RESUMO
Ubiquitin-dependent unfolding of the CMG helicase by VCP/p97 is required to terminate DNA replication. Other replisome components are not processed in the same fashion, suggesting that additional mechanisms underlie replication protein turnover. Here, we identify replisome factor interactions with a protein complex composed of AAA+ ATPases SPATA5-SPATA5L1 together with heterodimeric partners C1orf109-CINP (55LCC). An integrative structural biology approach revealed a molecular architecture of SPATA5-SPATA5L1 N-terminal domains interacting with C1orf109-CINP to form a funnel-like structure above a cylindrically shaped ATPase motor. Deficiency in the 55LCC complex elicited ubiquitin-independent proteotoxicity, replication stress, and severe chromosome instability. 55LCC showed ATPase activity that was specifically enhanced by replication fork DNA and was coupled to cysteine protease-dependent cleavage of replisome substrates in response to replication fork damage. These findings define 55LCC-mediated proteostasis as critical for replication fork progression and genome stability and provide a rationale for pathogenic variants seen in associated human neurodevelopmental disorders.
Assuntos
Adenosina Trifosfatases , Replicação do DNA , Instabilidade Genômica , Proteostase , Humanos , Adenosina Trifosfatases/metabolismo , Proteína com Valosina/metabolismo , Proteína com Valosina/genética , Células HEK293 , Proteínas de Ciclo Celular/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genéticaRESUMO
Transcription factors (TFs) regulate gene programs, thereby controlling diverse cellular processes and cell states. To comprehensively understand TFs and the programs they control, we created a barcoded library of all annotated human TF splice isoforms (>3,500) and applied it to build a TF Atlas charting expression profiles of human embryonic stem cells (hESCs) overexpressing each TF at single-cell resolution. We mapped TF-induced expression profiles to reference cell types and validated candidate TFs for generation of diverse cell types, spanning all three germ layers and trophoblasts. Targeted screens with subsets of the library allowed us to create a tailored cellular disease model and integrate mRNA expression and chromatin accessibility data to identify downstream regulators. Finally, we characterized the effects of combinatorial TF overexpression by developing and validating a strategy for predicting combinations of TFs that produce target expression profiles matching reference cell types to accelerate cellular engineering efforts.
Assuntos
Diferenciação Celular , Fatores de Transcrição , Humanos , Cromatina , Regulação da Expressão Gênica , Células-Tronco Embrionárias Humanas/metabolismo , Fatores de Transcrição/metabolismo , Atlas como AssuntoRESUMO
T cell-mediated immunity plays an important role in controlling SARS-CoV-2 infection, but the repertoire of naturally processed and presented viral epitopes on class I human leukocyte antigen (HLA-I) remains uncharacterized. Here, we report the first HLA-I immunopeptidome of SARS-CoV-2 in two cell lines at different times post infection using mass spectrometry. We found HLA-I peptides derived not only from canonical open reading frames (ORFs) but also from internal out-of-frame ORFs in spike and nucleocapsid not captured by current vaccines. Some peptides from out-of-frame ORFs elicited T cell responses in a humanized mouse model and individuals with COVID-19 that exceeded responses to canonical peptides, including some of the strongest epitopes reported to date. Whole-proteome analysis of infected cells revealed that early expressed viral proteins contribute more to HLA-I presentation and immunogenicity. These biological insights, as well as the discovery of out-of-frame ORF epitopes, will facilitate selection of peptides for immune monitoring and vaccine development.
Assuntos
Epitopos de Linfócito T/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Fases de Leitura Aberta/genética , Peptídeos/imunologia , Proteoma/imunologia , SARS-CoV-2/imunologia , Células A549 , Alelos , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , COVID-19/imunologia , COVID-19/virologia , Feminino , Células HEK293 , Humanos , Cinética , Masculino , Camundongos , Peptídeos/química , Linfócitos T/imunologiaRESUMO
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.
