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1.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33827988

RESUMEN

In order to understand the transmission and virulence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is necessary to understand the functions of each of the gene products encoded in the viral genome. One feature of the SARS-CoV-2 genome that is not present in related, common coronaviruses is ORF10, a putative 38-amino acid protein-coding gene. Proteomic studies found that ORF10 binds to an E3 ubiquitin ligase containing Cullin-2, Rbx1, Elongin B, Elongin C, and ZYG11B (CRL2ZYG11B). Since CRL2ZYG11B mediates protein degradation, one possible role for ORF10 is to "hijack" CRL2ZYG11B in order to target cellular, antiviral proteins for ubiquitylation and subsequent proteasomal degradation. Here, we investigated whether ORF10 hijacks CRL2ZYG11B or functions in other ways, for example, as an inhibitor or substrate of CRL2ZYG11B While we confirm the ORF10-ZYG11B interaction and show that the N terminus of ORF10 is critical for it, we find no evidence that ORF10 is functioning to inhibit or hijack CRL2ZYG11B Furthermore, ZYG11B and its paralog ZER1 are dispensable for SARS-CoV-2 infection in cultured cells. We conclude that the interaction between ORF10 and CRL2ZYG11B is not relevant for SARS-CoV-2 infection in vitro.


Asunto(s)
/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cullin/metabolismo , Complejos Multiproteicos/metabolismo , Sistemas de Lectura Abierta , Proteínas Virales/metabolismo , /genética , Proteínas de Ciclo Celular/genética , Proteínas Cullin/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Proteínas Virales/genética
2.
Nat Commun ; 12(1): 2160, 2021 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-33846341

RESUMEN

The dynamic assembly of the cell wall is key to the maintenance of cell shape during bacterial growth. Here, we present a method for the analysis of Escherichia coli cell wall growth at high spatial and temporal resolution, which is achieved by tracing the movement of fluorescently labeled cell wall-anchored flagellar motors. Using this method, we clearly identify the active and inert zones of cell wall growth during bacterial elongation. Within the active zone, the insertion of newly synthesized peptidoglycan occurs homogeneously in the axial direction without twisting of the cell body. Based on the measured parameters, we formulate a Bernoulli shift map model to predict the partitioning of cell wall-anchored proteins following cell division.


Asunto(s)
Pared Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Complejos Multiproteicos/metabolismo , División Celular , Escherichia coli/crecimiento & desarrollo , Flagelos/metabolismo , Fluorescencia , Peptidoglicano/metabolismo
3.
Nat Commun ; 12(1): 1796, 2021 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-33741907

RESUMEN

Most diseases disrupt multiple proteins, and drugs treat such diseases by restoring the functions of the disrupted proteins. How drugs restore these functions, however, is often unknown as a drug's therapeutic effects are not limited to the proteins that the drug directly targets. Here, we develop the multiscale interactome, a powerful approach to explain disease treatment. We integrate disease-perturbed proteins, drug targets, and biological functions into a multiscale interactome network. We then develop a random walk-based method that captures how drug effects propagate through a hierarchy of biological functions and physical protein-protein interactions. On three key pharmacological tasks, the multiscale interactome predicts drug-disease treatment, identifies proteins and biological functions related to treatment, and predicts genes that alter a treatment's efficacy and adverse reactions. Our results indicate that physical interactions between proteins alone cannot explain treatment since many drugs treat diseases by affecting the biological functions disrupted by the disease rather than directly targeting disease proteins or their regulators. We provide a general framework for explaining treatment, even when drugs seem unrelated to the diseases they are recommended for.


