ABSTRACT
Condensins play a key role in higher order chromosome organization. In budding yeast Saccharomyces cerevisiae, a condensin complex consists of five subunits: two conserved structural maintenance of chromosome subunits, Smc2 and Smc4, a kleisin Brn1, and two HEAT repeat subunits, Ycg1, which possesses a DNA binding activity, and Ycs4, which can transiently associate with Smc4 and thereby disrupt its association with the Smc2 head. We characterized here DNA binding activity of the non-SMC subunits using an agnostic, model-independent approach. To this end, we mapped the DNA interface of the complex using sulfo-NHS biotin labeling. Besides the known site on Ycg1, we found a patch of lysines at the C-terminal domain of Ycs4 that were protected from biotinylation in the presence of DNA. Point mutations at the predicted protein-DNA interface reduced both Ycs4 binding to DNA and the DNA stimulated ATPase activity of the reconstituted condensin, whereas overproduction of the mutant Ycs4 was detrimental for yeast viability. Notably, the DNA binding site on Ycs4 partially overlapped with its interface with SMC4, revealing an intricate interplay between DNA binding, engagement of the Smc2-Smc4 heads, and ATP hydrolysis and suggesting a mechanism for ATP-modulated loading and translocation of condensins on DNA.
Subject(s)
Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/physiology , DNA-Binding Proteins/metabolism , Multiprotein Complexes/metabolism , Multiprotein Complexes/physiology , Adenosine Triphosphatases/genetics , Binding Sites/genetics , Biotinylation , Cell Communication , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone , DNA/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Multiprotein Complexes/genetics , Nuclear Proteins , Phagocytosis , Point Mutation/genetics , Protein Domains/physiology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Ghrelin is a peptide hormone secreted primarily by the stomach that acts upon the growth hormone secretagogue receptor (GHSR1), a G protein-coupled receptor whose functions include growth hormone secretion, appetite regulation, energy expenditure, regulation of adiposity, and insulin release. Following the discovery that GHSR1a stimulates food intake, receptor antagonists were developed as potential therapies to regulate appetite. However, despite reductions in signalling, the desired effects on appetite were absent. Studies in the past 15 years have demonstrated GHSR1a can interact with other transmembrane proteins, either by direct binding (i.e. heteromerisation) or via signalling cross-talk. These interactions have various effects on GHSR1a signalling including preferential coupling to one pathway (i.e. biased signalling), coupling to a unique G protein (G protein switching), suppression of GHSR1a signalling, and enhancement of signalling by both receptors. While many of these interactions have been shown in cells overexpressing the proteins of interest and remain to be verified in tissues, substantial evidence exists showing that GHSR1a and the dopamine receptor D1 (DRD1) form heteromers, which promote synaptic plasticity and formation of hippocampal memory. Additionally, a reduction in GHSR1a-DRD1 complexes in favour of establishment of GHSR1a-Aß complexes correlates with Alzheimer's disease, indicating that GHSR1a heteromers may have pathological functions. Herein, we summarise the evidence published to date describing interactions between GHSR1a and transmembrane proteins, discuss the experimental strengths and limitations of these studies, describe the physiological evidence for each interaction, and address their potential as novel drug targets for appetite regulation, Alzheimer's disease, insulin secretion, and inflammation.
Subject(s)
Multiprotein Complexes/physiology , Protein Multimerization/physiology , Receptors, Ghrelin/physiology , Animals , Ghrelin/metabolism , Ghrelin/physiology , Humans , Multiprotein Complexes/metabolism , Protein Binding/physiology , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D1/physiology , Receptors, Ghrelin/metabolism , Signal Transduction/physiologyABSTRACT
Many proteins have been found to operate in a complex with various biomolecules such as proteins, nucleic acids, carbohydrates, or lipids. Protein complexes can be transient, stable or dynamic and their association is controlled under variable cellular conditions. Complexome profiling is a recently developed mass spectrometry-based method that combines mild separation techniques, native gel electrophoresis, and density gradient centrifugation with quantitative mass spectrometry to generate inventories of protein assemblies within a cell or subcellular fraction. This review summarizes applications of complexome profiling with respect to assembly ranging from single subunits to large macromolecular complexes, as well as their stability, and remodeling in health and disease.
