSD to the rest of Scap. In the presence of Insig, this platform undergoes a large rotation accompanied by rearrangement of Scap's transmembrane helices. We postulate that this conformational change halts Scap transport of SREBPs and inhibits cholesterol synthesis.
Subject(s)
Cholesterol/metabolism , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Amino Acid Sequence , Animals , Antibodies/metabolism , Chickens , Membrane Proteins/isolation & purification , Membrane Proteins/ultrastructure , Models, Biological , Models, Molecular , Protein Binding , Protein Domains , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Scap is a polytopic membrane protein that functions as a molecular machine to control the cholesterol content of membranes in mammalian cells. In the 21 years since our laboratory discovered Scap, we have learned how it binds sterol regulatory element-binding proteins (SREBPs) and transports them from the endoplasmic reticulum (ER) to the Golgi for proteolytic processing. Proteolysis releases the SREBP transcription factor domains, which enter the nucleus to promote cholesterol synthesis and uptake. When cholesterol in ER membranes exceeds a threshold, the sterol binds to Scap, triggering several conformational changes that prevent the Scap-SREBP complex from leaving the ER. As a result, SREBPs are no longer processed, cholesterol synthesis and uptake are repressed, and cholesterol homeostasis is restored. This review focuses on the four domains of Scap that undergo concerted conformational changes in response to cholesterol binding. The data provide a molecular mechanism for the control of lipids in cell membranes.
Subject(s)
Cholesterol/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Animals , Homeostasis , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Models, Biological , Models, Molecular , Protein Conformation , Protein Transport , Proteolysis , Receptors, LDL/metabolism , Sterol Regulatory Element Binding Proteins/metabolismABSTRACT
One-fourth of all deaths in industrialized countries result from coronary heart disease. A century of research has revealed the essential causative agent: cholesterol-carrying low-density lipoprotein (LDL). LDL is controlled by specific receptors (LDLRs) in liver that remove it from blood. Mutations that eliminate LDLRs raise LDL and cause heart attacks in childhood, whereas mutations that raise LDLRs reduce LDL and diminish heart attacks. If we are to eliminate coronary disease, lowering LDL should be the primary goal. Effective means to achieve this goal are currently available. The key questions are: who to treat, when to treat, and how long to treat.
Subject(s)
Cholesterol/metabolism , Coronary Vessels/pathology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Plaque, Atherosclerotic/drug therapy , Animals , Cardiovascular Diseases/drug therapy , Cardiovascular Diseases/genetics , Cardiovascular Diseases/pathology , Coronary Vessels/metabolism , Dietary Fats/metabolism , Humans , Plaque, Atherosclerotic/genetics , Plaque, Atherosclerotic/metabolism , Receptors, LDL/metabolismABSTRACT
Asteroids with diameters less than about 5 km have complex histories because they are small enough for radiative torques (that is, YORP, short for the Yarkovsky-O'Keefe-Radzievskii-Paddack effect)1 to be a notable factor in their evolution2. (152830) Dinkinesh is a small asteroid orbiting the Sun near the inner edge of the main asteroid belt with a heliocentric semimajor axis of 2.19 AU; its S-type spectrum3,4 is typical of bodies in this part of the main belt5. Here we report observations by the Lucy spacecraft6,7 as it passed within 431 km of Dinkinesh. Lucy revealed Dinkinesh, which has an effective diameter of only 720 m, to be unexpectedly complex. Of particular note is the presence of a prominent longitudinal trough overlain by a substantial equatorial ridge and the discovery of the first confirmed contact binary satellite, now named (152830) Dinkinesh I Selam. Selam consists of two near-equal-sized lobes with diameters of 210 m and 230 m. It orbits Dinkinesh at a distance of 3.1 km with an orbital period of about 52.7 h and is tidally locked. The dynamical state, angular momentum and geomorphologic observations of the system lead us to infer that the ridge and trough of Dinkinesh are probably the result of mass failure resulting from spin-up by YORP followed by the partial reaccretion of the shed material. Selam probably accreted from material shed by this event.
