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1.
Mol Cell ; 2024 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-39013473

RESUMO

The human silencing hub (HUSH) preserves genome integrity through the epigenetic repression of invasive genetic elements. However, despite our understanding of HUSH as an obligate complex of three subunits, only loss of MPP8 or Periphilin, but not TASOR, triggers interferon signaling following derepression of endogenous retroelements. Here, we resolve this paradox by characterizing a second HUSH complex that shares MPP8 and Periphilin but assembles around TASOR2, an uncharacterized paralog of TASOR. Whereas HUSH represses LINE-1 retroelements marked by the repressive histone modification H3K9me3, HUSH2 is recruited by the transcription factor IRF2 to repress interferon-stimulated genes. Mechanistically, HUSH-mediated retroelement silencing sequesters the limited pool of the shared subunits MPP8 and Periphilin, preventing TASOR2 from forming HUSH2 complexes and hence relieving the HUSH2-mediated repression of interferon-stimulated genes. Thus, competition between two HUSH complexes intertwines retroelement silencing with the induction of an immune response, coupling epigenetic and immune aspects of genome defense.

3.
Nat Cell Biol ; 25(10): 1535-1545, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37735597

RESUMO

Specificity within the ubiquitin-proteasome system is primarily achieved through E3 ubiquitin ligases, but for many E3s their substrates-and in particular the molecular features (degrons) that they recognize-remain largely unknown. Current approaches for assigning E3s to their cognate substrates are tedious and low throughput. Here we developed a multiplex CRISPR screening platform to assign E3 ligases to their cognate substrates at scale. A proof-of-principle multiplex screen successfully performed ~100 CRISPR screens in a single experiment, refining known C-degron pathways and identifying an additional pathway through which Cul2FEM1B targets C-terminal proline. Further, by identifying substrates for Cul1FBXO38, Cul2APPBP2, Cul3GAN, Cul3KLHL8, Cul3KLHL9/13 and Cul3KLHL15, we demonstrate that the approach is compatible with pools of full-length protein substrates of varying stabilities and, when combined with site-saturation mutagenesis, can assign E3 ligases to their cognate degron motifs. Thus, multiplex CRISPR screening will accelerate our understanding of how specificity is achieved within the ubiquitin-proteasome system.


Assuntos
Complexo de Endopeptidases do Proteassoma , Ubiquitina-Proteína Ligases , Ubiquitina-Proteína Ligases/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Ubiquitina/genética , Ubiquitina/metabolismo
4.
Mol Cell ; 82(2): 479-491.e7, 2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-34963054

RESUMO

Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.


Assuntos
Técnicas Biossensoriais , Epigênese Genética , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Lentivirus/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Montagem e Desmontagem da Cromatina , Epigenoma , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Lentivirus/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
5.
Nat Commun ; 11(1): 4940, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009411

RESUMO

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.


Assuntos
Elementos de DNA Transponíveis/genética , Epigênese Genética , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Sequência de Aminoácidos , Antígenos de Neoplasias/metabolismo , Sítios de Ligação , Éxons/genética , Genoma , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Lisina/metabolismo , Espectroscopia de Ressonância Magnética , Metilação , NAD/metabolismo , Proteínas Nucleares/química , Fosfoproteínas/metabolismo , Ligação Proteica , Domínios Proteicos , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Transcrição Gênica
6.
Nucleic Acids Res ; 48(18): 10313-10328, 2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-32976585