Subject(s)
Multiprotein Complexes/chemistry , Multiprotein Complexes/physiology , Proteins/chemistry , Proteins/physiology , Animals , HumansABSTRACT
The mammalian SWI/SNF complex, or BAF complex, has a conserved and direct role in antagonizing Polycomb-mediated repression. Yet, BAF also promotes repression by Polycomb in stem cells and cancer. How BAF both antagonizes and promotes Polycomb-mediated repression remains unknown. Here, we utilize targeted protein degradation to dissect the BAF-Polycomb axis in mouse embryonic stem cells on short timescales. We report that rapid BAF depletion redistributes Polycomb repressive complexes PRC1 and PRC2 from highly occupied domains, like Hox clusters, to weakly occupied sites normally opposed by BAF. Polycomb redistribution from highly repressed domains results in their decompaction, gain of active epigenomic features and transcriptional derepression. Surprisingly, through dose-dependent degradation of PRC1 and PRC2, we identify a conventional role for BAF in Polycomb-mediated repression, in addition to global Polycomb redistribution. These findings provide new mechanistic insight into the highly dynamic state of the Polycomb-Trithorax axis.
Subject(s)
Chromatin Assembly and Disassembly/physiology , Epigenetic Repression/physiology , Gene Expression Regulation/physiology , Multiprotein Complexes/physiology , Polycomb-Group Proteins/physiology , Animals , CRISPR-Cas Systems , Cells, Cultured , Chromatin Assembly and Disassembly/genetics , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/physiology , Embryonic Stem Cells/metabolism , Epigenesis, Genetic , Epigenetic Repression/genetics , Gene Editing , Gene Expression Regulation/genetics , Genes, Homeobox , Genome , HEK293 Cells , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Loss of Function Mutation , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proteolysis , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcription Factors/physiologyABSTRACT
Since its recent discovery, the subcortical maternal complex (SCMC) is emerging as a maternally inherited and crucial biological structure for the initial stages of embryogenesis in mammals. Uniquely expressed in oocytes and preimplantation embryos, where it localizes to the cell subcortex, this multiprotein complex is essential for early embryo development in the mouse and is functionally conserved across mammalian species, including humans. The complex has been linked to key processes leading the transition from oocyte to embryo, including meiotic spindle formation and positioning, regulation of translation, organelle redistribution, and epigenetic reprogramming. Yet, the underlying molecular mechanisms for these diverse functions are just beginning to be understood, hindered by unresolved interplay of SCMC components and variations in early lethal phenotypes. Here we review recent advances confirming involvement of the SCMC in human infertility, revealing an unexpected relationship with offspring health. Moreover, SCMC organization is being further revealed in terms of novel components and interactions with additional cell constituents. Collectively, this evidence prompts new avenues of investigation into possible roles during the process of oogenesis and the regulation of maternal transcript turnover during the oocyte to embryo transition.