Assuntos
Proteína C9orf72/metabolismo , Expansão das Repetições de DNA/genética , Degeneração Neural/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Axônios/metabolismo , Proteína C9orf72/genética , Morte Celular , Células Cultivadas , Córtex Cerebral/patologia , Cromatina/metabolismo , Dano ao DNA , Modelos Animais de Doenças , Drosophila , Camundongos Endogâmicos C57BL , Degeneração Neural/patologia , Estabilidade Proteica , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Espectrometria de Massas/métodos , Enzimas Multifuncionais/genética , Fosforilação , Proteínas/genética , Nucleotídeos de Purina/metabolismo , Purinas/metabolismoRESUMO
Gene expression in human tissue has primarily been studied on the transcriptional level, largely neglecting translational regulation. Here, we analyze the translatomes of 80 human hearts to identify new translation events and quantify the effect of translational regulation. We show extensive translational control of cardiac gene expression, which is orchestrated in a process-specific manner. Translation downstream of predicted disease-causing protein-truncating variants appears to be frequent, suggesting inefficient translation termination. We identify hundreds of previously undetected microproteins, expressed from lncRNAs and circRNAs, for which we validate the protein products in vivo. The translation of microproteins is not restricted to the heart and prominent in the translatomes of human kidney and liver. We associate these microproteins with diverse cellular processes and compartments and find that many locate to the mitochondria. Importantly, dozens of microproteins are translated from lncRNAs with well-characterized noncoding functions, indicating previously unrecognized biology.
Assuntos
Miocárdio/metabolismo , Biossíntese de Proteínas , Adolescente , Adulto , Idoso , Animais , Códon/genética , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fases de Leitura Aberta/genética , RNA Circular/genética , RNA Circular/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ribossomos/genética , Ribossomos/metabolismo , Adulto JovemRESUMO
Defects in nucleocytoplasmic transport have been identified as a key pathogenic event in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) mediated by a GGGGCC hexanucleotide repeat expansion in C9ORF72, the most common genetic cause of ALS/FTD. Furthermore, nucleocytoplasmic transport disruption has also been implicated in other neurodegenerative diseases with protein aggregation, suggesting a shared mechanism by which protein stress disrupts nucleocytoplasmic transport. Here, we show that cellular stress disrupts nucleocytoplasmic transport by localizing critical nucleocytoplasmic transport factors into stress granules, RNA/protein complexes that play a crucial role in ALS pathogenesis. Importantly, inhibiting stress granule assembly, such as by knocking down Ataxin-2, suppresses nucleocytoplasmic transport defects as well as neurodegeneration in C9ORF72-mediated ALS/FTD. Our findings identify a link between stress granule assembly and nucleocytoplasmic transport, two fundamental cellular processes implicated in the pathogenesis of C9ORF72-mediated ALS/FTD and other neurodegenerative diseases.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Esclerose Lateral Amiotrófica/patologia , Ataxina-2/metabolismo , Proteína C9orf72/genética , Demência Frontotemporal/patologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Idoso , Esclerose Lateral Amiotrófica/metabolismo , Arsenitos/toxicidade , Ataxina-2/antagonistas & inibidores , Ataxina-2/genética , Proteína C9orf72/metabolismo , Expansão das Repetições de DNA/genética , Feminino , Demência Frontotemporal/metabolismo , Células HEK293 , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Compostos de Sódio/toxicidade , alfa Carioferinas/antagonistas & inibidores , alfa Carioferinas/genética , alfa Carioferinas/metabolismo , beta Carioferinas/antagonistas & inibidores , beta Carioferinas/genética , beta Carioferinas/metabolismo , Proteína ran de Ligação ao GTP/antagonistas & inibidores , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
LINE-1 retrotransposition is tightly restricted by layers of regulatory control, with epigenetic pathways being the best characterized. Looking at post-transcriptional regulation, we now show that LINE-1 mRNA 3' ends are pervasively uridylated in various human cellular models and in mouse testes. TUT4 and TUT7 uridyltransferases catalyze the modification and function in cooperation with the helicase/RNPase MOV10 to counteract the RNA chaperone activity of the L1-ORF1p retrotransposon protein. Uridylation potently restricts LINE-1 retrotransposition by a multilayer mechanism depending on differential subcellular localization of the uridyltransferases. We propose that uridine residues added by TUT7 in the cytoplasm inhibit initiation of reverse transcription of LINE-1 mRNAs once they are reimported to the nucleus, whereas uridylation by TUT4, which is enriched in cytoplasmic foci, destabilizes mRNAs. These results provide a model for the post-transcriptional restriction of LINE-1, revealing a key physiological role for TUT4/7-mediated uridylation in maintaining genome stability.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , RNA Nucleotidiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Uridina/metabolismo , Animais , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Células HEK293 , Humanos , Camundongos , Proteínas Nucleares/genética , Ligação Proteica , RNA Helicases/antagonistas & inibidores , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , RNA Nucleotidiltransferases/antagonistas & inibidores , RNA Nucleotidiltransferases/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/genética , Retroelementos/genéticaRESUMO
Genomics has provided a detailed structural description of the cancer genome. Identifying oncogenic drivers that work primarily through dosage changes is a current challenge. Unrestrained proliferation is a critical hallmark of cancer. We constructed modular, barcoded libraries of human open reading frames (ORFs) and performed screens for proliferation regulators in multiple cell types. Approximately 10% of genes regulate proliferation, with most performing in an unexpectedly highly tissue-specific manner. Proliferation drivers in a given cell type showed specific enrichment in somatic copy number changes (SCNAs) from cognate tumors and helped predict aneuploidy patterns in those tumors, implying that tissue-type-specific genetic network architectures underlie SCNA and driver selection in different cancers. In vivo screening confirmed these results. We report a substantial contribution to the catalog of SCNA-associated cancer drivers, identifying 147 amplified and 107 deleted genes as potential drivers, and derive insights about the genetic network architecture of aneuploidy in tumors.
