RESUMO
CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease) defense systems have been naturally coopted for guide RNA-directed transposition on multiple occasions. In all cases, cooption occurred with diverse elements related to the bacterial transposon Tn7. Tn7 tightly controls transposition; the transposase is activated only when special targets are recognized by dedicated target-site selection proteins. Tn7 and the Tn7-like elements that coopted CRISPR-Cas systems evolved complementary targeting pathways: one that recognizes a highly conserved site in the chromosome and a second pathway that targets mobile plasmids capable of cell-to-cell transfer. Tn7 and Tn7-like elements deliver a single integration into the site they recognize and also control the orientation of the integration event, providing future potential for use as programmable gene-integration tools. Early work has shown that guide RNA-directed transposition systems can be adapted to diverse hosts, even within microbial communities, suggesting great potential for engineering these systems as powerful gene-editing tools.
Assuntos
Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , RNA Guia de Sistemas CRISPR-Cas , Transposases , Elementos de DNA Transponíveis/genética , RNA Guia de Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas/metabolismo , Transposases/metabolismo , Transposases/genética , Edição de Genes/métodos , Bactérias/genética , Plasmídeos/metabolismo , Plasmídeos/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente EspaçadasRESUMO
The CD4-binding site (CD4bs) is a conserved epitope on HIV-1 envelope (Env) that can be targeted by protective broadly neutralizing antibodies (bnAbs). HIV-1 vaccines have not elicited CD4bs bnAbs for many reasons, including the occlusion of CD4bs by glycans, expansion of appropriate naive B cells with immunogens, and selection of functional antibody mutations. Here, we demonstrate that immunization of macaques with a CD4bs-targeting immunogen elicits neutralizing bnAb precursors with structural and genetic features of CD4-mimicking bnAbs. Structures of the CD4bs nAb bound to HIV-1 Env demonstrated binding angles and heavy-chain interactions characteristic of all known human CD4-mimicking bnAbs. Macaque nAb were derived from variable and joining gene segments orthologous to the genes of human VH1-46-class bnAb. This vaccine study initiated in primates the B cells from which CD4bs bnAbs can derive, accomplishing the key first step in the development of an effective HIV-1 vaccine.
Assuntos
Vacinas contra a AIDS , HIV-1 , Animais , Humanos , Anticorpos Amplamente Neutralizantes , Antígenos CD4 , Moléculas de Adesão Celular , HIV-1/fisiologia , Macaca , Vacinas contra a AIDS/imunologiaRESUMO
The insulin receptor (IR) is a type II receptor tyrosine kinase that plays essential roles in metabolism, growth, and proliferation. Dysregulation of IR signaling is linked to many human diseases, such as diabetes and cancers. The resolution revolution in cryo-electron microscopy has led to the determination of several structures of IR with different numbers of bound insulin molecules in recent years, which have tremendously improved our understanding of how IR is activated by insulin. Here, we review the insulin-induced activation mechanism of IR, including (a) the detailed binding modes and functions of insulin at site 1 and site 2 and (b) the insulin-induced structural transitions that are required for IR activation. We highlight several other key aspects of the activation and regulation of IR signaling and discuss the remaining gaps in our understanding of the IR activation mechanism and potential avenues of future research.
Assuntos
Insulina , Receptor de Insulina , Humanos , Receptor de Insulina/genética , Receptor de Insulina/química , Receptor de Insulina/metabolismo , Microscopia Crioeletrônica , Insulina/química , Insulina/metabolismo , Transdução de Sinais , Receptores Proteína Tirosina Quinases/metabolismo , FosforilaçãoRESUMO
It is unknown whether pangolins, the most trafficked mammals, play a role in the zoonotic transmission of bat coronaviruses. We report the circulation of a novel MERS-like coronavirus in Malayan pangolins, named Manis javanica HKU4-related coronavirus (MjHKU4r-CoV). Among 86 animals, four tested positive by pan-CoV PCR, and seven tested seropositive (11 and 12.8%). Four nearly identical (99.9%) genome sequences were obtained, and one virus was isolated (MjHKU4r-CoV-1). This virus utilizes human dipeptidyl peptidase-4 (hDPP4) as a receptor and host proteases for cell infection, which is enhanced by a furin cleavage site that is absent in all known bat HKU4r-CoVs. The MjHKU4r-CoV-1 spike shows higher binding affinity for hDPP4, and MjHKU4r-CoV-1 has a wider host range than bat HKU4-CoV. MjHKU4r-CoV-1 is infectious and pathogenic in human airways and intestinal organs and in hDPP4-transgenic mice. Our study highlights the importance of pangolins as reservoir hosts of coronaviruses poised for human disease emergence.