ABSTRACT
Earth is the only planet known to have continents, although how they formed and evolved is unclear. Here using the oxygen isotope compositions of dated magmatic zircon, we show that the Pilbara Craton in Western Australia, Earth's best-preserved Archaean (4.0-2.5 billion years ago (Ga)) continental remnant, was built in three stages. Stage 1 zircons (3.6-3.4 Ga) form two age clusters with one-third recording submantle δ18O, indicating crystallization from evolved magmas derived from hydrothermally altered basaltic crust like that in modern-day Iceland1,2. Shallow melting is consistent with giant impacts that typified the first billion years of Earth history3-5. Giant impacts provide a mechanism for fracturing the crust and establishing prolonged hydrothermal alteration by interaction with the globally extensive ocean6-8. A giant impact at around 3.6 Ga, coeval with the oldest low-δ18O zircon, would have triggered massive mantle melting to produce a thick mafic-ultramafic nucleus9,10. A second low-δ18O zircon cluster at around 3.4 Ga is contemporaneous with spherule beds that provide the oldest material evidence for giant impacts on Earth11. Stage 2 (3.4-3.0 Ga) zircons mostly have mantle-like δ18O and crystallized from parental magmas formed near the base of the evolving continental nucleus12. Stage 3 (<3.0 Ga) zircons have above-mantle δ18O, indicating efficient recycling of supracrustal rocks. That the oldest felsic rocks formed at 3.9-3.5 Ga (ref. 13), towards the end of the so-called late heavy bombardment4, is not a coincidence.
ABSTRACT
Among the caspases that cause regulated cell death, a unique function for caspase-7 has remained elusive. Caspase-3 performs apoptosis, whereas caspase-7 is typically considered an inefficient back-up. Caspase-1 activates gasdermin D pores to lyse the cell; however, caspase-1 also activates caspase-7 for unknown reasons1. Caspases can also trigger cell-type-specific death responses; for example, caspase-1 causes the extrusion of intestinal epithelial cell (IECs) in response to infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium)2,3. Here we show in both organoids and mice that caspase-7-deficient IECs do not complete extrusion. Mechanistically, caspase-7 counteracts gasdermin D pores and preserves cell integrity by cleaving and activating acid sphingomyelinase (ASM), which thereby generates copious amounts of ceramide to enable enhanced membrane repair. This provides time to complete the process of IEC extrusion. In parallel, we also show that caspase-7 and ASM cleavage are required to clear Chromobacterium violaceum and Listeria monocytogenes after perforin-pore-mediated attack by natural killer cells or cytotoxic T lymphocytes, which normally causes apoptosis in infected hepatocytes. Therefore, caspase-7 is not a conventional executioner but instead is a death facilitator that delays pore-driven lysis so that more-specialized processes, such as extrusion or apoptosis, can be completed before cell death. Cells must put their affairs in order before they die.