Subject(s)
Blastocyst/ultrastructure , Embryonic Development , Multiprotein Complexes/physiology , Oocytes/ultrastructure , Aneuploidy , Animals , Blastocyst/metabolism , Congenital Abnormalities , Egg Proteins/physiology , Genomic Imprinting , Humans , Infertility/genetics , Mice , Multiprotein Complexes/ultrastructure , Mutation , Oocytes/metabolism , RNA Stability , RNA, Messenger/metabolismABSTRACT
Nuclear organisation shapes gene regulation; however, the principles by which three-dimensional genome architecture influences gene transcription are incompletely understood. Condensin is a key architectural chromatin constituent, best known for its role in mitotic chromosome condensation. Yet at least a subset of condensin is bound to DNA throughout the cell cycle. Studies in various organisms have reported roles for condensin in transcriptional regulation, but no unifying mechanism has emerged. Here, we use rapid conditional condensin depletion in the budding yeast Saccharomyces cerevisiae to study its role in transcriptional regulation. We observe a large number of small gene expression changes, enriched at genes located close to condensin-binding sites, consistent with a possible local effect of condensin on gene expression. Furthermore, nascent RNA sequencing reveals that transcriptional down-regulation in response to environmental stimuli, in particular to heat shock, is subdued without condensin. Our results underscore the multitude by which an architectural chromosome constituent can affect gene regulation and suggest that condensin facilitates transcriptional reprogramming as part of adaptation to environmental changes.
Subject(s)
Adenosine Triphosphatases/metabolism , DNA-Binding Proteins/metabolism , Gene Expression Regulation/physiology , Gene Expression/physiology , Multiprotein Complexes/metabolism , Adenosine Triphosphatases/physiology , Chromatin/metabolism , Chromosome Segregation/physiology , Chromosomes/metabolism , DNA/physiology , DNA-Binding Proteins/physiology , Gene Expression/genetics , Gene Expression Regulation/genetics , Mitosis/physiology , Multiprotein Complexes/physiology , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.
Subject(s)
Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/physiology , Biological Evolution , Chromosomes/ultrastructure , DNA-Binding Proteins/genetics , DNA-Binding Proteins/physiology , Eukaryota/genetics , Genome , Multiprotein Complexes/genetics , Multiprotein Complexes/physiology , Adenosine Triphosphatases/chemistry , Algorithms , Animals , Cell Nucleolus/ultrastructure , Cell Nucleus/ultrastructure , Centromere/ultrastructure , Chromosomes/chemistry , Chromosomes, Human/chemistry , Chromosomes, Human/ultrastructure , DNA-Binding Proteins/chemistry , Genome, Human , Genomics , Heterochromatin/ultrastructure , Humans , Interphase , Mitosis , Models, Biological , Multiprotein Complexes/chemistry , Telomere/ultrastructureABSTRACT
TMEM132D is a human gene identified with multiple risk alleles for panic disorders, anxiety and major depressive disorders. Defining a conserved family of transmembrane proteins, TMEM132D and its homologs are still of unknown molecular functions. By generating loss-of-function mutants of the sole TMEM132 ortholog in C. elegans, we identify abnormal morphologic phenotypes in the dopaminergic PDE neurons. Using a yeast two-hybrid screen, we find that NAP1 directly interacts with the cytoplasmic domain of human TMEM132D, and mutations in C. elegans tmem-132 that disrupt interaction with NAP1 cause similar morphologic defects in the PDE neurons. NAP1 is a component of the WAVE regulatory complex (WRC) that controls F-actin cytoskeletal dynamics. Decreasing activity of WRC rescues the PDE defects in tmem-132 mutants, whereas gain-of-function of TMEM132D in mammalian cells inhibits WRC, leading to decreased abundance of select WRC components, impaired actin nucleation and cell motility. We propose that metazoan TMEM132 family proteins play evolutionarily conserved roles in regulating NAP1 protein homologs to restrict inappropriate WRC activity, cytoskeletal and morphologic changes in the cell.
