ABSTRACT
Breast cancer (BC) comprises multiple distinct subtypes that differ genetically, pathologically, and clinically. Here, we describe a robust protocol for long-term culturing of human mammary epithelial organoids. Using this protocol, >100 primary and metastatic BC organoid lines were generated, broadly recapitulating the diversity of the disease. BC organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. DNA copy number variations as well as sequence changes were consistent within tumor-organoid pairs and largely retained even after extended passaging. BC organoids furthermore populated all major gene-expression-based classification groups and allowed in vitro drug screens that were consistent with in vivo xeno-transplantations and patient response. This study describes a representative collection of well-characterized BC organoids available for cancer research and drug development, as well as a strategy to assess in vitro drug response in a personalized fashion.
Subject(s)
Breast Neoplasms/pathology , Genetic Heterogeneity , Organoids/pathology , Tissue Banks , Animals , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Cells, Cultured , Drug Screening Assays, Antitumor/methods , Female , Humans , Mice , Mice, Nude , Organoids/drug effects , Precision Medicine/methodsABSTRACT
Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.
Subject(s)
Lipid Bilayers , Saccharomyces cerevisiae Proteins , Lipid Bilayers/metabolism , Saccharomyces cerevisiae/metabolism , Endoplasmic Reticulum Stress/physiology , Saccharomyces cerevisiae Proteins/metabolism , Unfolded Protein Response , Endoplasmic Reticulum/metabolism , Technology , Lipid MetabolismABSTRACT
Biomembranes are complex materials composed of lipids and proteins that compartmentalize biochemistry. They are actively remodeled in response to physical and metabolic cues, as well as during cell differentiation and stress. The concept of homeoviscous adaptation has become a textbook example of membrane responsiveness. Here, we discuss limitations and common misconceptions revolving around it. By highlighting key moments in the life cycle of a transmembrane protein, we illustrate that membrane thickness and a finely regulated membrane compressibility are crucial to facilitate proper membrane protein insertion, function, sorting, and inheritance. We propose that the unfolded protein response (UPR) provides a mechanism for endoplasmic reticulum (ER) membrane homeostasis by sensing aberrant transverse membrane stiffening and triggering adaptive responses that re-establish membrane compressibility.
Subject(s)
Endoplasmic Reticulum Stress , Unfolded Protein Response , Endoplasmic Reticulum Stress/physiology , Homeostasis/physiology , Endoplasmic Reticulum/metabolism , Membrane Proteins/metabolismABSTRACT
Complex metabolic diseases such as diabetes and non-alcoholic fatty liver disease have been associated with aberrant lipid metabolism and lipotoxicity. To maintain lipid homeostasis and escape lipotoxicity, cells deploy a plethora of mechanisms, the most fascinating of which relying on a sense-and-response circuit. New work by Volkmar et al reveals an auto-regulated pathway formed by a lipid hydrolase and a lipid-sensitive E3 ubiquitin ligase playing hide-and-seek to warrant membrane function in stressed cells.
Subject(s)
Liver , Ubiquitin , Endoplasmic Reticulum , Hydrolases/metabolism , Lipid Metabolism , Lipids , Liver/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Eukaryotic cells face the challenge of maintaining the complex composition of several coexisting organelles. The molecular mechanisms underlying the homeostasis of subcellular membranes and their adaptation during stress are only now starting to emerge. Here, we discuss three membrane property sensors of the endoplasmic reticulum (ER), namely OPI1, MGA2, and IRE1, each controlling a large cellular program impacting the lipid metabolic network. OPI1 coordinates the production of membrane and storage lipids, MGA2 regulates the production of unsaturated fatty acids required for membrane biogenesis, and IRE1 controls the unfolded protein response (UPR) to adjust ER size, protein folding, and the secretory capacity of the cell. Although these proteins use remarkably distinct sensing mechanisms, they are functionally connected via the ER membrane and cooperate to maintain membrane homeostasis. As a rationalization of the recently described mechanism of UPR activation by lipid bilayer stress, we propose that IRE1 can sense the protein-to-lipid ratio in the ER membrane to ensure a balanced production of membrane proteins and lipids.