Subject(s)
Caspase 7 , Perforin , Phosphate-Binding Proteins , Pore Forming Cytotoxic Proteins , Sphingomyelin Phosphodiesterase , Animals , Apoptosis , Caspase 7/metabolism , Chromobacterium/immunology , Epithelial Cells/cytology , Intestines/cytology , Killer Cells, Natural/immunology , Listeria monocytogenes/immunology , Mice , Organoids , Perforin/metabolism , Phosphate-Binding Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Sphingomyelin Phosphodiesterase/metabolism , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
Insig-1 and Insig-2 are endoplasmic reticulum (ER) proteins that inhibit lipid synthesis by blocking transport of sterol regulatory element-binding proteins (SREBP-1 and SREBP-2) from ER to Golgi. In the Golgi, SREBPs are processed proteolytically to release their transcription-activating domains, which enhance the synthesis of fatty acids, triglycerides, and cholesterol. Heretofore, the two Insigs have redundant functions, and there is no rationale for two isoforms. The current data identify a specific function for Insig-2. We show that eicosapentaenoic acid (EPA), a polyunsaturated fatty acid, inhibits fatty acid synthesis in human fibroblasts and rat hepatocytes by activating adenylate cyclase, which induces protein kinase A (PKA) to phosphorylate serine-106 in Insig-2. Phosphorylated Insig-2 inhibits the proteolytic processing of SREBP-1, thereby blocking fatty acid synthesis. Phosphorylated Insig-2 does not block the processing of SREBP-2, which activates cholesterol synthesis. Insig-1 lacks serine-106 and is not phosphorylated at this site. EPA inhibition of SREBP-1 processing was reduced by the replacement of serine-106 in Insig-2 with alanine or by treatment with KT5720, a PKA inhibitor. Inhibition did not occur in mutant human fibroblasts that possess Insig-1 but lack Insig-2. These data provide an Insig-2-specific mechanism for the long-known inhibition of fatty acid synthesis by polyunsaturated fatty acids.
Subject(s)
Cyclic AMP-Dependent Protein Kinases , Fibroblasts , Intracellular Signaling Peptides and Proteins , Membrane Proteins , Sterol Regulatory Element Binding Protein 1 , Humans , Membrane Proteins/metabolism , Membrane Proteins/genetics , Animals , Phosphorylation , Rats , Sterol Regulatory Element Binding Protein 1/metabolism , Sterol Regulatory Element Binding Protein 1/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Intracellular Signaling Peptides and Proteins/genetics , Cyclic AMP-Dependent Protein Kinases/metabolism , Fibroblasts/metabolism , Fatty Acids, Unsaturated/metabolism , Fatty Acids/metabolism , Fatty Acids/biosynthesis , Eicosapentaenoic Acid/pharmacology , Sterol Regulatory Element Binding Protein 2/metabolism , Hepatocytes/metabolismABSTRACT
ABSTRACT: Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.
Subject(s)
Cell Adhesion Molecules , Factor VIII , Kininogens , Lectins, C-Type , Receptors, Cell Surface , von Willebrand Factor , Humans , von Willebrand Factor/genetics , von Willebrand Factor/metabolism , Factor VIII/genetics , Factor VIII/metabolism , Polymorphism, Single Nucleotide , Human Umbilical Vein Endothelial Cells/metabolism , Mendelian Randomization Analysis , Genome-Wide Association Study , Thrombosis/genetics , Thrombosis/blood , Genetic Association Studies , Male , Endothelial Cells/metabolism , FemaleABSTRACT
M.R2k/b mice are identical to the MA/My parent strain aside from a 5.58-Mb C57L-derived region on chromosome 17 (Cmv5s) that causes increased susceptibility to acute murine CMV (MCMV) infection and the development of significant spleen tissue damage. Spleen pathology begins at the marginal zone (MZ), apparent by 2 d postinfection (dpi), and progresses throughout the red pulp by 4 dpi. To better understand how M.R2k/b mice respond to infection and how Cmv5s contributes to tissue damage in the spleen, we assessed the regulation of myeloid cells and inflammation during acute MCMV infection in MA/My and M.R2k/b mice. We found that Cmv5s drove increased neutrophil accumulation and cell death at the MZ, which corresponded with evidence of localized oxidative stress and increased overall spleen IL-6 and TGF-ß1 early during infection. Further assessment of MCMV infection dynamics at the early MZ revealed infected SIGNR1+ MZ macrophages as the first apparent cell type lost during infection in these mice and the likely target of early neutrophil recruitment. Spleen macrophages were also identified as the mediators of differential spleen IL-6 and TGF-ß1 between MA/My and M.R2k/b mice. Interrogation of MCMV progression past 2 dpi revealed substantial M.R2k/b F480+ red pulp macrophage loss along with buildup of oxidative stress and MZ macrophage debris that was not neutrophil dependent. Together we identify Cmv5s-driven macrophage loss and inflammation during acute MCMV infection corresponding with the spatial and temporal development of spleen tissue damage.