Subject(s)
Caenorhabditis elegans Proteins/physiology , Cytoskeleton/ultrastructure , Dopaminergic Neurons/ultrastructure , Membrane Proteins/metabolism , Morphogenesis/genetics , Neurogenesis/genetics , Sensory Receptor Cells/ultrastructure , Actins/metabolism , Animals , Biological Evolution , Caenorhabditis elegans/cytology , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Cell Shape , Conserved Sequence , Dopaminergic Neurons/metabolism , Gain of Function Mutation , Genes, Reporter , HEK293 Cells , Humans , Loss of Function Mutation , Multigene Family , Multiprotein Complexes/physiology , Panic Disorder/genetics , Protein Domains , Protein Interaction Mapping , Recombinant Fusion Proteins/metabolism , Sensory Receptor Cells/metabolism , Two-Hybrid System TechniquesABSTRACT
Biomolecular condensates are membraneless intracellular assemblies that often form via liquid-liquid phase separation and have the ability to concentrate biopolymers. Research over the past 10 years has revealed that condensates play fundamental roles in cellular organization and physiology, and our understanding of the molecular principles, components and forces underlying their formation has substantially increased. Condensate assembly is tightly regulated in the intracellular environment, and failure to control condensate properties, formation and dissolution can lead to protein misfolding and aggregation, which are often the cause of ageing-associated diseases. In this Review, we describe the mechanisms and regulation of condensate assembly and dissolution, highlight recent advances in understanding the role of biomolecular condensates in ageing and disease, and discuss how cellular stress, ageing-related loss of homeostasis and a decline in protein quality control may contribute to the formation of aberrant, disease-causing condensates. Our improved understanding of condensate pathology provides a promising path for the treatment of protein aggregation diseases.
Subject(s)
Aging , Macromolecular Substances/chemistry , Multiprotein Complexes/physiology , Protein Aggregation, Pathological/etiology , Stress, Physiological/physiology , Aging/metabolism , Aging/pathology , Animals , Cell Physiological Phenomena , Humans , Macromolecular Substances/metabolism , Protein Aggregates/physiology , Protein Aggregation, Pathological/metabolismABSTRACT
Biomolecular condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liquid-liquid phase separation (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomolecular condensation and highlight considerations for designing condensate reconstitution experiments. We focus on RNA properties such as composition, length, structure, modifications and expression level. These properties can modulate the biophysical features of native condensates, including their size, shape, viscosity, liquidity, surface tension and composition. We also discuss the role of RNA-protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be determined by RNA. Finally, we discuss the multifaceted cellular functions of biomolecular condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific molecules, and buffering noise and responding to stress.
Subject(s)
Macromolecular Substances/chemistry , Multiprotein Complexes/chemistry , Multiprotein Complexes/physiology , RNA/physiology , Animals , Cell Physiological Phenomena , Chemical Phenomena , Humans , Macromolecular Substances/metabolism , Multiprotein Complexes/metabolism , Protein Aggregates/physiology , RNA-Binding Proteins/metabolism , RNA-Binding Proteins/physiologyABSTRACT
Proteostasis needs to be tightly controlled to meet the cellular demand for correctly de novo folded proteins and to avoid protein aggregation. While a coupling between translation rate and co-translational folding, likely involving an interplay between the ribosome and its associated chaperones, clearly appears to exist, the underlying mechanisms and the contribution of ribosomal proteins remain to be explored. The ribosomal protein uL3 contains a long internal loop whose tip region is in close proximity to the ribosomal peptidyl transferase center. Intriguingly, the rpl3[W255C] allele, in which the residue making the closest contact to this catalytic site is mutated, affects diverse aspects of ribosome biogenesis and function. Here, we have uncovered, by performing a synthetic lethal screen with this allele, an unexpected link between translation and the folding of nascent proteins by the ribosome-associated Ssb-RAC chaperone system. Our results reveal that uL3 and Ssb-RAC cooperate to prevent 80S ribosomes from piling up within the 5' region of mRNAs early on during translation elongation. Together, our study provides compelling in vivo evidence for a functional connection between peptide bond formation at the peptidyl transferase center and chaperone-assisted de novo folding of nascent polypeptides at the solvent-side of the peptide exit tunnel.