Asunto(s)
Complejos Multiproteicos/metabolismo , Preparaciones Farmacéuticas/administración & dosificación , Mapas de Interacción de Proteínas/efectos de los fármacos , Proteínas/metabolismo , Algoritmos , Animales , Biología Computacional/métodos , Quimioterapia/métodos , Humanos , Modelos Teóricos , Unión Proteica/efectos de los fármacos , Mapeo de Interacción de Proteínas/métodos
4.
Mol Cell ; 81(6): 1246-1259.e8, 2021 03 18.
Artículo en Inglés | MEDLINE | ID: mdl-33548203

RESUMEN

The Integrator is a specialized 3' end-processing complex involved in cleavage and transcription termination of a subset of nascent RNA polymerase II transcripts, including small nuclear RNAs (snRNAs). We provide evidence of the modular nature of the Integrator complex by biochemically characterizing its two subcomplexes, INTS5/8 and INTS10/13/14. Using cryoelectron microscopy (cryo-EM), we determined a 3.5-Å-resolution structure of the INTS4/9/11 ternary complex, which constitutes Integrator's catalytic core. Our structure reveals the spatial organization of the catalytic nuclease INTS11, bound to its catalytically impaired homolog INTS9 via several interdependent interfaces. INTS4, a helical repeat protein, plays a key role in stabilizing nuclease domains and other components. In this assembly, all three proteins form a composite electropositive groove, suggesting a putative RNA binding path within the complex. Comparison with other 3' end-processing machineries points to distinct features and a unique architecture of the Integrator's catalytic module.


Asunto(s)
Complejos Multiproteicos , Terminación de la Transcripción Genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Microscopía por Crioelectrón , Células HEK293 , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo
5.
Int J Mol Sci ; 22(3)2021 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-33540902

RESUMEN

Human epidermal keratinocytes are constantly exposed to UV radiation. As a result, there is a significant need for safe and effective compounds to protect skin cells against this environmental damage. This study aimed to analyze the effect of phytocannabinoid-cannabinoid (CBD)-on the proteome of UVA/B irradiated keratinocytes. The keratinocytes were cultured in a three-dimensional (3D) system, designed to mimic epidermal conditions closely. The obtained results indicate that CBD protected against the harmful effects of UVA/B radiation. CBD decreased the expression of proinflammatory proteins, including TNFα/NFκB and IκBKB complex and decreased the expression of proteins involved in de novo protein biosynthesis, which are increased in UVA/B-irradiated cells. Additionally, CBD enhanced the UV-induced expression of 20S proteasome subunits. CBD also protected protein structures from 4-hydroxynonenal (HNE)-binding induced by UV radiation, which primarily affects antioxidant enzymes. CBD-through its antioxidant/anti-inflammatory activity and regulation of protein biosynthesis and degradation-protects skin cells against UVA/B-induced changes. In the future, its long-term use in epidermal cells should be investigated.


Asunto(s)
Cannabidiol/farmacología , Queratinocitos/efectos de los fármacos , Proteoma/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Rayos Ultravioleta , Aldehídos/farmacología , Antiinflamatorios/farmacología , Antioxidantes/farmacología , Cannabidiol/química , Técnicas de Cultivo de Célula , Células Cultivadas , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/efectos de la radiación , Humanos , Quinasa I-kappa B/metabolismo , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Estructura Molecular , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Análisis de Componente Principal , Proteoma/efectos de la radiación , Transducción de Señal/efectos de la radiación , Factor de Necrosis Tumoral alfa/metabolismo
6.
Nat Genet ; 53(3): 279-287, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33558757

RESUMEN

Chromatin accessibility is a hallmark of regulatory regions, entails transcription factor (TF) binding and requires nucleosomal reorganization. However, it remains unclear how dynamic this process is. In the present study, we use small-molecule inhibition of the catalytic subunit of the mouse SWI/SNF remodeler complex to show that accessibility and reduced nucleosome presence at TF-binding sites rely on persistent activity of nucleosome remodelers. Within minutes of remodeler inhibition, accessibility and TF binding decrease. Although this is irrespective of TF function, we show that the activating TF OCT4 (POU5F1) exhibits a faster response than the repressive TF REST. Accessibility, nucleosome depletion and gene expression are rapidly restored on inhibitor removal, suggesting that accessible chromatin is regenerated continuously and in a largely cell-autonomous fashion. We postulate that TF binding to chromatin and remodeler-mediated nucleosomal removal do not represent a stable situation, but instead accessible chromatin reflects an average of a dynamic process under continued renewal.