ABSTRACT
The MHC class I molecule H-2Dk conveys resistance to acute murine CMV infection in both C57L (H-2Dk transgenic) and MA/My mice. M.H2k/b mice are on an MA/My background aside from a C57L-derived region spanning the MHC (Cmv5s), which diminishes this resistance and causes significant spleen histopathology. To hone in on the effector elements within the Cmv5s interval, we generated several Cmv5-recombinant congenic mouse strains and screened them in vivo, allowing us to narrow the phenotype-associated interval >6-fold and segment the genetic mechanism to at least two independent loci within the MHC region. In addition, we sought to further characterize the Cmv5s-associated phenotypes in their temporal appearance and potential direct relationship to viral load. To this end, we found that Cmv5s histopathology and NK cell activation could not be fully mirrored in the MA/My mice with increased viral dose, and that marginal zone destruction was the first apparent Cmv5s phenotype, being reliably quantified as early as 2 d postinfection in the M.H2k/b mice, prior to divergence in viral load, weight loss, or NK cell phenotype. Finally, we further dissect NK cell involvement, finding no intrinsic differences in NK cell function, despite increased upregulation of activation markers and checkpoint receptors. In conclusion, these data dissect the genetic and immunologic underpinnings of Cmv5 and reveal a model in which polymorphism within the MHC region of the genome leads to the development of tissue damage and corrupts protective NK cell immunity during acute viral infection.
Subject(s)
Cytomegalovirus Infections , Muromegalovirus , Mice , Animals , Histocompatibility Antigens Class I/genetics , Killer Cells, Natural , Lymphoid Tissue , Mice, Inbred C57BLABSTRACT
BACKGROUND: Remdesivir improves clinical outcomes in patients hospitalized with moderate-to-severe coronavirus disease 2019 (Covid-19). Whether the use of remdesivir in symptomatic, nonhospitalized patients with Covid-19 who are at high risk for disease progression prevents hospitalization is uncertain. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving nonhospitalized patients with Covid-19 who had symptom onset within the previous 7 days and who had at least one risk factor for disease progression (age ≥60 years, obesity, or certain coexisting medical conditions). Patients were randomly assigned to receive intravenous remdesivir (200 mg on day 1 and 100 mg on days 2 and 3) or placebo. The primary efficacy end point was a composite of Covid-19-related hospitalization or death from any cause by day 28. The primary safety end point was any adverse event. A secondary end point was a composite of a Covid-19-related medically attended visit or death from any cause by day 28. RESULTS: A total of 562 patients who underwent randomization and received at least one dose of remdesivir or placebo were included in the analyses: 279 patients in the remdesivir group and 283 in the placebo group. The mean age was 50 years, 47.9% of the patients were women, and 41.8% were Hispanic or Latinx. The most common coexisting conditions were diabetes mellitus (61.6%), obesity (55.2%), and hypertension (47.7%). Covid-19-related hospitalization or death from any cause occurred in 2 patients (0.7%) in the remdesivir group and in 15 (5.3%) in the placebo group (hazard ratio, 0.13; 95% confidence interval [CI], 0.03 to 0.59; P = 0.008). A total of 4 of 246 patients (1.6%) in the remdesivir group and 21 of 252 (8.3%) in the placebo group had a Covid-19-related medically attended visit by day 28 (hazard ratio, 0.19; 95% CI, 0.07 to 0.56). No patients had died by day 28. Adverse events occurred in 42.3% of the patients in the remdesivir group and in 46.3% of those in the placebo group. CONCLUSIONS: Among nonhospitalized patients who were at high risk for Covid-19 progression, a 3-day course of remdesivir had an acceptable safety profile and resulted in an 87% lower risk of hospitalization or death than placebo. (Funded by Gilead Sciences; PINETREE ClinicalTrials.gov number, NCT04501952; EudraCT number, 2020-003510-12.).