Subject(s)
Molecular Chaperones/physiology , Multiprotein Complexes/physiology , Peptide Chain Elongation, Translational/physiology , Protein Folding , Proteostasis/physiology , Ribosomes/metabolism , Saccharomyces cerevisiae Proteins/physiology , Saccharomyces cerevisiae/metabolism , Alleles , Loss of Function Mutation , Molecular Chaperones/genetics , Mutation, Missense , Peptidyl Transferases/physiology , Point Mutation , Recombinant Proteins/metabolism , Ribosomal Proteins/genetics , Ribosomal Proteins/physiology , Ribosomes/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
Biomolecular condensates are found throughout eukaryotic cells, including in the nucleus, in the cytoplasm and on membranes. They are also implicated in a wide range of cellular functions, organizing molecules that act in processes ranging from RNA metabolism to signalling to gene regulation. Early work in the field focused on identifying condensates and understanding how their physical properties and regulation arise from molecular constituents. Recent years have brought a focus on understanding condensate functions. Studies have revealed functions that span different length scales: from molecular (modulating the rates of chemical reactions) to mesoscale (organizing large structures within cells) to cellular (facilitating localization of cellular materials and homeostatic responses). In this Roadmap, we discuss representative examples of biochemical and cellular functions of biomolecular condensates from the recent literature and organize these functions into a series of non-exclusive classes across the different length scales. We conclude with a discussion of areas of current interest and challenges in the field, and thoughts about how progress may be made to further our understanding of the widespread roles of condensates in cell biology.
Subject(s)
Macromolecular Substances , Multiprotein Complexes/physiology , Animals , Biochemical Phenomena , Cell Physiological Phenomena , Cytoplasm/chemistry , Cytoplasm/genetics , Cytoplasm/metabolism , Eukaryotic Cells/chemistry , Eukaryotic Cells/metabolism , Eukaryotic Cells/physiology , Humans , Macromolecular Substances/chemistry , Macromolecular Substances/metabolism , Multiprotein Complexes/chemistry , Organelles/chemistry , Organelles/genetics , Organelles/metabolism , Protein Aggregates/physiologyABSTRACT
CONTEXT: Low-grade chronic inflammation is commonly seen in polycystic ovary syndrome (PCOS) patients with elevated levels of inflammatory cytokines in the endometrium. OBJECTIVE: This work aimed to increase the limited understanding of the mechanisms underlying cytokine synthesis and increased endometrial inflammation in PCOS patients. METHODS: Endometrial biopsy samples were collected from non-PCOS (nâ =â 17) and PCOS (nâ =â 22) patients either during the proliferative phase of the menstrual cycle or with hyperplasia. Endometrial explants were prepared from PCOS patients and underwent pharmacological manipulation in vitro. The expression and localization of toll-like receptor 2 (TLR2)/4, key elements of innate immune signal transduction and nuclear factor κB (NFκB) signaling pathways, and multiple cytokines were comprehensively evaluated by Western blotting, immunohistochemistry, and immunofluorescence in endometrial tissues. RESULTS: We demonstrated the distribution of protein expression and localization associated with the significantly increased androgen receptor, TLR2, and TLR4-mediated activation of interferon regulatory factor-7 (IRF-7) and NFκB signaling, cytokine production, and endometrial inflammation in PCOS patients compared to non-PCOS patients with and without endometrial hyperplasia. In vitro experiments showed that 5-dihydrotestosterone (DHT) enhanced androgen receptor, TLR4, IRF-7, and p-NFκB p65 protein expression along with increased interferon α (IFNα) and IFNÉ£ abundance. The effects of DHT on IRF-7, p-NFκB p65, and IFN abundance were abolished by flutamide, an antiandrogen. Although 17ß-estradiol (E2) decreased p-IRF-7 expression with little effect on TLR-mediated IRF7 and NFκB signaling or on cytokine protein levels, exposure to metformin alone or in combination with E2 suppressed interleukin-1 receptor-associated kinase 4 (IRAK4), p-IRF-7, IRF-7, IκB kinase α (IKKα), p-NFκB p65, IFNÉ£, and tumor necrosis factor α protein expression. CONCLUSION: Cytokine synthesis and increased endometrial inflammation in PCOS patients are coupled to androgen-induced TLR4/IRF-7/NFκB signaling, which is inhibited by metformin treatment.