Asunto(s)
Cromatina/metabolismo , Complejos Multiproteicos/metabolismo , Factores de Transcripción/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Animales , Sitios de Unión , Línea Celular/efectos de los fármacos , Cromatina/genética , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Ensamble y Desensamble de Cromatina/fisiología , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Histonas/genética , Histonas/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Complejos Multiproteicos/efectos de los fármacos , Complejos Multiproteicos/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Receptores Estrogénicos/genética , Receptores Estrogénicos/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética
7.
Nat Genet ; 53(3): 269-278, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33558760

RESUMEN

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1-8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9-19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20-24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities.


Asunto(s)
Cromatina/genética , ADN Helicasas/metabolismo , Complejos Multiproteicos/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Acetilación , Animales , Línea Celular , Cromatina/metabolismo , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Drosophila/citología , Elementos de Facilitación Genéticos , Técnicas de Inactivación de Genes , Histonas/genética , Histonas/metabolismo , Humanos , Complejos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Factores de Transcripción/genética
8.
Nat Struct Mol Biol ; 28(3): 249-257, 2021 03.
Artículo en Inglés | MEDLINE | ID: mdl-33558762

RESUMEN

Human serine palmitoyltransferase (SPT) complex catalyzes the initial and rate-limiting step in the de novo biosynthesis of all sphingolipids. ORMDLs regulate SPT function, with human ORMDL3 being related to asthma. Here we report three high-resolution cryo-EM structures: the human SPT complex, composed of SPTLC1, SPTLC2 and SPTssa; the SPT-ORMDL3 complex; and the SPT-ORMDL3 complex bound to two substrates, PLP-L-serine (PLS) and a non-reactive palmitoyl-CoA analogue. SPTLC1 and SPTLC2 form a dimer of heterodimers as the catalytic core. SPTssa participates in acyl-CoA coordination, thereby stimulating the SPT activity and regulating the substrate selectivity. ORMDL3 is located in the center of the complex, serving to stabilize the SPT assembly. Our structural and biochemical analyses provide a molecular basis for the assembly and substrate selectivity of the SPT and SPT-ORMDL3 complexes, and lay a foundation for mechanistic understanding of sphingolipid homeostasis and for related therapeutic drug development.


Asunto(s)
Microscopía por Crioelectrón , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos , Serina C-Palmitoiltransferasa/química , Serina C-Palmitoiltransferasa/metabolismo , Acilcoenzima A/química , Acilcoenzima A/metabolismo , Acilcoenzima A/ultraestructura , Sitios de Unión , Biocatálisis , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Reproducibilidad de los Resultados , Serina/química , Serina/metabolismo , Serina C-Palmitoiltransferasa/genética , Serina C-Palmitoiltransferasa/ultraestructura , Especificidad por Sustrato
9.
Nature ; 590(7844): 163-169, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33408415

RESUMEN

The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.


Asunto(s)
Microscopía por Crioelectrón , Mitocondrias/química , Mitocondrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/ultraestructura , Mitocondrias/ultraestructura , Proteínas Mitocondriales/química , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/ultraestructura , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura
10.
Nat Commun ; 12(1): 583, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495445

RESUMEN

We have recently described the development of a series of small-molecule inhibitors of human tumour necrosis factor (TNF) that stabilise an open, asymmetric, signalling-deficient form of the soluble TNF trimer. Here, we describe the generation, characterisation, and utility of a monoclonal antibody that selectively binds with high affinity to the asymmetric TNF trimer-small molecule complex. The antibody helps to define the molecular dynamics of the apo TNF trimer, reveals the mode of action and specificity of the small molecule inhibitors, acts as a chaperone in solving the human TNF-TNFR1 complex crystal structure, and facilitates the measurement of small molecule target occupancy in complex biological samples. We believe this work defines a role for monoclonal antibodies as tools to facilitate the discovery and development of small-molecule inhibitors of protein-protein interactions.