Subject(s)
Adenosine Monophosphate/analogs & derivatives , Alanine/analogs & derivatives , Antiviral Agents/therapeutic use , COVID-19 Drug Treatment , Adenosine Monophosphate/adverse effects , Adenosine Monophosphate/therapeutic use , Adult , Aged , Aged, 80 and over , Alanine/adverse effects , Alanine/therapeutic use , Antiviral Agents/adverse effects , COVID-19/complications , COVID-19/mortality , Comorbidity , Disease Progression , Double-Blind Method , Female , Hospitalization/statistics & numerical data , Humans , Male , Middle Aged , Outpatients , SARS-CoV-2/drug effects , Time-to-Treatment , Viral LoadABSTRACT
Plate tectonics is among the most important geological processes on Earth, but its emergence and evolution remain unclear. Here we extrapolate models of present-day plate tectonics to the past and propose that since about three billion years ago the rise of continents and the accumulation of sediments at continental edges and in trenches has provided lubrication for the stabilization of subduction and has been crucial in the development of plate tectonics on Earth. We conclude that the two largest surface erosion and subduction lubrication events occurred after the Palaeoproterozoic Huronian global glaciations (2.45 to 2.2 billion years ago), leading to the formation of the Columbia supercontinent, and after the Neoproterozoic 'snowball' Earth glaciations (0.75 to 0.63 billion years ago). The snowball Earth event followed the 'boring billion'-a period of reduced plate tectonic activity about 1.75 to 0.75 billion years ago that was probably caused by a shortfall of sediments in trenches-and it kick-started the modern episode of active plate tectonics.
ABSTRACT
Earth's mantle convection, which facilitates planetary heat loss, is manifested at the surface as present-day plate tectonics1. When plate tectonics emerged and how it has evolved through time are two of the most fundamental and challenging questions in Earth science1-4. Metamorphic rocks-rocks that have experienced solid-state mineral transformations due to changes in pressure (P) and temperature (T)-record periods of burial, heating, exhumation and cooling that reflect the tectonic environments in which they formed5,6. Changes in the global distribution of metamorphic (P, T) conditions in the continental crust through time might therefore reflect the secular evolution of Earth's tectonic processes. On modern Earth, convergent plate margins are characterized by metamorphic rocks that show a bimodal distribution of apparent thermal gradients (temperature change with depth; parameterized here as metamorphic T/P) in the form of paired metamorphic belts5, which is attributed to metamorphism near (low T/P) and away from (high T/P) subduction zones5,6. Here we show that Earth's modern plate tectonic regime has developed gradually with secular cooling of the mantle since the Neoarchaean era, 2.5 billion years ago. We evaluate the emergence of bimodal metamorphism (as a proxy for secular change in plate tectonics) using a statistical evaluation of the distributions of metamorphic T/P through time. We find that the distribution of metamorphic T/P has gradually become wider and more distinctly bimodal from the Neoarchaean era to the present day, and the average metamorphic T/P has decreased since the Palaeoproterozoic era. Our results contrast with studies that inferred an abrupt transition in tectonic style in the Neoproterozoic era (about 0.7 billion years ago1,7,8) or that suggested that modern plate tectonics has operated since the Palaeoproterozoic era (about two billion years ago9-12) at the latest.