Assuntos
Infecções por Coronavirus , Coronavirus , Dipeptidil Peptidase 4 , Pangolins , Animais , Humanos , Camundongos , Quirópteros , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/virologia , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Endopeptidases/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Peptídeo Hidrolases/metabolismo , Receptores Virais/metabolismo , Internalização do Vírus , Coronavirus/fisiologiaRESUMO
The generation of distinct messenger RNA isoforms through alternative RNA processing modulates the expression and function of genes, often in a cell-type-specific manner. Here, we assess the regulatory relationships between transcription initiation, alternative splicing, and 3' end site selection. Applying long-read sequencing to accurately represent even the longest transcripts from end to end, we quantify mRNA isoforms in Drosophila tissues, including the transcriptionally complex nervous system. We find that in Drosophila heads, as well as in human cerebral organoids, 3' end site choice is globally influenced by the site of transcription initiation (TSS). "Dominant promoters," characterized by specific epigenetic signatures including p300/CBP binding, impose a transcriptional constraint to define splice and polyadenylation variants. In vivo deletion or overexpression of dominant promoters as well as p300/CBP loss disrupted the 3' end expression landscape. Our study demonstrates the crucial impact of TSS choice on the regulation of transcript diversity and tissue identity.
Assuntos
Processamento Alternativo , Isoformas de RNA , Sítio de Iniciação de Transcrição , Humanos , Poliadenilação , Regiões Promotoras Genéticas , Isoformas de RNA/metabolismo , RNA Mensageiro/metabolismoRESUMO
HIV-1-infected cells that persist despite antiretroviral therapy (ART) are frequently considered "transcriptionally silent," but active viral gene expression may occur in some cells, challenging the concept of viral latency. Applying an assay for profiling the transcriptional activity and the chromosomal locations of individual proviruses, we describe a global genomic and epigenetic map of transcriptionally active and silent proviral species and evaluate their longitudinal evolution in persons receiving suppressive ART. Using genome-wide epigenetic reference data, we show that proviral transcriptional activity is associated with activating epigenetic chromatin features in linear proximity of integration sites and in their inter- and intrachromosomal contact regions. Transcriptionally active proviruses were actively selected against during prolonged ART; however, this pattern was violated by large clones of virally infected cells that may outcompete negative selection forces through elevated intrinsic proliferative activity. Our results suggest that transcriptionally active proviruses are dynamically evolving under selection pressure by host factors.
Assuntos
HIV-1/genética , Provírus/genética , Transcrição Gênica , Idoso , Sequência de Bases , Evolução Biológica , Cromatina/metabolismo , Células Clonais , DNA Viral/genética , Epigênese Genética/efeitos dos fármacos , Feminino , Humanos , Ionomicina/farmacologia , Masculino , Pessoa de Meia-Idade , Filogenia , Provírus/efeitos dos fármacos , RNA Viral/genética , Acetato de Tetradecanoilforbol/farmacologia , Transcrição Gênica/efeitos dos fármacos , Integração Viral/genética , Latência Viral/efeitos dos fármacos , Latência Viral/genéticaRESUMO
Bacterial cytoplasmic membrane vesicles provide a unique experimental system for studying active transport. Vesicles are prepared by lysis of osmotically sensitized cells (i.e., protoplasts or spheroplasts) and comprise osmotically intact, unit-membrane-bound sacs that are approximately 0.5-1.0 µm in diameter and devoid of internal structure. Their metabolic activities are restricted to those provided by the enzymes of the membrane itself, and each vesicle is functional. The energy source for accumulation of a particular substrate can be determined by studying which compounds or experimental conditions drive solute accumulation, and metabolic conversion of the transported substrate or the energy source is minimal. These properties of the vesicle system constitute a considerable advantage over intact cells, as the system provides clear definition of the reactions involved in the transport process. This discussion is not intended as a general review but is concerned with respiration-dependent active transport in membrane vesicles from Escherichia coli. Emphasis is placed on experimental observations demonstrating that respiratory energy is converted primarily into work in the form of a solute concentration gradient that is driven by a proton electrochemical gradient, as postulated by the chemiosmotic theory of Peter Mitchell.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Escherichia coli/metabolismo , Biologia Molecular/história , Transporte Biológico , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Membrana Celular/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , História do Século XX , História do Século XXI , Humanos , Ácido Láctico/metabolismo , Masculino , Estados UnidosRESUMO
The N6-methyladenosine (m6A) RNA modification is used widely to alter the fate of mRNAs. Here we demonstrate that the C. elegans writer METT-10 (the ortholog of mouse METTL16) deposits an m6A mark on the 3' splice site (AG) of the S-adenosylmethionine (SAM) synthetase pre-mRNA, which inhibits its proper splicing and protein production. The mechanism is triggered by a rich diet and acts as an m6A-mediated switch to stop SAM production and regulate its homeostasis. Although the mammalian SAM synthetase pre-mRNA is not regulated via this mechanism, we show that splicing inhibition by 3' splice site m6A is conserved in mammals. The modification functions by physically preventing the essential splicing factor U2AF35 from recognizing the 3' splice site. We propose that use of splice-site m6A is an ancient mechanism for splicing regulation.