Subject(s)
Membrane Glycoproteins/metabolism , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Repressor Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription Factors/metabolism , Animals , Biological Transport , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum/physiology , Endoplasmic Reticulum Stress/physiology , Endoribonucleases/metabolism , Homeostasis/physiology , Humans , Lipid Metabolism/physiology , Models, Biological , Protein Folding , Saccharomyces cerevisiae/metabolism , Signal Transduction , Unfolded Protein Response/genetics , Unfolded Protein Response/physiologyABSTRACT
The unfolded protein response (UPR) is a conserved homeostatic program that is activated by misfolded proteins in the lumen of the endoplasmic reticulum (ER). Recently, it became evident that aberrant lipid compositions of the ER membrane, referred to as lipid bilayer stress, are equally potent in activating the UPR. The underlying molecular mechanism, however, remained unclear. We show that the most conserved transducer of ER stress, Ire1, uses an amphipathic helix (AH) to sense membrane aberrancies and control UPR activity. In vivo and in vitro experiments, together with molecular dynamics (MD) simulations, identify the physicochemical properties of the membrane environment that control Ire1 oligomerization. This work establishes the molecular mechanism of UPR activation by lipid bilayer stress.
Subject(s)
Endoplasmic Reticulum Stress , Endoplasmic Reticulum/metabolism , Intracellular Membranes/metabolism , Lipid Bilayers/metabolism , Membrane Glycoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Unfolded Protein Response , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/genetics , Molecular Dynamics Simulation , Mutation , Protein Conformation, alpha-Helical , Protein Folding , Protein Serine-Threonine Kinases/chemistry , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Structure-Activity Relationship , Time FactorsABSTRACT
Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.
Subject(s)
Cystic Fibrosis , Lipid A , Pseudomonas Infections , Pseudomonas aeruginosa , Pseudomonas aeruginosa/immunology , Humans , Lipid A/metabolism , Lipid A/immunology , Cystic Fibrosis/microbiology , Cystic Fibrosis/immunology , Cystic Fibrosis/metabolism , Pseudomonas Infections/immunology , Pseudomonas Infections/microbiology , Pseudomonas Infections/metabolism , Toll-Like Receptor 4/metabolism , Toll-Like Receptor 4/immunology , Cytokines/metabolism , Macrophages/immunology , Macrophages/metabolism , Macrophages/microbiology , Bronchoalveolar Lavage Fluid/immunology , Lung/microbiology , Lung/immunology , Lung/metabolismABSTRACT
Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.
Subject(s)
Anti-Bacterial Agents , Colistin , Disease Models, Animal , Immunity, Innate , Klebsiella Infections , Klebsiella pneumoniae , Lipid A , Animals , Klebsiella pneumoniae/immunology , Klebsiella pneumoniae/drug effects , Colistin/pharmacology , Lipid A/immunology , Mice , Klebsiella Infections/immunology , Klebsiella Infections/microbiology , Humans , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial/genetics , Pneumonia, Bacterial/immunology , Pneumonia, Bacterial/microbiology , Mice, Inbred C57BL , Cytokines/metabolism , Bronchoalveolar Lavage Fluid/immunology , Bronchoalveolar Lavage Fluid/microbiology , FemaleABSTRACT
The protection afforded by acellular pertussis vaccines wanes over time, and there is a need to develop improved vaccine formulations. Options to improve the vaccines involve the utilization of different adjuvants and administration via different routes. While intramuscular (IM) vaccination provides a robust systemic immune response, intranasal (IN) vaccination theoretically induces a localized immune response within the nasal cavity. In the case of a Bordetella pertussis infection, IN vaccination results in an immune response that is similar to natural infection, which provides the longest duration of protection. Current acellular formulations utilize an alum adjuvant, and antibody levels wane over time. To overcome the current limitations with the acellular vaccine, we incorporated a novel TLR4 agonist, BECC438b, into both IM and IN acellular formulations to determine its ability to protect against infection in a murine airway challenge model. Following immunization and challenge, we observed that DTaP + BECC438b reduced bacterial burden within the lung and trachea for both administration routes when compared with mock-vaccinated and challenged (MVC) mice. Interestingly, IN administration of DTaP + BECC438b induced a Th1-polarized immune response, while IM vaccination polarized toward a Th2 immune response. RNA sequencing analysis of the lung demonstrated that DTaP + BECC438b activates biological pathways similar to natural infection. Additionally, IN administration of DTaP + BECC438b activated the expression of genes involved in a multitude of pathways associated with the immune system. Overall, these data suggest that BECC438b adjuvant and the IN vaccination route can impact efficacy and responses of pertussis vaccines in pre-clinical mouse models.