ABSTRACT
Wild-type (WT) mice maintain viable levels of blood glucose even when adipose stores are depleted by 6 d of 60% calorie restriction followed by a 23-h fast (hereafter designated as "starved" mice). Survival depends on ghrelin, an octanoylated peptide hormone. Mice that lack ghrelin suffer lethal hypoglycemia when subjected to the same starvation regimen. Ghrelin is known to stimulate secretion of growth hormone (GH), which in turn stimulates secretion of IGF-1 (insulin-like growth factor-1). In the current study, we found that starved ghrelin-deficient mice had a 90% reduction in plasma IGF-1 when compared with starved WT mice. Injection of IGF-1 in starved ghrelin-deficient mice caused a twofold increase in glucose production and raised blood glucose to levels seen in starved WT mice. Increased glucose production was accompanied by increases in plasma glycerol, fatty acids and ketone bodies, and hepatic triglycerides. All of these increases were abolished when the mice were treated with atglistatin, an inhibitor of adipose tissue triglyceride lipase. We conclude that IGF-1 stimulates adipose tissue lipolysis in starved mice and that this lipolysis supplies energy and substrates that restore hepatic gluconeogenesis. This action of IGF-1 in starved mice is in contrast to its known action in inhibiting adipose tissue lipase in fed mice. Surprisingly, the ghrelin-dependent maintenance of plasma IGF-1 in starved mice was not mediated by GH. Direct injection of GH into starved ghrelin-deficient mice failed to increase plasma IGF-1. These data call attention to an unsuspected role of IGF-1 in the adaptation to starvation.
Subject(s)
Blood Glucose , Insulin-Like Growth Factor I , Starvation , Adaptation, Physiological , Adipose Tissue/drug effects , Adipose Tissue/enzymology , Adipose Tissue/metabolism , Animals , Blood Glucose/metabolism , Fatty Acids/blood , Ghrelin/metabolism , Gluconeogenesis , Glycerol/blood , Growth Hormone/metabolism , Insulin-Like Growth Factor I/analysis , Insulin-Like Growth Factor I/metabolism , Ketone Bodies/blood , Lipase/antagonists & inhibitors , Lipase/metabolism , Lipolysis , Liver/metabolism , Mice , Phenylurea Compounds/pharmacology , Starvation/blood , Starvation/metabolism , Triglycerides/metabolismABSTRACT
Low-density lipoprotein (LDL) delivers cholesterol to mammalian cells through receptor-mediated endocytosis. The LDL cholesterol is liberated in lysosomes and transported to the plasma membrane (PM) and from there to the endoplasmic reticulum (ER). Excess ER cholesterol is esterified with a fatty acid for storage as cholesteryl esters. Recently, we showed that PM-to-ER transport of LDL cholesterol requires phosphatidylserine (PS). Others showed that PM-to-ER transport of cholesterol derived from other sources requires Asters (also called GRAMD1s), a family of three ER proteins that bridge between the ER and PM by binding to PS. Here, we use a cholesterol esterification assay and other measures of ER cholesterol delivery to demonstrate that Asters participate in PM-to-ER transport of LDL cholesterol in Chinese hamster ovary cells. Knockout of the gene encoding PTDSS1, the major PS-synthesizing enzyme, lowered LDL-stimulated cholesterol esterification by 85%, whereas knockout of all three Aster genes lowered esterification by 65%. The reduction was even greater (94%) when the genes encoding PTDSS1 and the three Asters were knocked out simultaneously. We conclude that Asters participate in LDL cholesterol delivery from PM to ER, and their action depends in large part, but not exclusively, on PS. The data also indicate that PS participates in another delivery pathway, so far undefined, that is independent of Asters.