Assuntos
Aneuploidia , Neoplasias/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Mapeamento Cromossômico , Cromossomos/genética , Fator de Transcrição E2F1/antagonistas & inibidores , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Feminino , Biblioteca Gênica , Genômica , Humanos , Queratinas/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Oncogenes , Fases de Leitura Aberta/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Eukaryotic translation is tightly regulated to ensure that protein production occurs at the right time and place. Recent studies on abnormal repeat proteins, especially in age-dependent neurodegenerative diseases caused by nucleotide repeat expansion, have highlighted or identified two forms of unconventional translation initiation: usage of AUG-like sites (near cognates) or repeat-associated non-AUG (RAN) translation. We discuss how repeat proteins may differ due to not just unconventional initiation, but also ribosomal frameshifting and/or imperfect repeat DNA replication, expansion, and repair, and we highlight how research on translation of repeats may uncover insights into the biology of translation and its contribution to disease.
Assuntos
Doenças Neurodegenerativas/genética , Biossíntese de Proteínas , Animais , Códon de Iniciação , Mudança da Fase de Leitura do Gene Ribossômico , Humanos , Doenças Neurodegenerativas/metabolismo , Fases de Leitura Aberta , Sequências Reguladoras de Ácido Ribonucleico , Expansão das Repetições de TrinucleotídeosRESUMO
Synaptic function in neurons is modulated by local translation of mRNAs that are transported to distal portions of axons and dendrites. The metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is broadly expressed across cell types, almost exclusively as a nuclear long noncoding RNA. We found that in differentiating neurons, a portion of Malat1 RNA redistributes to the cytoplasm. Depletion of Malat1 using antisense oligonucleotides (ASOs) stimulates the expression of particular pre- and postsynaptic proteins, implicating Malat1 in their regulation. Neuronal Malat1 is localized in puncta of both axons and dendrites that costain with Staufen1 protein, similar to neuronal RNA granules formed by locally translated mRNAs. Ribosome profiling of cultured mouse cortical neurons identified ribosome footprints within a 5' region of Malat1 containing short open reading frames. The upstream-most reading frame (M1) of the Malat1 locus was linked to the GFP-coding sequence in mouse embryonic stem cells. When these gene-edited cells were differentiated into glutamatergic neurons, the M1-GFP fusion protein was expressed. Antibody staining for the M1 peptide confirmed its presence in wild-type neurons and showed that M1 expression was enhanced by synaptic stimulation with KCl. Our results indicate that Malat1 serves as a cytoplasmic coding RNA in the brain that is both modulated by and modulates synaptic function.
Assuntos
Citoplasma , Neurônios , RNA Longo não Codificante , RNA Mensageiro , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Camundongos , Neurônios/metabolismo , Citoplasma/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Células Cultivadas , Diferenciação Celular , Peptídeos/metabolismo , Peptídeos/genéticaRESUMO
Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Proteína C9orf72 , Nucléolo Celular/metabolismo , Grânulos Citoplasmáticos/metabolismo , Expansão das Repetições de DNA , Dipeptídeos/genética , Drosophila melanogaster/genética , Demência Frontotemporal/genética , Humanos , Membranas Intracelulares/metabolismo , Poro Nuclear/metabolismo , Peptídeos/genética , Peptídeos/metabolismo , Proteínas/genéticaRESUMO
Mitochondrial ribosomes translate membrane integral core subunits of the oxidative phosphorylation system encoded by mtDNA. These translation products associate with nuclear-encoded, imported proteins to form enzyme complexes that produce ATP. Here, we show that human mitochondrial ribosomes display translational plasticity to cope with the supply of imported nuclear-encoded subunits. Ribosomes expressing mitochondrial-encoded COX1 mRNA selectively engage with cytochrome c oxidase assembly factors in the inner membrane. Assembly defects of the cytochrome c oxidase arrest mitochondrial translation in a ribosome nascent chain complex with a partially membrane-inserted COX1 translation product. This complex represents a primed state of the translation product that can be retrieved for assembly. These findings establish a mammalian translational plasticity pathway in mitochondria that enables adaptation of mitochondrial protein synthesis to the influx of nuclear-encoded subunits.