Subject(s)
Diphtheria-Tetanus-acellular Pertussis Vaccines , Whooping Cough , Animals , Mice , Whooping Cough/prevention & control , Toll-Like Receptor 4 , Pertussis Vaccine , Diphtheria-Tetanus-Pertussis Vaccine , Bordetella pertussis , Adjuvants, Immunologic , Immunity , Antibodies, BacterialABSTRACT
Structural elucidation of Gram-negative bacterial lipid A traditionally requires chemical extraction followed by tandem MS data in the negative ion mode. Previously, we reported FLAT and FLATn as methods to rapidly determine the structure of lipid A without chromatographic techniques. In this work, we extend the capability and effectiveness of these techniques to elucidate the chemical structure in a de novo manner by including the use of positive ion mode (FLAT+ and FLATn+) spectral approaches. Advantages of positive mode analysis of lipid A include the generation of more interpretable and informative fragmentation patterns that include the identification of diagnostic fragments, including selective dissociation of a glycosidic bond between two glucosamine units and the selective dissociation at the secondary acyl chain in 2'-N, allowing for the determination of the composition of fatty acids. As a proof of principle, we present here two previously uncharacterized structures of lipid A from Roseomonas mucosa (R. mucosa) and Moraxella canis (M. canis). In R. mucosa, we determined the lipid A structure with a nonconventional backbone of-ß-1,6 linked 2,3-dideoxy-2,3-diamno-d-glucopyranose further modified with galacturonic acid in the place of typical 1-phosphate, and in M. canis, we assigned a single discrete structure using the specific fragmentation patterns of terminal phosphate groups present in lipid A. Therefore, FLATn+, in combination with FLAT and FLATn, provides a multimodal structural platform for rapid structure characterization of unusual and complex lipid A structures from a single colony.
ABSTRACT
Rapid diagnostic tests (RDTs) for bloodstream infections have the potential to reduce time to appropriate antimicrobial therapy and improve patient outcomes. Previously, an in-house, lipid-based, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) method, Fast Lipid Analysis Technique (FLAT MS), has shown promise as a rapid pathogen identification method. In this study, FLAT MS for direct from blood culture identification was evaluated and compared to FDA-cleared identification methods using the Benefit-risk Evaluation Framework (BED-FRAME) analysis. FLAT MS was evaluated and compared to Bruker Sepsityper and bioMérieux BioFire FilmArray BCID2 using results from a previous study. For this study, 301 positive blood cultures were collected from the University of Maryland Medical Center. The RDTs were compared by their sensitivities, time-to-results, hands-on time, and BED-FRAME analysis. The overall sensitivity of all platforms compared to culture results from monomicrobial-positive blood cultures was 88.3%. However, the three RDTs differed in their accuracy for identifying Gram-positive bacteria, Gram-negative bacteria, and yeast. Time-to-results for FLAT MS, Sepsityper, and BioFire BCID2 were all approximately one hour. Hands-on times for FLAT MS, Sepsityper, and BioFire BCID2 were 10 (±1.3), 40 (±2.8), and 5 (±0.25) minutes, respectively. BED-FRAME demonstrated that each RDT had utility at different pathogen prevalence and relative importance. BED-FRAME is a useful tool that can used to determine which RDT is best for a healthcare center.
Subject(s)
Bacteremia , Sepsis , Humans , Bacteremia/diagnosis , Bacteremia/microbiology , Rapid Diagnostic Tests , Bacteriological Techniques/methods , Sepsis/diagnosis , Blood Culture , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , LipidsABSTRACT
The development of novel therapeutic approaches is crucial in the fight against multi-drug resistant (MDR) bacteria, particularly gram-negative species. Small molecule adjuvants that enhance the activity of otherwise gram-positive selective antibiotics against gram-negative bacteria have the potential to expand current treatment options. We have previously reported adjuvants based upon a 2-aminoimidazole (2-AI) scaffold that potentiate macrolide antibiotics against several gram-negative pathogens. Herein, we report the discovery and structure-activity relationship (SAR) investigation of an additional class of macrolide adjuvants based upon a 2-aminobenzimidazole (2-ABI) scaffold. The lead compound lowers the minimum inhibitory concentration (MIC) of clarithromycin (CLR) from 512 to 2â µg/mL at 30â µM against Klebsiella pneumoniae 2146, and from 32 to 2â µg/mL at 5â µM, against Acinetobacter baumannii 5075. Preliminary investigation into the mechanism of action suggests that the compounds are binding to lipopolysaccharide (LPS) in K.â pneumoniae, and modulating lipooligosaccharide (LOS) biosynthesis, assembly, or transport in A.â baumannii.
