RESUMEN
α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.
Asunto(s)
Enfermedad de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Vesículas Sinápticas/metabolismo , Lisofosfatidilcolinas/metabolismo , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Fosfolípidos/metabolismoRESUMEN
Muscle cell fusion is a multistep process where the final step of the reaction drives progression beyond early hemifusion events to complete fusion. This step requires activity of the muscle-specific fusogen Myomerger, a single-pass transmembrane protein containing 84 amino acids with an ectodomain that includes two α-helices. Previous studies have demonstrated that Myomerger acts by destabilizing membranes through generation of elastic stresses in the outer leaflet of the plasma membrane. An obvious question is how such destabilizing activity might be regulated to avoid membrane and cellular damage, and how the two juxtaposed helices cooperate in fusion. Using cellular fusion assays and in vitro liposome assays, we report that the two helices possess unique characteristics, both of which are needed for full activity of the protein. We demonstrate that externalized phosphatidylserine (PS), a lipid previously implicated in myoblast fusion, has a determinant role in the regulation of Myomerger activity. The membrane-proximal, amphipathic Helix-1 is normally disordered and its α-helical structure is induced by PS, making membrane interactions more efficacious. The distal, more hydrophobic Helix-2 is intrinsically ordered, possesses an ability to insert into membranes, and augments the membrane-stressing effects of Helix-1. These data reveal that Myomerger fusogenic activity is an exquisitely orchestrated event involving its two ectodomain helices, which are controlled by membrane lipid composition, providing an explanation as to how its membrane-stressing activity is spatially and temporally regulated during the final step of myoblast fusion.
Asunto(s)
Fusión Celular , Proteínas de la Membrana , Mioblastos , Fosfatidilserinas , Animales , Línea Celular , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mioblastos/fisiologíaRESUMEN
Structured illumination microscopy (SIM) is a super-resolution technology for imaging living cells and has been used for studying the dynamics of lysosomes and mitochondria. Recently, new probes and analyzing methods have been developed for SIM imaging, enabling the quantitative analysis of these subcellular structures and their interactions. This review provides an overview of the working principle and advances of SIM, as well as the organelle-targeting principles and types of fluorescence probes, including small molecules, metal complexes, nanoparticles, and fluorescent proteins. Additionally, quantitative methods based on organelle morphology and distribution are outlined. Finally, the review provides an outlook on the current challenges and future directions for improving the combination of SIM imaging and image analysis to further advance the study of organelles. We hope that this review will be useful for researchers working in the field of organelle research and help to facilitate the development of SIM imaging and analysis techniques.
RESUMEN
α-Synuclein (α-Syn) is an intrinsically disordered protein whose aggregation is associated with Parkinson's disease, dementia, and other neurodegenerative diseases known as synucleinopathies. However, the functional role of α-Syn is still unclear, although it has been shown to be involved in the regulation of neurotransmitter release via the interaction with synaptic vesicles (SVs), vesicle clustering, and SNARE complex assembly. Fatty acids have significant occupancy in synaptic vesicles; and recent studies suggest the interaction of fatty acids with α-Syn affect the formation of (pathological) aggregates, but it is less clear how fatty acids affects the functional role of α-Syn including α-Syn-membrane interactions, in particular with (SV-like) vesicles. Here, we report the concentration dependent effect of docosahexaenoic acid (DHA) in synaptic-like vesicle clustering via α-Syn interaction. Through molecular dynamics simulation, we revealed that DHA promoted vesicle clustering is due to the electrostatic interaction between DHA in the membrane and the N-terminal region of α-Syn. Moreover, this increased electrostatic interaction arises from a change in the macroscopic properties of the protein-membrane interface induced by (preferential solvation of) DHA. Our results provide insight as to how DHA regulates vesicle clustering mediated by α-Syn and may further be useful to understand its physiological as well as pathological role. Description: In physiological environments, α-Synuclein (α-Syn) localizes at nerve termini and synaptic vesicles and interacts with anionic phospholipid membranes to promote vesicle clustering. Docosahexaenoic acid (DHA) increases binding affinity between α-Syn and lipid membranes by increasing electrostatic interaction energy through modulating the local and global membrane environment and conformational properties of α-Syn.