Asunto(s)
Anticuerpos Monoclonales/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Bibliotecas de Moléculas Pequeñas/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Anticuerpos Monoclonales/farmacología , Células Cultivadas , Cristalografía por Rayos X , Epítopos/química , Epítopos/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Complejos Multiproteicos/química , Unión Proteica/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Receptores Tipo I de Factores de Necrosis Tumoral/química , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Factor de Necrosis Tumoral alfa/química
11.
Nat Commun ; 12(1): 563, 2021 01 25.
Artículo en Inglés | MEDLINE | ID: mdl-33495456

RESUMEN

Myosin-7a, despite being monomeric in isolation, plays roles in organizing actin-based cell protrusions such as filopodia, microvilli and stereocilia, as well as transporting cargoes within them. Here, we identify a binding protein for Drosophila myosin-7a termed M7BP, and describe how M7BP assembles myosin-7a into a motile complex that enables cargo translocation and actin cytoskeletal remodeling. M7BP binds to the autoinhibitory tail of myosin-7a, extending the molecule and activating its ATPase activity. Single-molecule reconstitution show that M7BP enables robust motility by complexing with myosin-7a as 2:2 translocation dimers in an actin-regulated manner. Meanwhile, M7BP tethers actin, enhancing complex's processivity and driving actin-filament alignment during processive runs. Finally, we show that myosin-7a-M7BP complex assembles actin bundles and filopodia-like protrusions while migrating along them in living cells. Together, these findings provide insights into the mechanisms by which myosin-7a functions in actin protrusions.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Miosina VIIa/metabolismo , Animales , Proteínas Portadoras/genética , Línea Celular , Movimiento Celular/genética , Movimiento Celular/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Microscopía Fluorescente/métodos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Miosina VIIa/química , Miosina VIIa/genética , Unión Proteica , Multimerización de Proteína , Seudópodos/genética , Seudópodos/fisiología , Estereocilios/genética , Estereocilios/fisiología
12.
Science ; 371(6524): 67-71, 2021 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-33384373

RESUMEN

Open (O) and closed (C) topologies of HORMA-domain proteins are respectively associated with inactive and active states of fundamental cellular pathways. The HORMA protein O-MAD2 converts to C-MAD2 upon binding CDC20. This is rate limiting for assembly of the mitotic checkpoint complex (MCC), the effector of a checkpoint required for mitotic fidelity. A catalyst assembled at kinetochores accelerates MAD2:CDC20 association through a poorly understood mechanism. Using a reconstituted SAC system, we discovered that CDC20 is an impervious substrate for which access to MAD2 requires simultaneous docking on several sites of the catalytic complex. Our analysis indicates that the checkpoint catalyst is substrate assisted and promotes MCC assembly through spatially and temporally coordinated conformational changes in both MAD2 and CDC20. This may define a paradigm for other HORMA-controlled systems.


Asunto(s)
Proteínas Cdc20/metabolismo , Cinetocoros/metabolismo , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Mad2/metabolismo , Complejos Multiproteicos/metabolismo , Biocatálisis , Proteínas de Ciclo Celular/metabolismo , Células HeLa , Humanos , Proteínas Mad2/genética , Mutación , Proteínas de Unión a Poli-ADP-Ribosa/metabolismo , Proteínas Serina-Treonina Quinasas , Huso Acromático/metabolismo
13.
Methods Mol Biol ; 2200: 425-440, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33175391

RESUMEN

Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to the environment. However, a main limitation of traditional approaches to study protein interactions, such as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of interactions include many of the protein complexes that mediate the perception and response to cellular stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells, including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins (within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the study of proteomes in numerous organisms, its application to plant systems has only recently been realized. In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.


Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Biotina/química , Complejos Multiproteicos , Mapeo de Interacción de Proteínas , Proteómica , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Biotinilación , Cromatografía de Afinidad , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Unión Proteica
14.
Nature ; 590(7846): 498-503, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33361816

RESUMEN

Histone methyltransferases of the nuclear receptor-binding SET domain protein (NSD) family, including NSD1, NSD2 and NSD3, have crucial roles in chromatin regulation and are implicated in oncogenesis1,2. NSD enzymes exhibit an autoinhibitory state that is relieved by binding to nucleosomes, enabling dimethylation of histone H3 at Lys36 (H3K36)3-7. However, the molecular basis that underlies this mechanism is largely unknown. Here we solve the cryo-electron microscopy structures of NSD2 and NSD3 bound to mononucleosomes. We find that binding of NSD2 and NSD3 to mononucleosomes causes DNA near the linker region to unwrap, which facilitates insertion of the catalytic core between the histone octamer and the unwrapped segment of DNA. A network of DNA- and histone-specific contacts between NSD2 or NSD3 and the nucleosome precisely defines the position of the enzyme on the nucleosome, explaining the specificity of methylation to H3K36. Intermolecular contacts between NSD proteins and nucleosomes are altered by several recurrent cancer-associated mutations in NSD2 and NSD3. NSDs that contain these mutations are catalytically hyperactive in vitro and in cells, and their ectopic expression promotes the proliferation of cancer cells and the growth of xenograft tumours. Together, our research provides molecular insights into the nucleosome-based recognition and histone-modification mechanisms of NSD2 and NSD3, which could lead to strategies for therapeutic targeting of proteins of the NSD family.


Asunto(s)
N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/química , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/metabolismo , Proteínas Represoras/metabolismo , Sitios de Unión , Biocatálisis , Línea Celular Tumoral , Proliferación Celular , Microscopía por Crioelectrón , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/ultraestructura , Histonas/ultraestructura , Humanos , Metilación , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/ultraestructura , Mutación , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/patología , Proteínas Nucleares/genética , Proteínas Nucleares/ultraestructura , Nucleosomas/ultraestructura , Fenotipo , Unión Proteica , Proteínas Represoras/genética , Proteínas Represoras/ultraestructura
15.
Methods Mol Biol ; 2217: 71-81, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215378

RESUMEN

The in situ proximity ligation assay (PLA) is capable of detecting single protein events such as protein protein-interactions and posttranslational modifications (e.g., protein phosphorylation) in tissue and cell samples prepared for analysis by immunofluorescent or immunohistochemical microscopy. The targets are detected using two primary antibodies which must be from different host species. A pair of secondary antibodies (PLA probes) conjugated to complementary oligonucleotides is applied to the sample, and a signal is generated only when the two PLA probes are in close proximity by their binding to the two primary antibodies that have bound to their targets in close proximity. The signal from each pair of PLA probes is visualized as an individual fluorescent spot. These PLA signals can be quantified (counted) using image analysis software (ImageJ), and also assigned to a specific subcellular location based on microscopy image overlays. In principle, in situ PLA offers a relatively simple and sensitive technique to analyze interactions among any proteins for which suitable antibodies are available. Integrin-mediated focal adhesions (FAs) are large multiprotein complexes consisting of more than 150 proteins, also known as the integrin adhesome, which link the extracellular matrix (ECM) to the actin cytoskeleton and regulate the functioning of mechanosignaling pathways. The in situ PLA approach is well suited for examining the spatiotemporal aspects of protein posttranslational modifications and protein interactions occurring in dynamic multiprotein complexes such as integrin mediated focal adhesions.