Subject(s)
Acinetobacter baumannii , Anti-Bacterial Agents , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Gram-Negative Bacteria , Benzimidazoles/pharmacology , Macrolides , Microbial Sensitivity TestsABSTRACT
Maintaining a fluid bilayer is essential for cell signaling and survival. Lipid saturation is a key factor determining lipid packing and membrane fluidity, and it must be tightly controlled to guarantee organelle function and identity. A dedicated eukaryotic mechanism of lipid saturation sensing, however, remains elusive. Here we show that Mga2, a transcription factor conserved among fungi, acts as a lipid-packing sensor in the ER membrane to control the production of unsaturated fatty acids. Systematic mutagenesis, molecular dynamics simulations, and electron paramagnetic resonance spectroscopy identify a pivotal role of the oligomeric transmembrane helix (TMH) of Mga2 for intra-membrane sensing, and they show that the lipid environment controls the proteolytic activation of Mga2 by stabilizing alternative rotational orientations of the TMH region. This work establishes a eukaryotic strategy of lipid saturation sensing that differs significantly from the analogous bacterial mechanism relying on hydrophobic thickness.
Subject(s)
Endoplasmic Reticulum/metabolism , Fatty Acids/metabolism , Intracellular Membranes/metabolism , Membrane Fluidity , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Transcription Factors/metabolism , Electron Spin Resonance Spectroscopy , Fatty Acid Desaturases/genetics , Fatty Acid Desaturases/metabolism , Gene Expression Regulation, Fungal , Membrane Proteins/chemistry , Membrane Proteins/genetics , Molecular Dynamics Simulation , Mutation , Protein Conformation, alpha-Helical , Protein Stability , Proteolysis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Signal Transduction , Stearoyl-CoA Desaturase , Structure-Activity Relationship , Transcription Factors/chemistry , Transcription Factors/genetics , Transcriptional ActivationABSTRACT
BACKGROUND: Obesity and type 2 diabetes mellitus (T2DM) are associated with an increased risk of severe outcomes from infectious diseases, including coronavirus disease 2019. These conditions are also associated with distinct responses to immunization, including an impaired response to widely used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines. OBJECTIVE: We sought to establish a connection between reduced immunization efficacy via modeling the effects of metabolic diseases on vaccine immunogenicity that is essential for the development of more effective vaccines for this distinct vulnerable population. METHODS: A murine model of diet-induced obesity and insulin resistance was used to model the effects of comorbid T2DM and obesity on vaccine immunogenicity and protection. RESULTS: Mice fed a high-fat diet (HFD) developed obesity, hyperinsulinemia, and glucose intolerance. Relative to mice fed a normal diet, HFD mice vaccinated with a SARS-CoV-2 mRNA vaccine exhibited significantly lower anti-spike IgG titers, predominantly in the IgG2c subclass, associated with a lower type 1 response, along with a 3.83-fold decrease in neutralizing titers. Furthermore, enhanced vaccine-induced spike-specific CD8+ T-cell activation and protection from lung infection against SARS-CoV-2 challenge were seen only in mice fed a normal diet but not in HFD mice. CONCLUSIONS: The study demonstrated impaired immunity following SARS-CoV-2 mRNA immunization in a murine model of comorbid T2DM and obesity, supporting the need for further research into the basis for impaired anti-SARS-CoV-2 immunity in T2DM and investigation of novel approaches to enhance vaccine immunogenicity among those with metabolic diseases.