Asunto(s)
Ácidos Docosahexaenoicos , alfa-Sinucleína , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Ácidos Docosahexaenoicos/metabolismo , Electricidad Estática , Fosfolípidos/química , Vesículas Sinápticas/metabolismoRESUMEN
The protein tyrosine phosphatase SHP2 mediates multiple signal transductions in various cellular pathways, controlled by a variety of upstream inputs. SHP2 dysregulation is causative of different types of cancers and developmental disorders, making it a promising drug target. However, how SHP2 is modulated by its different regulators remains largely unknown. Here, we use single-molecule fluorescence resonance energy transfer and molecular dynamics simulations to investigate this question. We identify a partially open, semiactive conformation of SHP2 that is intermediate between the known open and closed states. We further demonstrate a "multiple gear" regulatory mechanism, in which different activators (e.g., insulin receptor substrate-1 and CagA), oncogenic mutations (e.g., E76A), and allosteric inhibitors (e.g., SHP099) can shift the equilibrium of the three conformational states and regulate SHP2 activity to different levels. Our work reveals the essential role of the intermediate state in fine-tuning the activity of SHP2, which may provide new opportunities for drug development for relevant cancers.
Asunto(s)
Calgranulina A/metabolismo , Proteínas Sustrato del Receptor de Insulina/metabolismo , Piperidinas/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/química , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Pirimidinas/metabolismo , Regulación Alostérica , Humanos , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Conformación Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genéticaRESUMEN
Critical challenges remain in trauma emergency and surgical procedures involving liver bleeding, particularly in perforating wounds that cannot be pressed and large wounds that cannot be sewn. Self-assembling peptide hydrogels are particularly attractive due to their intrinsic biocompatibility and programmability. Herein, we develop a nano-band-aid (NBA) through a three-stage self-assembly strategy of two functionalized peptides, which were first coassembled into nanofibers and then woven to a meshlike network driven by Ca2+. Then, catalyzed by blood coagulation factor XIIIa (FXIIIa), NBA underwent a third stage, self-assembly into a densely compacted physical barrier to stop and control the bleeding. As expected, NBA rapidly and efficiently stopped the bleeding in rat liver scratches while effectively reducing the inflammation around the wound and promoting the wound healing. This bionic self-assembly strategy will provide a clinically potential peptide-based treatment for fatal liver bleeding and reinvigorate efforts to develop self-assembling peptide hydrogels as hemostatic agents.
Asunto(s)
Biónica , Hemostasis , Animales , Hidrogeles , Hígado , Péptidos , RatasRESUMEN
In nature, cells rely on a structural framework called the "cytoskeleton" to maintain their shape and polarity. Based on this, herein a new class of cell-mimicking nanomedicine using bionic skeletons constituted by the oligomeric Au(I)-peptide complex is developed. The peptide function of degrading pathological MDM2 and MDMX is used to synthesize an oligomeric Au(I)-PMIV precursor capable of self-assembling into a clustered spherical bionic skeleton. Through coating by erythrocyte membrane, an erythrocyte-mimicking nano-cell (Nery-PMIV) is developed with depressed macrophage uptakes, increased colloidal stability, and prolonged blood circulation. Nery-PMIV potently restores p53 and p73 in vitro and in vivo by degrading MDM2/MDMX. More importantly, Nery-PMIV effectively augments antitumor immunity elicited by anti-PD1 therapy in a murine orthotopic allograft model for LUAD and a humanized patient-derived xenograft (PDX) mouse model for LUAD, while maintaining a favorable safety profile. Taken together, this work not only presents evidence showing that MDM2/MDMX degradation is a potentially viable therapeutic paradigm to synergize anti-PD1 immunotherapy toward LUAD carrying wild-type p53; it also suggests that cell-mimicking nanoparticles with applicable bionic skeletons hold tremendous promise in offering new therapies to revolutionize nanomedicine in the treatment of a myriad of human diseases.