Asunto(s)
Adhesiones Focales/metabolismo , Inmunohistoquímica/métodos , Cadenas alfa de Integrinas/metabolismo , Integrina beta1/metabolismo , Complejos Multiproteicos/metabolismo , Oligonucleótidos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestructura , Anticuerpos/química , Matriz Extracelular/metabolismo , Matriz Extracelular/ultraestructura , Adhesiones Focales/ultraestructura , Mucosa Gástrica/metabolismo , Mucosa Gástrica/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Cadenas alfa de Integrinas/química , Integrina beta1/química , Microscopía Fluorescente , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Complejos Multiproteicos/química , Músculo Liso/metabolismo , Músculo Liso/ultraestructura , Oligonucleótidos/síntesis química , Unión Proteica
16.
Methods Mol Biol ; 2217: 149-179, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215381

RESUMEN

Cell-surface adhesion receptors mediate interactions with the extracellular matrix (ECM) to control many fundamental aspects of cell behavior, including cell migration, survival, and proliferation. Integrin adhesion receptors recruit structural and signaling proteins to form multimolecular adhesion complexes that link the plasma membrane to the actomyosin cytoskeleton. The assembly and turnover of adhesion complexes are tightly regulated, governed in part by the networks of physical protein interactions and functional signaling associations between components of the adhesome. Proteomic profiling of adhesion complexes has begun to reveal their molecular complexity and diversity. To interrogate the composition of cell-ECM adhesions, we detail herein an approach for the network analysis of adhesion complex proteomes. Integration of these proteomic data with adhesome databases in the context of predicted protein interactions enables the mapping of experimentally defined adhesion complex networks. Computational analysis of resultant network models can identify subnetworks of putative functionally linked adhesion protein communities. This approach provides a framework to predict functional adhesion protein relationships and generate new mechanistic hypotheses for further experimental testing.


Asunto(s)
Biología Computacional/métodos , Integrinas/metabolismo , Complejos Multiproteicos/metabolismo , Redes Neurales de la Computación , Mapeo de Interacción de Proteínas/métodos , Proteoma/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animales , Adhesión Celular , Movimiento Celular , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Humanos , Integrinas/genética , Complejos Multiproteicos/genética , Unión Proteica , Proteoma/genética , Programas Informáticos
17.
Methods Mol Biol ; 2217: 285-300, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33215387

RESUMEN

The extracellular matrix (ECM) is the noncellular compartment of living organisms and is formed of a complex network of cross-linked proteins, which is collectively known as the matrisome. Apart from providing the structure for an organism, cells interact and thereby communicate with the ECM. Cells interact with their surrounding ECM using cell-surface receptors, such as integrins. Upon integrin engagement with the ECM, cytoskeletal proteins are recruited to integrins and form a molecular protein complex known as the integrin adhesome. Global descriptions of the matrisome and integrin adhesome have been proposed using in silico bioinformatics approaches, as well as through biochemical enrichment of matrisome and adhesome fractions coupled with mass spectrometry-based proteomic analyses, providing inventories of their compositions in different contexts. Here, methods are described for the computational downstream analyses of matrisome and adhesome mass spectrometry datasets that are accessible to wet lab biologists, which include comparing datasets to in silico descriptions, generating interaction networks and performing functional ontological analyses.


Asunto(s)
Biología Computacional/métodos , Proteínas de la Matriz Extracelular/metabolismo , Matriz Extracelular/metabolismo , Redes Reguladoras de Genes , Integrinas/metabolismo , Complejos Multiproteicos/metabolismo , Animales , Adhesión Celular , Células Cultivadas , Bases de Datos Genéticas , Matriz Extracelular/química , Proteínas de la Matriz Extracelular/clasificación , Proteínas de la Matriz Extracelular/genética , Ontología de Genes , Humanos , Integrinas/clasificación , Integrinas/genética , Espectrometría de Masas , Ratones , Anotación de Secuencia Molecular , Familia de Multigenes , Complejos Multiproteicos/clasificación , Complejos Multiproteicos/genética , Unión Proteica
18.
Methods Mol Biol ; 2192: 69-73, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33230766