Assuntos
Adenosina/análogos & derivados , Sítios de Splice de RNA/genética , Splicing de RNA/genética , Fator de Processamento U2AF/metabolismo , Adenosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Sequência Conservada/genética , Dieta , Células HeLa , Humanos , Íntrons/genética , Metionina Adenosiltransferase , Metilação , Metiltransferases/química , Camundongos , Mutação/genética , Conformação de Ácido Nucleico , Ligação Proteica , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Nuclear Pequeno , S-Adenosilmetionina , Transcriptoma/genéticaRESUMO
Chromosome loops shift dynamically during development, homeostasis, and disease. CCCTC-binding factor (CTCF) is known to anchor loops and construct 3D genomes, but how anchor sites are selected is not yet understood. Here, we unveil Jpx RNA as a determinant of anchor selectivity. Jpx RNA targets thousands of genomic sites, preferentially binding promoters of active genes. Depleting Jpx RNA causes ectopic CTCF binding, massive shifts in chromosome looping, and downregulation of >700 Jpx target genes. Without Jpx, thousands of lost loops are replaced by de novo loops anchored by ectopic CTCF sites. Although Jpx controls CTCF binding on a genome-wide basis, it acts selectively at the subset of developmentally sensitive CTCF sites. Specifically, Jpx targets low-affinity CTCF motifs and displaces CTCF protein through competitive inhibition. We conclude that Jpx acts as a CTCF release factor and shapes the 3D genome by regulating anchor site usage.
Assuntos
Fator de Ligação a CCCTC/metabolismo , Cromossomos/metabolismo , RNA Longo não Codificante/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias , Camundongos , Ligação ProteicaRESUMO
Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3' extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5' end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP.
Assuntos
Vírus da Influenza A/genética , Transcrição Gênica/genética , Replicação Viral/genética , Domínio Catalítico , Simulação por Computador , Microscopia Crioeletrônica/métodos , Genoma Viral/genética , Humanos , Vírus da Influenza A/metabolismo , Influenza Humana/genética , Influenza Humana/virologia , Nucleotidiltransferases/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Relação Estrutura-AtividadeRESUMO
Autophagy is a conserved catabolic homeostasis process central for cellular and organismal health. During autophagy, small single-membrane phagophores rapidly expand into large double-membrane autophagosomes to encapsulate diverse cargoes for degradation. It is thought that autophagic membranes are mainly derived from preformed organelle membranes. Instead, here we delineate a pathway that expands the phagophore membrane by localized phospholipid synthesis. Specifically, we find that the conserved acyl-CoA synthetase Faa1 accumulates on nucleated phagophores and locally activates fatty acids (FAs) required for phagophore elongation and autophagy. Strikingly, using isotopic FA tracing, we directly show that Faa1 channels activated FAs into the synthesis of phospholipids and promotes their assembly into autophagic membranes. Indeed, the first committed steps of de novo phospholipid synthesis at the ER, which forms stable contacts with nascent autophagosomes, are essential for autophagy. Together, our work illuminates how cells spatially tune synthesis and flux of phospholipids for autophagosome biogenesis during autophagy.