Asunto(s)
Adenocarcinoma , Proteínas Proto-Oncogénicas c-mdm2 , Animales , Biomimética , Proteínas de Ciclo Celular , Eritrocitos/metabolismo , Inmunoterapia , Ratones , Péptidos/metabolismo , Conducta Predatoria , Unión Proteica , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Esqueleto/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Complexin-1 (Cpx) and α-synuclein (α-Syn) are involved in neurotransmitter release through an interaction with synaptic vesicles (SVs). Recent studies demonstrated that Cpx and α-Syn preferentially associate with highly curved membranes, like SVs, to correctly position them for fusion. Here, based on recent experimental results, to further propose a possible explanation for this mechanism, we performed in silico simulations probing interactions between Cpx or α-Syn and membranes of varying curvature. We found that the preferential association is attributed to smaller, curved membranes containing more packing defects that expose hydrophobic acyl tails, which may favorably interact with hydrophobic residues of Cpx and α-Syn. The number of membrane defects is proportional to the curvature and the size can be regulated by cholesterol.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Vesículas Sinápticas/metabolismo , alfa-Sinucleína/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/química , Colesterol/química , Enlace de Hidrógeno , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Proteínas del Tejido Nervioso/química , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Unión Proteica , Vesículas Sinápticas/química , alfa-Sinucleína/químicaRESUMEN
Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17-SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17-SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome-endolysosome fusion.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Autofagia , Endosomas/metabolismo , Lisosomas/metabolismo , Fusión de Membrana , Fagosomas/metabolismo , Proteínas Relacionadas con la Autofagia , Células HEK293 , Células HeLa , Humanos , Fagosomas/química , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Qa-SNARE/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismoRESUMEN
Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.
Asunto(s)
Exocitosis , Neuronas/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Sinaptotagminas/química , Sinaptotagminas/metabolismo , Animales , Sitios de Unión/genética , Calcio/química , Calcio/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Electrones , Hipocampo/citología , Rayos Láser , Magnesio/química , Magnesio/metabolismo , Fusión de Membrana , Ratones , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Neuronas/química , Neuronas/citología , Proteínas SNARE/genética , Transmisión Sináptica , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Sinaptotagminas/genéticaRESUMEN
SLC25A46 mutations have been found to lead to mitochondrial hyper-fusion and reduced mitochondrial respiratory function, which results in optic atrophy, cerebellar atrophy, and other clinical symptoms of mitochondrial disease. However, it is generally believed that mitochondrial fusion is attributable to increased mitochondrial oxidative phosphorylation (OXPHOS), which is inconsistent with the decreased OXPHOS of highly-fused mitochondria observed in previous studies. In this paper, we have used the live-cell nanoscope to observe and quantify the structure of mitochondrial cristae, and the behavior of mitochondria and lysosomes in patient-derived SLC25A46 mutant fibroblasts. The results show that the cristae have been markedly damaged in the mutant fibroblasts, but there is no corresponding increase in mitophagy. This study suggests that severely damaged mitochondrial cristae might be the predominant cause of reduced OXPHOS in SLC25A46 mutant fibroblasts. This study demonstrates the utility of nanoscope-based imaging for realizing the sub-mitochondrial morphology, mitophagy and mitochondrial dynamics in living cells, which may be particularly valuable for the quick evaluation of pathogenesis of mitochondrial morphological abnormalities.