Assuntos
Ciclo-Oxigenase 1/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Membrana/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , DNA Mitocondrial/genética , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Fosforilação Oxidativa , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , RNA Mitocondrial , Ribossomos/metabolismoRESUMO
Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/metabolismo , Peptídeos/metabolismo , Proteínas/metabolismo , Esclerose Lateral Amiotrófica/genética , Proteína C9orf72 , Expansão das Repetições de DNA , Dipeptídeos/química , Dipeptídeos/genética , Demência Frontotemporal/genética , Células HeLa , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Peptídeos/química , Peptídeos/genética , Domínios Proteicos , Proteínas/genéticaRESUMO
Dissecting the evolution of memory B cells (MBCs) against SARS-CoV-2 is critical for understanding antibody recall upon secondary exposure. Here, we used single-cell sequencing to profile SARS-CoV-2-reactive B cells in 38 COVID-19 patients. Using oligo-tagged antigen baits, we isolated B cells specific to the SARS-CoV-2 spike, nucleoprotein (NP), open reading frame 8 (ORF8), and endemic human coronavirus (HCoV) spike proteins. SARS-CoV-2 spike-specific cells were enriched in the memory compartment of acutely infected and convalescent patients several months post symptom onset. With severe acute infection, substantial populations of endemic HCoV-reactive antibody-secreting cells were identified and possessed highly mutated variable genes, signifying preexisting immunity. Finally, MBCs exhibited pronounced maturation to NP and ORF8 over time, especially in older patients. Monoclonal antibodies against these targets were non-neutralizing and non-protective in vivo. These findings reveal antibody adaptation to non-neutralizing intracellular antigens during infection, emphasizing the importance of vaccination for inducing neutralizing spike-specific MBCs.
Assuntos
Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , COVID-19/imunologia , Interações Hospedeiro-Patógeno/imunologia , Epitopos Imunodominantes/imunologia , SARS-CoV-2/imunologia , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/genética , Linfócitos B/metabolismo , Biologia Computacional/métodos , Reações Cruzadas/imunologia , Mapeamento de Epitopos , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Interações Hospedeiro-Patógeno/genética , Humanos , Epitopos Imunodominantes/genética , Memória Imunológica , Masculino , Testes de Neutralização , Análise de Célula Única/métodos , Glicoproteína da Espícula de Coronavírus/imunologia , TranscriptomaRESUMO
DNA-induced liquid-liquid phase separation of cyclic GMP-AMP synthase (cGAS) triggers a potent response to detect pathogen infection and promote innate immune signaling. Whether and how pathogens manipulate cGAS-DNA condensation to mediate immune evasion is unknown. We report the identification of a structurally related viral tegument protein family, represented by ORF52 and VP22 from gamma- and alpha-herpesvirinae, respectively, that employs a conserved mechanism to restrict cGAS-DNA phase separation. ORF52/VP22 proteins accumulate into, and effectively disrupt, the pre-formed cGAS-DNA condensation both in vitro and in cells. The inhibition process is dependent on DNA-induced liquid-liquid phase separation of the viral protein rather than a direct interaction with cGAS. Moreover, highly abundant ORF52 proteins carried within viral particles are able to target cGAS-DNA phase separation in early infection stage. Our results define ORF52/VP22-type tegument proteins as a family of inhibitors targeting cGAS-DNA phase separation and demonstrate a mechanism for how viruses overcome innate immunity.