Subject(s)
Cholesterol, LDL/metabolism , Membrane Proteins/metabolism , Phosphatidylserines/metabolism , Animals , Biological Transport , CHO Cells , Cell Membrane/metabolism , Cholesterol/metabolism , Cholesterol Esters/metabolism , Cricetinae , Cricetulus , Endocytosis , Endoplasmic Reticulum/metabolism , Lysosomes/metabolismABSTRACT
The Rhodopsin family of G-proteincoupled receptors (GPCRs) comprises the targets of nearly a third of all pharmaceuticals. Despite structural water present in GPCR X-ray structures, the physiological relevance of these solvent molecules to rhodopsin signaling remains unknown. Here, we show experimental results consistent with the idea that rhodopsin activation in lipid membranes is coupled to bulk water movements into the protein. To quantify hydration changes, we measured reversible shifting of the metarhodopsin equilibrium due to osmotic stress using an extensive series of polyethylene glycol (PEG) osmolytes. We discovered clear evidence that light activation entails a large influx of bulk water (â¼80100 molecules) into the protein, giving insight into GPCR activation mechanisms. Various size polymer osmolytes directly control rhodopsin activation, in which large solutes are excluded from rhodopsin and dehydrate the protein, favoring the inactive state. In contrast, small osmolytes initially forward shift the activation equilibrium until a quantifiable saturation point is reached, similar to gain-of-function protein mutations. For the limit of increasing osmolyte size, a universal response of rhodopsin to osmotic stress is observed, suggesting it adopts a dynamic, hydrated sponge-like state upon photoactivation. Our results demand a rethinking of the role of water dynamics in modulating various intermediates in the GPCR energy landscape. We propose that besides bound water, an influx of bulk water plays a necessary role in establishing the active GPCR conformation that mediates signaling.
Subject(s)
Receptors, G-Protein-Coupled , Rhodopsin , Protein Conformation , Receptors, G-Protein-Coupled/metabolism , Rhodopsin/metabolism , Solvents/chemistry , Water/chemistryABSTRACT
BACKGROUND: Recent cases of clinical failure in malaria patients in the United Kingdom (UK) treated with artemether-lumefantrine have implications for malaria chemotherapy worldwide. METHODS: Parasites were isolated from an index case of confirmed Plasmodium falciparum treatment failure after standard treatment, and from comparable travel-acquired UK malaria cases. Drug susceptibility in vitro and genotypes at 6 resistance-associated loci were determined for all parasite isolates and compared with clinical outcomes for each parasite donor. RESULTS: A traveler, who returned to the UK from Uganda in 2022 with Plasmodium falciparum malaria, twice failed treatment with full courses of artemether-lumefantrine. Parasites from the patient exhibited significantly reduced susceptibility to artemisinin (ring-stage survival, 17.3% [95% confidence interval {CI}, 13.6%-21.1%]; P < .0001) and lumefantrine (effective concentration preventing 50% of growth = 259.4â nM [95% CI, 130.6-388.2â nM]; P = .001). Parasite genotyping identified an allele of pfk13 encoding both the A675V variant in the Pfk13 propeller domain and a novel L145V nonpropeller variant. In vitro susceptibility testing of 6 other P. falciparum lines of Ugandan origin identified reduced susceptibility to artemisinin and lumefantrine in 1 additional line, also from a 2022 treatment failure case. These parasites did not harbor a pfk13 propeller domain variant but rather the novel nonpropeller variant T349I. Variant alleles of pfubp1, pfap2mu, and pfcoronin were also identified among the 7 parasite lines. CONCLUSIONS: We confirm, in a documented case of artemether-lumefantrine treatment failure imported from Uganda, the presence of pfk13 mutations encoding L145V and A675V. Parasites with reduced susceptibility to both artemisinin and lumefantrine may be emerging in Uganda.