Asunto(s)
Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Dinámicas Mitocondriales/fisiología , Mitofagia/fisiología , Proliferación Celular , Fibroblastos/metabolismo , Humanos , Lisosomas/metabolismo , Enfermedades Mitocondriales/genética , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación , Proteínas de Transporte de Fosfato/genética , Proteínas de Transporte de Fosfato/metabolismoRESUMEN
Membrane fusion is essential for eukaryotic life, requiring SNARE proteins to zipper up in an α-helical bundle to pull two membranes together. Here, we show that vesicle fusion can be suppressed by phosphorylation of core conserved residues inside the SNARE domain. We took a proteomics approach using a PKCB knockout mast cell model and found that the key mast cell secretory protein VAMP8 becomes phosphorylated by PKC at multiple residues in the SNARE domain. Our data suggest that VAMP8 phosphorylation reduces vesicle fusion in vitro and suppresses secretion in living cells, allowing vesicles to dock but preventing fusion with the plasma membrane. Markedly, we show that the phosphorylation motif is absent in all eukaryotic neuronal VAMPs, but present in all other VAMPs. Thus, phosphorylation of SNARE domains is a general mechanism to restrict how much cells secrete, opening the door for new therapeutic strategies for suppression of secretion.
Asunto(s)
Proteína Quinasa C/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas R-SNARE/metabolismo , Vesículas Secretoras/metabolismo , Animales , Línea Celular , Mastocitos/fisiología , Fosforilación , Proteómica , RatasRESUMEN
Toxic abnormal aggregation of α-synuclein (α-Syn) is a feature of Parkinson's disease. Several biochemical and biophysical studies have demonstrated that many post-translational modifications (PTM) of α-Syn could distinctly alleviate its oligomerization-mediated toxicity. Recently, a compelling link is emerging between the PTM O-GlcNAcylation (O-GlcNAc) and protein aggregation, yet the underlying molecular mechanism remains unclear. Based on the all-atom molecular dynamics simulations, we found that O-GlcNAc modifications can suppress the process of oligomerization of α-Syn aggregates via a steric effect-the additional O-linked glycosyl group disrupts the formation of hydrogen bonds (H-bonds) between α-Syn monomers. Besides, we proposed a theoretical model to further capture the physical mechanism of α-Syn aggregation/disaggregation in the absence/presence of O-GlcNAc-modified α-Syn. Our findings unveil the molecular mechanism of the O-GlcNAc-induced inhibition of α-Syn oligomerization, which may help to understand how O-GlcNAc prevents the oligomerization of other proteins and provides the guideline for the development of O-GlcNAc-based therapeutic strategies in neurodegenerative diseases.
Asunto(s)
Enfermedades Neurodegenerativas/tratamiento farmacológico , Procesamiento Proteico-Postraduccional , alfa-Sinucleína/metabolismo , Acilación , Modelos Biológicos , Polimerizacion , Agregación Patológica de Proteínas/metabolismo , alfa-Sinucleína/químicaRESUMEN
Currently, photosensitizers (PSs) that are microenvironment responsive and hypoxia active are scarcely available and urgently desired for antitumor photodynamic therapy (PDT). Presented herein is the design of a redox stimuli activatable metal-free photosensitizer (aPS), also functioning as a pre-photosensitizer as it is converted to a PS by the mutual presence of glutathione (GSH) and hydrogen peroxide (H2 O2 ) with high specificity on a basis of domino reactions on the benzothiadiazole ring. Superior to traditional PSs, the activated aPS contributed to efficient generation of reactive oxygen species including singlet oxygen and superoxide ion through both type 1 and type 2 pathways, alleviating the aerobic requirement for PDT. Equipped with a triphenylphosphine ligand for mitochondria targeting, mito aPS showed excellent phototoxicity to tumor cells with low light fluence under both normoxic and hypoxic conditions, after activation by intracellular GSH and H2 O2 . The mito aPS was also compatible to near infrared PDT with two photon excitation (800â nm) for extensive bioapplications.