Subject(s)
Androgens/pharmacology , Endometrium/drug effects , Metformin/pharmacology , Polycystic Ovary Syndrome , Adult , Cytokines/biosynthesis , Endometrial Hyperplasia/metabolism , Endometrial Hyperplasia/pathology , Endometrium/metabolism , Endometrium/pathology , Female , Humans , Interferon Regulatory Factor-7/metabolism , Menstrual Cycle/drug effects , Menstrual Cycle/metabolism , Multiprotein Complexes/metabolism , Multiprotein Complexes/physiology , NF-kappa B/metabolism , Polycystic Ovary Syndrome/metabolism , Polycystic Ovary Syndrome/pathology , Protein Binding , Signal Transduction/drug effects , Signal Transduction/physiology , Toll-Like Receptor 4/metabolismABSTRACT
Several adaptor molecules bind to cytoplasmic tails of ß-integrins and facilitate bidirectional signaling, which is critical in thrombosis and hemostasis. Interfering with integrin-adaptor interactions spatially or temporally to inhibit thrombosis without affecting hemostasis is an attractive strategy for the development of safe antithrombotic drugs. We show for the first time that the 14-3-3ζ-c-Src-integrin-ß3 complex is formed during platelet activation. 14-3-3ζ-c-Src interaction is mediated by the -PIRLGLALNFSVFYYE- fragment (PE16) on the 14-3-3ζ and SH2-domain on c-Src, whereas the 14-3-3ζ-integrin-ß3 interaction is mediated by the -ESKVFYLKMKGDYYRYL- fragment (EL17) on the 14-3-3ζ and -KEATSTF- fragment (KF7) on the ß3-integrin cytoplasmic tail. The EL17-motif inhibitor, or KF7 peptide, interferes with the formation of the 14-3-3ζ-c-Src-integrin-ß3 complex and selectively inhibits ß3 outside-in signaling without affecting the integrin-fibrinogen interaction, which suppresses thrombosis without causing significant bleeding. This study characterized a previously unidentified 14-3-3ζ-c-Src-integrin-ß3 complex in platelets and provided a novel strategy for the development of safe and effective antithrombotic treatments.
Subject(s)
14-3-3 Proteins/metabolism , Integrin beta3/metabolism , Platelet Activation , Proto-Oncogene Proteins pp60(c-src)/metabolism , 14-3-3 Proteins/genetics , Adult , Animals , Female , HEK293 Cells , Humans , Integrin beta3/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Multiprotein Complexes/metabolism , Multiprotein Complexes/physiology , Platelet Activation/genetics , Proto-Oncogene Proteins pp60(c-src)/genetics , Signal Transduction/physiologyABSTRACT
Vesicle fusion is mediated by assembly of SNARE proteins between opposing membranes. While previous work suggested an active role of SNARE transmembrane domains (TMDs) in promoting membrane merger (Dhara et al., 2016), the underlying mechanism remained elusive. Here, we show that naturally-occurring v-SNARE TMD variants differentially regulate fusion pore dynamics in mouse chromaffin cells, indicating TMD flexibility as a mechanistic determinant that facilitates transmitter release from differentially-sized vesicles. Membrane curvature-promoting phospholipids like lysophosphatidylcholine or oleic acid profoundly alter pore expansion and fully rescue the decelerated fusion kinetics of TMD-rigidifying VAMP2 mutants. Thus, v-SNARE TMDs and phospholipids cooperate in supporting membrane curvature at the fusion pore neck. Oppositely, slowing of pore kinetics by the SNARE-regulator complexin-2 withstands the curvature-driven speeding of fusion, indicating that pore evolution is tightly coupled to progressive SNARE complex formation. Collectively, TMD-mediated support of membrane curvature and SNARE force-generated membrane bending promote fusion pore formation and expansion.