Subject(s)
COVID-19 , Diabetes Mellitus, Type 2 , Insulin Resistance , Viral Vaccines , Animals , Humans , Mice , COVID-19 Vaccines , SARS-CoV-2 , COVID-19/prevention & control , Disease Models, Animal , Immunogenicity, Vaccine , Diet , Obesity , RNA, Messenger , Antibodies, Viral , Antibodies, NeutralizingABSTRACT
Upon nutrient limitation, budding yeast of Saccharomyces cerevisiae shift from fast growth (the log stage) to quiescence (the stationary stage). This shift is accompanied by liquid-liquid phase separation in the membrane of the vacuole, an endosomal organelle. Recent work indicates that the resulting micrometer-scale domains in vacuole membranes enable yeast to survive periods of stress. An outstanding question is which molecular changes might cause this membrane phase separation. Here, we conduct lipidomics of vacuole membranes in both the log and stationary stages. Isolation of pure vacuole membranes is challenging in the stationary stage, when lipid droplets are in close contact with vacuoles. Immuno-isolation has previously been shown to successfully purify log-stage vacuole membranes with high organelle specificity, but it was not previously possible to immuno-isolate stationary-stage vacuole membranes. Here, we develop Mam3 as a bait protein for vacuole immuno-isolation, and demonstrate low contamination by non-vacuolar membranes. We find that stationary-stage vacuole membranes contain surprisingly high fractions of phosphatidylcholine lipids (â¼40%), roughly twice as much as log-stage membranes. Moreover, in the stationary stage, these lipids have higher melting temperatures, due to longer and more saturated acyl chains. Another surprise is that no significant change in sterol content is observed. These lipidomic changes, which are largely reflected on the whole-cell level, fit within the predominant view that phase separation in membranes requires at least three types of molecules to be present: lipids with high melting temperatures, lipids with low melting temperatures, and sterols.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolism , Lipidomics , Vacuoles/metabolism , Saccharomyces cerevisiae Proteins/metabolism , LipidsABSTRACT
Lipopolysaccharides (LPSs) are a hallmark virulence factor of Gram-negative bacteria. They are complex, structurally heterogeneous mixtures due to variations in number, type, and position of their simplest units: fatty acids and monosaccharides. Thus, LPS structural characterization by traditional mass spectrometry (MS) methods is challenging. Here, we describe the benefits of field asymmetric ion mobility spectrometry (FAIMS) for analysis of an intact R-type lipopolysaccharide complex mixture (lipooligosaccharide; LOS). Structural characterization was performed using Escherichia coli J5 (Rc mutant) LOS, a TLR4 agonist widely used in glycoconjugate vaccine research. FAIMS gas-phase fractionation improved the (S/N) ratio and number of detected LOS species. Additionally, FAIMS allowed the separation of overlapping isobars facilitating their tandem MS characterization and unequivocal structural assignments. In addition to FAIMS gas-phase fractionation benefits, extra sorting of the structurally related LOS molecules was further accomplished using Kendrick mass defect (KMD) plots. Notably, a custom KMD base unit of [Na-H] created a highly organized KMD plot that allowed identification of interesting and novel structural differences across the different LOS ion families, i.e., ions with different acylation degrees, oligosaccharides composition, and chemical modifications. Defining the composition of a single LOS ion by tandem MS along with the organized KMD plot structural network was sufficient to deduce the composition of 181 LOS species out of 321 species present in the mixture. The combination of FAIMS and KMD plots allowed in-depth characterization of the complex LOS mixture and uncovered a wealth of novel information about its structural variations.
Subject(s)
Ion Mobility Spectrometry , Lipopolysaccharides , Humans , Lipopolysaccharides/chemistry , Cefotaxime , Tandem Mass Spectrometry , Ions/chemistry , Escherichia coliABSTRACT
PURPOSE: The SF3B splicing complex is composed of SF3B1-6 and PHF5A. We report a developmental disorder caused by de novo variants in PHF5A. METHODS: Clinical, genomic, and functional studies using subject-derived fibroblasts and a heterologous cellular system were performed. RESULTS: We studied 9 subjects with congenital malformations, including preauricular tags and hypospadias, growth abnormalities, and developmental delay who had de novo heterozygous PHF5A variants, including 4 loss-of-function (LOF), 3 missense, 1 splice, and 1 start-loss variant. In subject-derived fibroblasts with PHF5A LOF variants, wild-type and variant PHF5A mRNAs had a 1:1 ratio, and PHF5A mRNA levels were normal. Transcriptome sequencing revealed alternative promoter use and downregulated genes involved in cell-cycle regulation. Subject and control fibroblasts had similar amounts of PHF5A with the predicted wild-type molecular weight and of SF3B1-3 and SF3B6. SF3B complex formation was unaffected in 2 subject cell lines. CONCLUSION: Our data suggest the existence of feedback mechanisms in fibroblasts with PHF5A LOF variants to maintain normal levels of SF3B components. These compensatory mechanisms in subject fibroblasts with PHF5A or SF3B4 LOF variants suggest disturbed autoregulation of mutated splicing factor genes in specific cell types, that is, neural crest cells, during embryonic development rather than haploinsufficiency as pathomechanism.