Asunto(s)
Glutatión/metabolismo , Peróxido de Hidrógeno/metabolismo , Fotoquimioterapia/métodos , Animales , Línea Celular Tumoral , Diseño de Fármacos , Humanos , Hipoxia/metabolismo , Mitocondrias , Neoplasias/terapia , Fosfinas , Fármacos Fotosensibilizantes/síntesis química , Fármacos Fotosensibilizantes/farmacología , Oxígeno Singlete/metabolismoRESUMEN
It is of great significance to track the platinum drugs in real time with super-resolution to elucidate their mechanism of action, such as their behavior and distribution in live cells. Such information is required for further drug development. However, it is always challenging to design platinum complexes suitable for such research. Herein, we design a luminescent building block (L) for metal complexes and a dinuclear platinum complex (Pt2 L) for super-resolution imaging. Because of its super-large Stokes shift and excellent photophysical properties, Pt2 L is capable of serving as an ideal candidate for super-resolution imaging with extremely low luminescence background and high photobleaching resistance. Moreover, upon light stimulation, a matter flux of Pt2 L escaping from autolysosomes to nucleus was observed, which represents a new transportation path. Utilizing the photoactivated escape properties, we can regulate the nuclear accessibility of Pt2 L form autolysosomes with photo-selectivity, which provides a new way to improve the targeting of platinum drugs.
Asunto(s)
Color , Lisosomas/metabolismo , Compuestos de Platino/química , Células A549 , Transporte Biológico , Núcleo Celular/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente/métodos , Mitocondrias/metabolismo , Compuestos de Platino/metabolismoRESUMEN
We here review primary methods used in quantifying and mapping 5-hydroxymethylcytosine (5hmC), including global quantification, restriction enzyme-based detection, and methods involving DNA-enrichment strategies and the genome-wide sequencing of 5hmC. As discovered in the mammalian genome in 2009, 5hmC, oxidized from 5-methylcytosine (5mC) by ten-eleven translocation (TET) dioxygenases, is increasingly being recognized as a biomarker in biological processes from development to pathogenesis, as its various detection methods have shown. We focus in particular on an ultrasensitive single-molecule imaging technique that can detect and quantify 5hmC from trace samples and thus offer information regarding the distance-based relationship between 5hmC and 5mC when used in combination with fluorescence resonance energy transfer.
Asunto(s)
5-Metilcitosina/análogos & derivados , Epigénesis Genética , 5-Metilcitosina/análisis , 5-Metilcitosina/inmunología , 5-Metilcitosina/metabolismo , Animales , Especificidad de Anticuerpos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Mapeo Cromosómico , ADN/química , ADN/genética , Enzimas de Restricción del ADN , Transferencia Resonante de Energía de Fluorescencia , Marcadores Genéticos , Glicosilación , Humanos , Espectrometría de Masas , Análisis de Secuencia de ADN , Imagen Individual de Molécula/métodosRESUMEN
Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress.
Asunto(s)
G-Cuádruplex , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , ADN/metabolismo , ADN de Cadena Simple/metabolismo , Genoma Humano , Humanos , Unión Proteica , Células Sf9 , Telómero/químicaRESUMEN
The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.
Asunto(s)
5-Metilcitosina/metabolismo , Citosina/análogos & derivados , Metilación de ADN/fisiología , Epigénesis Genética/fisiología , Imagen Molecular , Animales , Citosina/metabolismo , RatonesRESUMEN
Complexin activates Ca(2+)-triggered neurotransmitter release and regulates spontaneous release in the presynaptic terminal by cooperating with the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and the Ca(2+)-sensor synaptotagmin. The N-terminal domain of complexin is important for activation, but its molecular mechanism is still poorly understood. Here, we observed that a split pair of N-terminal and central domain fragments of complexin is sufficient to activate Ca(2+)-triggered release using a reconstituted single-vesicle fusion assay, suggesting that the N-terminal domain acts as an independent module within the synaptic fusion machinery. The N-terminal domain can also interact independently with membranes, which is enhanced by a cooperative interaction with the neuronal SNARE complex. We show by mutagenesis that membrane binding of the N-terminal domain is essential for activation of Ca(2+)-triggered fusion. Consistent with the membrane-binding property, the N-terminal domain can be substituted by the influenza virus hemagglutinin fusion peptide, and this chimera also activates Ca(2+)-triggered fusion. Membrane binding of the N-terminal domain of complexin therefore cooperates with the other fusogenic elements of the synaptic fusion machinery during Ca(2+)-triggered release.