Assuntos
Autofagia/fisiologia , Ácidos Graxos/metabolismo , Fagossomos/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Coenzima A Ligases/metabolismo , Retículo Endoplasmático/metabolismo , Metabolismo dos Lipídeos , Proteínas de Membrana/metabolismo , Fagossomos/fisiologia , Fosfolipídeos/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Broadly neutralizing antibodies (bNAbs) represent a promising approach to prevent and treat HIV-1 infection. However, viral escape through mutation of the HIV-1 envelope glycoprotein (Env) limits clinical applications. Here we describe 1-18, a new VH1-46-encoded CD4 binding site (CD4bs) bNAb with outstanding breadth (97%) and potency (GeoMean IC50 = 0.048 µg/mL). Notably, 1-18 is not susceptible to typical CD4bs escape mutations and effectively overcomes HIV-1 resistance to other CD4bs bNAbs. Moreover, mutational antigenic profiling uncovered restricted pathways of HIV-1 escape. Of most promise for therapeutic use, even 1-18 alone fully suppressed viremia in HIV-1-infected humanized mice without selecting for resistant viral variants. A 2.5-Å cryo-EM structure of a 1-18-BG505SOSIP.664 Env complex revealed that these characteristics are likely facilitated by a heavy-chain insertion and increased inter-protomer contacts. The ability of 1-18 to effectively restrict HIV-1 escape pathways provides a new option to successfully prevent and treat HIV-1 infection.
Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Antígenos CD4/metabolismo , Células CHO , Estudos de Coortes , Cricetulus , Epitopos/imunologia , Feminino , Células HEK293 , Infecções por HIV/prevenção & controle , Infecções por HIV/virologia , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Mutação , Ligação Proteica/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
During autophagy, vesicle dynamics and cargo recruitment are driven by numerous adaptors and receptors that become tethered to the phagophore through interactions with lipidated ATG8/LC3 decorating the expanding membrane. Most currently described ATG8-binding proteins exploit a well-defined ATG8-interacting motif (AIM, or LC3-interacting region [LIR]) that contacts a hydrophobic patch on ATG8 known as the LIR/AIM docking site (LDS). Here we describe a new class of ATG8 interactors that exploit ubiquitin-interacting motif (UIM)-like sequences for high-affinity binding to an alternative ATG8 interaction site. Assays with candidate UIM-containing proteins together with unbiased screens identified a large collection of UIM-based ATG8 interactors in plants, yeast, and humans. Analysis of a subset also harboring ubiquitin regulatory X (UBX) domains revealed a role for UIM-directed autophagy in clearing non-functional CDC48/p97 complexes, including some impaired in human disease. With this new class of adaptors and receptors, we greatly extend the reach of selective autophagy and identify new factors regulating autophagic vesicle dynamics.
Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Família da Proteína 8 Relacionada à Autofagia/química , Sítios de Ligação , Humanos , Proteínas Associadas aos Microtúbulos/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de SequênciaRESUMO
Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modeling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighboring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations.
Assuntos
Processamento Alternativo/genética , Splicing de RNA/genética , Receptor fas/genética , Animais , Éxons/genética , Técnicas Genéticas , Genética , Genótipo , Humanos , Íntrons/genética , Camundongos , Modelos Teóricos , Mutação/genética , Fenótipo , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Mensageiro/metabolismoRESUMO
To maintain an asymmetric distribution of ions across membranes, protein pumps displace ions against their concentration gradient by using chemical energy. Here, we describe a functionally analogous but topologically opposite process that applies to the lipid transfer protein (LTP) oxysterol-binding protein (OSBP). This multidomain protein exchanges cholesterol for the phosphoinositide phosphatidylinositol 4-phosphate [PI(4)P] between two apposed membranes. Because of the subsequent hydrolysis of PI(4)P, this counterexchange is irreversible and contributes to the establishment of a cholesterol gradient along organelles of the secretory pathway. The facts that some natural anti-cancer molecules block OSBP and that many viruses hijack the OSBP cycle for the formation of intracellular replication organelles highlight the importance and potency of OSBP-mediated lipid exchange. The architecture of some LTPs is similar to that of OSBP, suggesting that the principles of the OSBP cycle-burning PI(4)P for the vectorial transfer of another lipid-might be general.
Assuntos
Colesterol/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Receptores de Esteroides/metabolismo , Transporte Biológico Ativo , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Humanos , Ligantes , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Modelos Moleculares , Oxisteróis/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores de Esteroides/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicação Viral/fisiologiaRESUMO
Continuously evolving influenza viruses cause seasonal epidemics and pose global pandemic threats. Although viral neuraminidase (NA) is an effective drug and vaccine target, our understanding of the NA antigenic landscape still remains incomplete. Here, we describe NA-specific human antibodies that target the underside of the NA globular head domain, inhibit viral propagation of a wide range of human H3N2, swine-origin variant H3N2, and H2N2 viruses, and confer both pre- and post-exposure protection against lethal H3N2 infection in mice. Cryo-EM structures of two such antibodies in complex with NA reveal non-overlapping epitopes covering the underside of the NA head. These sites are highly conserved among N2 NAs yet inaccessible unless the NA head tilts or dissociates. Our findings help guide the development of effective countermeasures against ever-changing influenza viruses by identifying hidden conserved sites of vulnerability on the NA underside.