Subject(s)
Antimalarials , Artemisinins , Malaria, Falciparum , Malaria , Humans , Lumefantrine/pharmacology , Lumefantrine/therapeutic use , Plasmodium falciparum , Antimalarials/pharmacology , Antimalarials/therapeutic use , Artemether, Lumefantrine Drug Combination/pharmacology , Artemether, Lumefantrine Drug Combination/therapeutic use , Uganda , Drug Resistance , Artemether/pharmacology , Artemether/therapeutic use , Artemisinins/pharmacology , Artemisinins/therapeutic use , Malaria/drug therapy , Malaria, Falciparum/drug therapy , Malaria, Falciparum/parasitology , Treatment Failure , United Kingdom , Protozoan Proteins/geneticsABSTRACT
BACKGROUND: Antithrombin, PC (protein C), and PS (protein S) are circulating natural anticoagulant proteins that regulate hemostasis and of which partial deficiencies are causes of venous thromboembolism. Previous genetic association studies involving antithrombin, PC, and PS were limited by modest sample sizes or by being restricted to candidate genes. In the setting of the Cohorts for Heart and Aging Research in Genomic Epidemiology consortium, we meta-analyzed across ancestries the results from 10 genome-wide association studies of plasma levels of antithrombin, PC, PS free, and PS total. METHODS: Study participants were of European and African ancestries, and genotype data were imputed to TOPMed, a dense multiancestry reference panel. Each of the 10 studies conducted a genome-wide association studies for each phenotype and summary results were meta-analyzed, stratified by ancestry. Analysis of antithrombin included 25 243 European ancestry and 2688 African ancestry participants, PC analysis included 16 597 European ancestry and 2688 African ancestry participants, PSF and PST analysis included 4113 and 6409 European ancestry participants. We also conducted transcriptome-wide association analyses and multiphenotype analysis to discover additional associations. Novel genome-wide association studies and transcriptome-wide association analyses findings were validated by in vitro functional experiments. Mendelian randomization was performed to assess the causal relationship between these proteins and cardiovascular outcomes. RESULTS: Genome-wide association studies meta-analyses identified 4 newly associated loci: 3 with antithrombin levels (GCKR, BAZ1B, and HP-TXNL4B) and 1 with PS levels (ORM1-ORM2). transcriptome-wide association analyses identified 3 newly associated genes: 1 with antithrombin level (FCGRT), 1 with PC (GOLM2), and 1 with PS (MYL7). In addition, we replicated 7 independent loci reported in previous studies. Functional experiments provided evidence for the involvement of GCKR, SNX17, and HP genes in antithrombin regulation. CONCLUSIONS: The use of larger sample sizes, diverse populations, and a denser imputation reference panel allowed the detection of 7 novel genomic loci associated with plasma antithrombin, PC, and PS levels.
Subject(s)
Protein C , Protein S , Protein C/genetics , Protein S/genetics , Genome-Wide Association Study , Antithrombins , Transcriptome , Anticoagulants , Antithrombin III/genetics , Polymorphism, Single NucleotideABSTRACT
The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface of infected cells where these complexes bind Ly49 inhibitory receptors (IRs) and prevent NK cell attack. Nonetheless, certain self-MHC I-binding Ly49 activating and inhibitory receptors are able to promote robust NK cell expansion and antiviral immunity during MCMV infection. A basis for MHC I-dependent NK cell sensing of MCMV-infected targets and control of MCMV infection however remains unclear. In this study, we discovered that the Ly49R activation receptor is selectively triggered during MCMV infection on antiviral NK cells licensed by the Ly49G2 IR. Ly49R activating receptor recognition of MCMV-infected targets is dependent on MHC I Dk and MCMV gp34 expression. Remarkably, although Ly49R is critical for Ly49G2-dependent antiviral immunity, blockade of the activation receptor in Ly49G2-deficient mice has no impact on virus control, suggesting that paired Ly49G2 MCMV sensing might enable Ly49R+ NK cells to better engage viral targets. Indeed, MCMV gp34 facilitates Ly49G2 binding to infected cells, and the IR is required to counter gp34-mediated immune evasion. A specific requirement for Ly49G2 in antiviral immunity is further explained by its capacity to license cytokine receptor signaling pathways and enhance Ly49R+ NK cell proliferation during infection. These findings advance our understanding of the molecular basis for functionally disparate self-receptor enhancement of antiviral NK cell immunity.
Subject(s)
Muromegalovirus , Animals , Antiviral Agents/metabolism , Carrier Proteins/metabolism , Histocompatibility Antigens Class I , Immune Evasion , Mice , Mice, Inbred C57BL , NK Cell Lectin-Like Receptor Subfamily A/metabolism , Receptors, Cytokine/metabolism , Receptors, Natural Killer Cell/metabolismABSTRACT
LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3beta-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3beta-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.