RESUMEN

The incorporation of nucleoside analogs is a useful tool to study the various functions of DNA and RNA. These analogs can be detected directly by fluorescence or by immunolabeling, allowing to visualize, track, or measure the nucleic acid molecules in which they have been incorporated. In this chapter, methodologies to measure human mitochondrial transcription are described. The nascent RNA that is transcribed from mitochondrial DNA (mtDNA) has been shown to assemble into large ribonucleoprotein complexes that form discrete foci. These structures were called mitochondrial RNA granules (MRGs) and can be observed in vitro by the incorporation of a 5-Bromouridine (BrU), which is subsequently visualized by fluorescent immunolabeling. Here, a combined protocol for the MRGs detection is detailed, consisting of BrU labeling and visualization of one of their bona fide protein components, Fas-activated serine-threonine kinase domain 2 (FASTKD2). Based on immunodetection, the half-life and kinetics of the MRGs under various experimental conditions can further be determined by chasing the BrU pulse with an excess of Uridine.


Asunto(s)
Bromouracilo/análogos & derivados , Inmunohistoquímica/métodos , Complejos Multiproteicos/metabolismo , ARN Mitocondrial/metabolismo , Ribonucleoproteínas/metabolismo , Uridina/análogos & derivados , Bromouracilo/metabolismo , ADN Mitocondrial/metabolismo , Semivida , Células HeLa , Humanos , Cinética , Complejos Multiproteicos/química , Proteínas Serina-Treonina Quinasas/metabolismo , Ribonucleoproteínas/química , Transcripción Genética , Uridina/metabolismo
19.
Methods Mol Biol ; 2192: 287-311, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33230780

RESUMEN

Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique optimized for the analysis of the five components of the mitochondrial oxidative phosphorylation (OXPHOS) system. BN-PAGE is based on the preservation of the interactions between the individual subunits within the integral complexes. To achieve this, the complexes are extracted from the mitochondrial inner membrane using mild detergents and separated by electrophoresis in the absence of denaturing agents. The electrophoretic procedures can then be combined with a variety of downstream detection techniques. Since its development in the 1990s, BN-PAGE has been applied in the study of mitochondria from all kinds of organisms and extensive amounts of data have been produced using this technique, being key for the understanding of many aspects of OXPHOS physiopathology.


Asunto(s)
Electroforesis en Gel Bidimensional/métodos , Mitocondrias/química , Complejos Multiproteicos/química , Electroforesis en Gel de Poliacrilamida Nativa/métodos , Fosforilación Oxidativa , Animales , Donantes de Sangre , Humanos , Leucocitos/citología , Hígado/citología , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Complejos Multiproteicos/metabolismo , Miocardio/citología
20.
Methods Mol Biol ; 2247: 287-301, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33301124

RESUMEN

The need to describe and understand signaling pathways in live cell is seen as a primary route to identifying and developing targeted medicines. Signaling cascade is also seen as a complex communication and involves interactions between multiple interconnecting proteins. Where subcellularly and how different proteins interact need to be preserved during investigation. Furthermore, these complex events occurring simultaneously may lead to a single or multiple end point or cell function such as protein synthesis, cell cytoskeleton formation, DNA damage repair, or autophagy. There is therefore a need of real-time noninvasive methods for protein assays to enable direct visualization of the interactions in their natural environment and hence overcome the limitations of methods that rely on invasive cell disruption techniques. Förster resonance energy transfer (FRET) coupled with fluorescence lifetime imaging microscopy (FLIM) is an advanced imaging method to observe protein-protein interactions at nanometer scale inside single living cells in real-time. Here we describe the development and use of two-channel pulsed interleave excitation (PIE) for multiple protein interactions in the mTORC1 pathway. The proteins were first tagged with multiple color fluorescent protein derivatives. The FRET-FLIM combination means that the information gained from using standard steady-state FRET between interacting proteins is considerably improved by monitoring changes in the excited-state lifetime of the donor fluorophore where its quenching in the presence of the acceptor is evidence for a direct physical interaction.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , Complejos Multiproteicos/metabolismo , Algoritmos , ADN/química , Modelos Teóricos , Unión Proteica
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