Assuntos
Vacinas contra Influenza , Influenza Humana , Infecções por Orthomyxoviridae , Humanos , Animais , Camundongos , Suínos , Proteínas Virais/genética , Neuraminidase , Vírus da Influenza A Subtipo H3N2 , Anticorpos Monoclonais , Anticorpos AntiviraisRESUMO
Endoplasmic reticulum (ER) membrane contact sites (MCSs) mark positions where endosomes undergo fission for cargo sorting. To define the role of ER at this unique MCS, we targeted a promiscuous biotin ligase to cargo-sorting domains on endosome buds. This strategy identified the ER membrane protein TMCC1, a member of a conserved protein family. TMCC1 concentrates at the ER-endosome MCSs that are spatially and temporally linked to endosome fission. When TMCC1 is depleted, endosome morphology is normal, buds still form, but ER-associated bud fission and subsequent cargo sorting to the Golgi are impaired. We find that the endosome-localized actin regulator Coronin 1C is required for ER-associated fission of actin-dependent cargo-sorting domains. Coronin 1C is recruited to endosome buds independently of TMCC1, while TMCC1/ER recruitment requires Coronin 1C. This link between TMCC1 and Coronin 1C suggests that the timing of TMCC1-dependent ER recruitment is tightly regulated to occur after cargo has been properly sequestered into the bud.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Animais , Células COS , Canais de Cálcio , Chlorocebus aethiops , Retículo Endoplasmático/fisiologia , Endossomos/fisiologia , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/fisiologia , Microtúbulos/metabolismo , Transporte Proteico/fisiologiaRESUMO
Nonalcoholic fatty liver disease (NAFLD) progresses to nonalcoholic steatohepatitis (NASH) in response to elevated endoplasmic reticulum (ER) stress. Whereas the onset of simple steatosis requires elevated de novo lipogenesis, progression to NASH is triggered by accumulation of hepatocyte-free cholesterol. We now show that caspase-2, whose expression is ER-stress inducible and elevated in human and mouse NASH, controls the buildup of hepatic-free cholesterol and triglycerides by activating sterol regulatory element-binding proteins (SREBP) in a manner refractory to feedback inhibition. Caspase-2 colocalizes with site 1 protease (S1P) and cleaves it to generate a soluble active fragment that initiates SCAP-independent SREBP1/2 activation in the ER. Caspase-2 ablation or pharmacological inhibition prevents diet-induced steatosis and NASH progression in ER-stress-prone mice. Caspase-2 inhibition offers a specific and effective strategy for preventing or treating stress-driven fatty liver diseases, whereas caspase-2-generated S1P proteolytic fragments, which enter the secretory pathway, are potential NASH biomarkers.
Assuntos
Caspase 2/fisiologia , Lipogênese/fisiologia , Pró-Proteína Convertases/fisiologia , Serina Endopeptidases/fisiologia , Animais , Colesterol/metabolismo , Retículo Endoplasmático/fisiologia , Estresse do Retículo Endoplasmático/fisiologia , Fígado Gorduroso/fisiopatologia , Células HEK293 , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pró-Proteína Convertases/metabolismo , Serina Endopeptidases/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Triglicerídeos/metabolismoRESUMO
Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs-a long ncRNA, a circular RNA, and two microRNAs-using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.
Assuntos
Encéfalo/metabolismo , Redes Reguladoras de Genes , RNA não Traduzido/metabolismo , Animais , Citoplasma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Metabolite-protein interactions control a variety of cellular processes, thereby playing a major role in maintaining cellular homeostasis. Metabolites comprise the largest fraction of molecules in cells, but our knowledge of the metabolite-protein interactome lags behind our understanding of protein-protein or protein-DNA interactomes. Here, we present a chemoproteomic workflow for the systematic identification of metabolite-protein interactions directly in their native environment. The approach identified a network of known and novel interactions and binding sites in Escherichia coli, and we demonstrated the functional relevance of a number of newly identified interactions. Our data enabled identification of new enzyme-substrate relationships and cases of metabolite-induced remodeling of protein complexes. Our metabolite-protein interactome consists of 1,678 interactions and 7,345 putative binding sites. Our data reveal functional and structural principles of chemical communication, shed light on the prevalence and mechanisms of enzyme promiscuity, and enable extraction of quantitative parameters of metabolite binding on a proteome-wide scale.