Subject(s)
Exocytosis , Membrane Fusion , Multiprotein Complexes/physiology , Neurotransmitter Agents/physiology , Phospholipids/metabolism , SNARE Proteins/physiology , Vesicle-Associated Membrane Protein 2/physiology , Animals , Calcium/physiology , Cell Membrane/metabolism , Cells, Cultured , Chromaffin Cells , Kinetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutant Proteins/physiology , Protein Binding , Protein Domains , Secretory Vesicles/physiologyABSTRACT
Photoreceptors possess ribbon synapses distinct from the conventional synapses in the brain. Little is known about the function of the BBSome, a complex integral in ciliary and intracellular trafficking, in ribbon synaptic formation. We performed immunohistochemistry using retinas from Bardet-Biedl Syndrome (BBS) mouse models and found that BBS mutant animals have significantly fewer ribbon synapses in the outer plexiform layer and increased ectopic synapses in the outer nuclear layer compared to controls. Many ectopic synapses in BBS mutant retinas are associated with horizontal cell axonal processes that aberrantly intrude into the outer nuclear layer. To determine whether this horizontal cell phenotype is a consequence of retinal degeneration, we examined this phenotype in mice with photoreceptor-specific inactivation of the BBSome induced by Cre recombinase driven by the rhodopsin promoter. At three months of age, despite retinal degeneration, Bbs8floxed/floxed; Rho-Cre+ mice lack the aberrant intrusion of horizontal cell processes. At 6 months, some horizontal cell processes intrude into the outer nuclear layer in Bbs8floxed/floxed; Rho-Cre+ mice, but the phenotype does not recapitulate the phenotypic severity observed in young congenital BBS mutant mice. Therefore, the lack of BBSome function negatively impacts retinal synaptogenesis, and causes horizontal cell defects in a potentially cell-autonomous fashion.
Subject(s)
Multiprotein Complexes/physiology , Photoreceptor Cells/physiology , Retina/pathology , Synapses/pathology , Animals , Bardet-Biedl Syndrome , Cilia/physiology , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/physiology , Female , Male , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Neurogenesis , Photoreceptor Cells/pathology , Proteins/genetics , Proteins/physiology , Retinal Degeneration/pathologyABSTRACT
Human cytomegalovirus (HCMV) is an important ubiquitous opportunistic pathogen that belongs to the betaherpesviridae. Primary HCMV infection is generally asymptomatic in immunocompetent individuals. In contrast, HCMV infection causes serious disease in immunocompromised patients and is the leading cause of congenital viral infection. Although they are effective, the use of conventional molecules is limited by the emergence of resistance and by their toxicity. New antivirals targeting other replication steps and inducing fewer adverse effects are therefore needed. During HCMV replication, DNA packaging is performed by the terminase complex, which cleaves DNA to package the virus genome into the capsid. With no counterpart in mammalian cells, these terminase proteins are ideal targets for highly specific antivirals. A new terminase inhibitor, letermovir, recently proved effective against HCMV in phase III clinical trials. However, its mechanism of action is unclear and it has no significant activity against other herpesvirus or non-human CMV.
TITLE: Le complexe terminase, une cible de choix dans le traitement de l'infection à cytomégalovirus humain. ABSTRACT: Le cytomégalovirus humain (CMVH) est un pathogène opportuniste majeur en cas d'immunodépression et représente la principale cause d'infection congénitale d'origine virale. Bien qu'efficace, l'utilisation des molécules conventionnelles est limitée par leur toxicité et par l'émergence de résistance du virus, rendant nécessaire le développement de nouveaux traitements. Lors de la réplication du CMVH, l'encapsidation de l'ADN est réalisée par le complexe terminase qui clive l'ADN pour empaqueter le génome dans la capside. L'absence d'homologues dans les cellules des mammifères rend les protéines du complexe terminase des cibles idéales pour des antiviraux spécifiques. Une nouvelle molécule, le letermovir, cible une étape exclusivement virale en interagissant avec le complexe terminase. Son efficacité a été prouvée lors d'essais cliniques de phase III. Néanmoins, son mécanisme d'action n'est, à ce jour, pas élucidé et aucune activité n'est observée contre les autres herpèsvirus.