Subject(s)
Craniofacial Abnormalities , Hypospadias , Male , Humans , Hypospadias/genetics , RNA Splicing Factors/genetics , RNA Splicing , Transcription Factors/genetics , Transcription Factors/metabolism , Trans-Activators/genetics , RNA-Binding Proteins/geneticsABSTRACT
Lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria is critical for the assembly of their cell envelopes. LPS synthesized in the cytoplasmic leaflet of the inner membrane is flipped to the periplasmic leaflet by MsbA, an ATP-binding cassette transporter. Despite substantial efforts, the structural mechanisms underlying MsbA-driven LPS flipping remain elusive. Here we use single-particle cryo-electron microscopy to elucidate the structures of lipid-nanodisc-embedded MsbA in three functional states. The 4.2 Å-resolution structure of the transmembrane domains of nucleotide-free MsbA reveals that LPS binds deep inside MsbA at the height of the periplasmic leaflet, establishing extensive hydrophilic and hydrophobic interactions with MsbA. Two sub-nanometre-resolution structures of MsbA with ADP-vanadate and ADP reveal an unprecedented closed and an inward-facing conformation, respectively. Our study uncovers the structural basis for LPS recognition, delineates the conformational transitions of MsbA to flip LPS, and paves the way for structural characterization of other lipid flippases.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , ATP-Binding Cassette Transporters/ultrastructure , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Cryoelectron Microscopy , Escherichia coli , Lipopolysaccharides/metabolism , ATP-Binding Cassette Transporters/chemistry , Adenosine Diphosphate/chemistry , Adenosine Diphosphate/metabolism , Bacterial Proteins/chemistry , Biological Transport , Cell Membrane/chemistry , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Escherichia coli/cytology , Escherichia coli/enzymology , Escherichia coli/ultrastructure , Hydrophobic and Hydrophilic Interactions , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Models, Molecular , Nanostructures/chemistry , Nanostructures/ultrastructure , Periplasm/chemistry , Periplasm/metabolism , Periplasm/ultrastructure , Protein Binding , Protein DomainsABSTRACT
Immune evasion through membrane remodeling is a hallmark of Yersinia pestis pathogenesis. Yersinia remodels its membrane during its life cycle as it alternates between mammalian hosts (37 °C) and ambient (21 °C to 26 °C) temperatures of the arthropod transmission vector or external environment. This shift in growth temperature induces changes in number and length of acyl groups on the lipid A portion of lipopolysaccharide (LPS) for the enteric pathogens Yersinia pseudotuberculosis (Ypt) and Yersinia enterocolitica (Ye), as well as the causative agent of plague, Yersinia pestis (Yp). Addition of a C16 fatty acid (palmitate) to lipid A by the outer membrane acyltransferase enzyme PagP occurs in immunostimulatory Ypt and Ye strains, but not in immune-evasive Yp Analysis of Yp pagP gene sequences identified a single-nucleotide polymorphism that results in a premature stop in translation, yielding a truncated, nonfunctional enzyme. Upon repair of this polymorphism to the sequence present in Ypt and Ye, lipid A isolated from a Yp pagP+ strain synthesized two structures with the C16 fatty acids located in acyloxyacyl linkage at the 2' and 3' positions of the diglucosamine backbone. Structural modifications were confirmed by mass spectrometry and gas chromatography. With the genotypic restoration of PagP enzymatic activity in Yp, a significant increase in lipid A endotoxicity mediated through the MyD88 and TRIF/TRAM arms of the TLR4-signaling pathway was observed. Discovery and repair of an evolutionarily lost lipid A modifying enzyme provides evidence of lipid A as a crucial determinant in Yp infectivity, pathogenesis, and host innate immune evasion.