ABSTRACT
Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.
ABSTRACT
The composition of the gastrointestinal microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine, are elevated in high-fat diet-induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon-like peptide 1. Obese Fpr1 knockout mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota. Overall, we describe a new mechanism by which the gut microbiota can modulate glucose metabolism, providing a potential approach for the treatment of metabolic disease.
Subject(s)
Glucagon-Like Peptide 1/metabolism , Microbiota/physiology , Obesity/metabolism , Oligopeptides/metabolism , Animals , Cells, Cultured , Chemotaxis/drug effects , Chromatography, Liquid , Diet, High-Fat , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Glucose/pharmacology , Glucose Intolerance , In Situ Hybridization, Fluorescence , Insulin/metabolism , Male , Mass Spectrometry , Mice , Mice, Knockout , Mice, Obese , Obesity/chemically inducedABSTRACT
BACKGROUND: Recent studies suggest that the principal active ingredient in phosphatidylcholine-containing injectable fat-reduction formulations is actually deoxycholate (DC). This bile acid acts as a detergent to rapidly disrupt cell membranes. Thus, it is not obvious why DC would preferentially target fat. OBJECTIVE: To investigate possible mechanisms for the selectivity of DC for fat tissue using in vivo and in vitro models. METHODS AND MATERIALS: Histology, drug distribution studies, and cell viability assays were used to examine possible mechanisms contributing to DC selectivity. RESULTS: In vitro, DC caused the lysis of all cell types tested within the tested concentration range. DC injected into fat tissue caused adipocyte death, whereas other cell types appeared less affected. Physiological concentrations of albumin or protein-rich tissues decrease the ability of DC to lyse cells. Furthermore, DC relocated to the gastrointestinal tract in animals within hours of injection. This suggests that similar mechanisms may be present in humans. CONCLUSION: We report observations that provide a possible explanation for the in vivo preferential fat targeting by DC. Fat tissue, being deficient in cell-associated proteins and interstitial albumin, may be unable to sufficiently neutralize the detergent activity of DC, possibly making fat uniquely sensitive to DC.
Subject(s)
Cholagogues and Choleretics/pharmacology , Deoxycholic Acid/pharmacology , Muscle, Skeletal/drug effects , Skin/drug effects , Subcutaneous Fat/drug effects , Adipocytes/drug effects , Animals , Cell Culture Techniques , Cholagogues and Choleretics/administration & dosage , Deoxycholic Acid/administration & dosage , Fibroblasts/drug effects , Humans , Injections, Subcutaneous , Keratinocytes/drug effects , Mice , Mice, Obese , Models, Animal , Muscle Cells/drug effects , Muscle, Skeletal/metabolism , Muscle, Skeletal/pathology , Skin/metabolism , Skin/pathology , Subcutaneous Fat/metabolism , Subcutaneous Fat/pathologyABSTRACT
Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs through two mechanisms in vertebrate cells: (1) phosphorylation and removal of cohesin from chromosome arms by mitotic kinases, including Plk1, during prophase, and (2) cleavage of centromeric cohesin by separase at the metaphase-anaphase transition. Bub1 and the MEI-S332/Shugoshin (Sgo1) family of proteins protect centromeric cohesin from mitotic kinases during prophase. We show that human Sgo1 binds to protein phosphatase 2A (PP2A). PP2A localizes to centromeres in a Bub1-dependent manner. The Sgo1-PP2A interaction is required for centromeric localization of Sgo1 and proper chromosome segregation in human cells. Depletion of Plk1 by RNA interference (RNAi) restores centromeric localization of Sgo1 and prevents chromosome missegregation in cells depleted of PP2A_Aalpha. Our findings suggest that Bub1 targets PP2A to centromeres, which in turn maintains Sgo1 at centromeres by counteracting Plk1-mediated chromosome removal of Sgo1.
Subject(s)
Cell Cycle Proteins/metabolism , Centromere/metabolism , Chromosome Segregation , Phosphoprotein Phosphatases/metabolism , Gene Expression , HeLa Cells , Humans , Mitosis , Mutation/genetics , Protein Binding , Protein Kinases/metabolism , Protein Phosphatase 2 , Protein Serine-Threonine Kinases/deficiency , Protein Serine-Threonine Kinases/metabolism , Protein Structure, Tertiary , Protein Subunits , Protein Transport , Proto-Oncogene Proteins/deficiency , Proto-Oncogene Proteins/metabolism , RNA Interference , Tumor Cells, Cultured , Polo-Like Kinase 1ABSTRACT
Matching the codon usage of recombinant genes to that of the expression host is a common strategy for increasing the expression of heterologous proteins in bacteria. However, while developing a cytoplasmic expression system for Fusarium solani cutinase in Escherichia coli, we found that altering codons to those preferred by E. coli led to significantly lower expression compared to the wild-type fungal gene, despite the presence of several rare E. coli codons in the fungal sequence. On the other hand, expression in the E. coli periplasm using a bacterial PhoA leader sequence resulted in high levels of expression for both the E. coli optimized and wild-type constructs. Sequence swapping experiments as well as calculations of predicted mRNA secondary structure provided support for the hypothesis that differential cytoplasmic expression of the E. coli optimized versus wild-type cutinase genes is due to differences in 5(') mRNA secondary structures. In particular, our results indicate that increased stability of 5(') mRNA secondary structures in the E. coli optimized transcript prevents efficient translation initiation in the absence of the phoA leader sequence. These results underscore the idea that potential 5(') mRNA secondary structures should be considered along with codon usage when designing a synthetic gene for high level expression in E. coli.