Subject(s)
Antiviral Agents/therapeutic use , Cytomegalovirus Infections/drug therapy , Endodeoxyribonucleases/antagonists & inhibitors , Molecular Targeted Therapy/methods , Cytomegalovirus/pathogenicity , Cytomegalovirus/physiology , Cytomegalovirus Infections/immunology , Endodeoxyribonucleases/physiology , Humans , Immunocompromised Host , Molecular Targeted Therapy/trends , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/physiology , Virus Assembly/drug effects , Virus Assembly/physiology , Virus Replication/drug effectsABSTRACT
The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas.
Subject(s)
Apoptosis/genetics , B-Lymphocytes/enzymology , Cell Transformation, Neoplastic/genetics , Lymphoma, Large B-Cell, Diffuse/etiology , Multiprotein Complexes/physiology , Ubiquitin-Protein Ligases/genetics , Animals , B-Lymphocytes/pathology , Carrier Proteins/physiology , DNA Damage , Gene Expression Regulation, Neoplastic , Heterografts , Humans , Intracellular Signaling Peptides and Proteins/physiology , Lymphoma, Large B-Cell, Diffuse/enzymology , Lymphoma, Large B-Cell, Diffuse/genetics , Mice , Mice, Transgenic , Mutation, Missense , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/physiology , NF-kappa B/metabolism , Neoplasm Transplantation , Polymorphism, Single Nucleotide , Polyubiquitin/biosynthesis , Protein Processing, Post-Translational , Transcription Factors/physiology , Transcriptome , Ubiquitin-Protein Ligases/analysis , Ubiquitin-Protein Ligases/physiology , Ubiquitination , Ubiquitins/physiologyABSTRACT
BACKGROUND: Studies have linked mutations in genes encoding the eight-protein exocyst protein complex to kidney disease, but the underlying mechanism is unclear. Because Drosophila nephrocytes share molecular and structural features with mammalian podocytes, they provide an efficient model for studying this issue. METHODS: We silenced genes encoding exocyst complex proteins specifically in Drosophila nephrocytes and studied the effects on protein reabsorption by lacuna channels and filtration by the slit diaphragm. We performed nephrocyte functional assays, carried out super-resolution confocal microscopy of slit diaphragm proteins, and used transmission electron microscopy to analyze ultrastructural changes. We also examined the colocalization of slit diaphragm proteins with exocyst protein Sec15 and with endocytosis and recycling regulators Rab5, Rab7, and Rab11. RESULTS: Silencing exocyst genes in nephrocytes led to profound changes in structure and function. Abolition of cellular accumulation of hemolymph proteins with dramatically reduced lacuna channel membrane invaginations offered a strong indication of reabsorption defects. Moreover, the slit diaphragm's highly organized surface structure-essential for filtration-was disrupted, and key proteins were mislocalized. Ultrastructural analysis revealed that exocyst gene silencing led to the striking appearance of novel electron-dense structures that we named "exocyst rods," which likely represent accumulated membrane proteins following defective exocytosis or recycling. The slit diaphragm proteins partially colocalized with Sec15, Rab5, and Rab11. CONCLUSIONS: Our findings suggest that the slit diaphragm of Drosophila nephrocytes requires balanced endocytosis and recycling to maintain its structural integrity and that impairment of the exocyst complex leads to disruption of the slit diaphragm and nephrocyte malfunction. This model may help identify therapeutic targets for treating kidney diseases featuring molecular defects in vesicle endocytosis, exocytosis, and recycling.