Assuntos
Alphaherpesvirinae , Betaherpesvirinae , DNA , Infecções por Herpesviridae , Evasão da Resposta Imune , Nucleotidiltransferases , Proteínas Estruturais Virais , Alphaherpesvirinae/química , Alphaherpesvirinae/genética , Alphaherpesvirinae/imunologia , Betaherpesvirinae/química , Betaherpesvirinae/genética , Betaherpesvirinae/imunologia , DNA/química , DNA/genética , DNA/imunologia , Células HEK293 , Células HeLa , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/imunologia , Humanos , Imunidade Inata , Nucleotidiltransferases/química , Nucleotidiltransferases/genética , Nucleotidiltransferases/imunologia , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/imunologiaRESUMO
A deficient interferon (IFN) response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has been implicated as a determinant of severe coronavirus disease 2019 (COVID-19). To identify the molecular effectors that govern IFN control of SARS-CoV-2 infection, we conducted a large-scale gain-of-function analysis that evaluated the impact of human IFN-stimulated genes (ISGs) on viral replication. A limited subset of ISGs were found to control viral infection, including endosomal factors inhibiting viral entry, RNA binding proteins suppressing viral RNA synthesis, and a highly enriched cluster of endoplasmic reticulum (ER)/Golgi-resident ISGs inhibiting viral assembly/egress. These included broad-acting antiviral ISGs and eight ISGs that specifically inhibited SARS-CoV-2 and SARS-CoV-1 replication. Among the broad-acting ISGs was BST2/tetherin, which impeded viral release and is antagonized by SARS-CoV-2 Orf7a protein. Overall, these data illuminate a set of ISGs that underlie innate immune control of SARS-CoV-2/SARS-CoV-1 infection, which will facilitate the understanding of host determinants that impact disease severity and offer potential therapeutic strategies for COVID-19.
Assuntos
Antígenos CD/genética , Interações Hospedeiro-Patógeno/genética , Fatores Reguladores de Interferon/genética , Interferon Tipo I/genética , SARS-CoV-2/genética , Proteínas Virais/genética , Animais , Antígenos CD/química , Antígenos CD/imunologia , Sítios de Ligação , Linhagem Celular Tumoral , Chlorocebus aethiops , Retículo Endoplasmático/genética , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/virologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/imunologia , Regulação da Expressão Gênica , Complexo de Golgi/genética , Complexo de Golgi/imunologia , Complexo de Golgi/virologia , Células HEK293 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunidade Inata , Fatores Reguladores de Interferon/classificação , Fatores Reguladores de Interferon/imunologia , Interferon Tipo I/imunologia , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/imunologia , Transdução de Sinais , Células Vero , Proteínas Virais/química , Proteínas Virais/imunologia , Internalização do Vírus , Liberação de Vírus/genética , Liberação de Vírus/imunologia , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
Initiation is the rate-limiting step in translation, and its dysregulation is vital for carcinogenesis, including hematopoietic malignancy. Thus, discovery of novel translation initiation regulators may provide promising therapeutic targets. Here, combining Ribo-seq, mass spectrometry, and RNA-seq datasets, we discovered an oncomicropeptide, APPLE (a peptide located in ER), encoded by a non-coding RNA transcript in acute myeloid leukemia (AML). APPLE is overexpressed in various subtypes of AML and confers a poor prognosis. The micropeptide is enriched in ribosomes and regulates the initiation step to enhance translation and to maintain high rates of oncoprotein synthesis. Mechanically, APPLE promotes PABPC1-eIF4G interaction and facilitates mRNA circularization and eIF4F initiation complex assembly to support a specific pro-cancer translation program. Targeting APPLE exhibited broad anti-cancer effects in vitro and in vivo. This study not only reports a previously unknown function of micropeptides but also provides new opportunities for targeting the translation machinery in cancer cells.
Assuntos
Fator de Iniciação 4F em Eucariotos/química , Fator de Iniciação Eucariótico 4G/metabolismo , Neoplasias Hematológicas/metabolismo , Peptídeos/química , Biossíntese de Proteínas , Animais , Progressão da Doença , Genoma Humano , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fases de Leitura Aberta , Polirribossomos/química , RNA Mensageiro/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Ribossomos/metabolismo , Sensibilidade e Especificidade , Resultado do TratamentoRESUMO
DNA repair through homologous recombination (HR) is of vital importance for maintaining genome stability and preventing tumorigenesis. RAD51 is the core component of HR, catalyzing the strand invasion and homology search. Here, we highlight recent findings on FIRRM and FIGNL1 as regulators of the dynamics of RAD51.
Assuntos
Recombinação Homóloga , Rad51 Recombinase , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Humanos , Reparo do DNA/genética , Instabilidade Genômica/genética , AnimaisRESUMO
Lipid droplets (LDs) form inter-organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance ß-oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria-LD and ER-LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER-LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production.