RESUMO

Transcription of integrated DNA from viruses or transposable elements is tightly regulated to prevent pathogenesis. The Human Silencing Hub (HUSH), composed of Periphilin, TASOR and MPP8, silences transcriptionally active viral and endogenous transgenes. HUSH recruits effectors that alter the epigenetic landscape and chromatin structure, but how HUSH recognizes target loci and represses their expression remains unclear. We identify the physicochemical properties of Periphilin necessary for HUSH assembly and silencing. A disordered N-terminal domain (NTD) and structured C-terminal domain are essential for silencing. A crystal structure of the Periphilin-TASOR minimal core complex shows Periphilin forms an α-helical homodimer, bound by a single TASOR molecule. The NTD forms insoluble aggregates through an arginine/tyrosine-rich sequence reminiscent of low-complexity regions from self-associating RNA-binding proteins. Residues required for TASOR binding and aggregation were required for HUSH-dependent silencing and genome-wide deposition of repressive mark H3K9me3. The NTD was functionally complemented by low-complexity regions from certain RNA-binding proteins and proteins that form condensates or fibrils. Our work suggests the associative properties of Periphilin promote HUSH aggregation at target loci.


Assuntos
Antígenos de Neoplasias/ultraestrutura , Proteínas Nucleares/ultraestrutura , Proteínas de Ligação a RNA/química , Transcrição Gênica , Antígenos de Neoplasias/química , Antígenos de Neoplasias/genética , Cristalografia por Raios X , Elementos de DNA Transponíveis/genética , Epigênese Genética/genética , Inativação Gênica , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fosfoproteínas/química , Fosfoproteínas/genética , Agregados Proteicos/genética , Ligação Proteica/genética , Conformação Proteica em alfa-Hélice , Domínios Proteicos/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Vírus/genética
7.
Am J Hum Genet ; 107(2): 352-363, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32693025

RESUMO

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.


Assuntos
Adenosina Trifosfatases/genética , Anormalidades Craniofaciais/genética , Transtornos do Crescimento/genética , Mutação/genética , Transtornos do Neurodesenvolvimento/genética , Fatores de Transcrição/genética , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Doenças Genéticas Inatas/genética , Heterozigoto , Humanos , Lactente , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Pessoa de Meia-Idade , Fenótipo , Adulto Jovem
8.
Nat Protoc ; 14(1): 153-170, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30518911

RESUMO

Alterations in chromatin structure play a major role in the epigenetic regulation of gene expression. Here, we describe a step-by-step protocol for differential viral accessibility (DIVA), a method for identifying changes in chromatin accessibility genome-wide. Commonly used methods for mapping accessible genomic loci have strong preferences toward detecting 'open' chromatin found at regulatory regions but are not well suited to studying chromatin accessibility in gene bodies and intergenic regions. DIVA overcomes this limitation, enabling a broader range of sites to be interrogated. Conceptually, DIVA is similar to ATAC-seq in that it relies on the integration of exogenous DNA into the genome to map accessible chromatin, except that chromatin architecture is probed through mapping integration sites of exogenous lentiviruses. An isogenic pair of cell lines are transduced with a lentiviral vector, followed by PCR amplification and Illumina sequencing of virus-genome junctions; the resulting sequences define a set of unique lentiviral integration sites, which are compared to determine whether genomic loci exhibit significantly altered accessibility between experimental and control cells. Experienced researchers will take 6 d to generate lentiviral stocks and transduce the target cells, a further 5 d to prepare the Illumina sequencing libraries and a few hours to perform the bioinformatic analysis.


Assuntos
Cromatina/química , Mapeamento Cromossômico/métodos , DNA Viral/genética , Genoma Humano , Lentivirus/genética , Integração Viral , Cromatina/virologia , Mapeamento Cromossômico/estatística & dados numéricos , Loci Gênicos , Biblioteca Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase , Transdução Genética
9.
Genome Res ; 28(6): 836-845, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29728366

RESUMO

Retrotransposons encompass half of the human genome and contribute to the formation of heterochromatin, which provides nuclear structure and regulates gene expression. Here, we asked if the human silencing hub (HUSH) complex is necessary to silence retrotransposons and whether it collaborates with TRIM28 and the chromatin remodeler ATRX at specific genomic loci. We show that the HUSH complex contributes to de novo repression and DNA methylation of an SVA retrotransposon reporter. By using naïve versus primed mouse pluripotent stem cells, we reveal a critical role for the HUSH complex in naïve cells, implicating it in programming epigenetic marks in development. Although the HUSH component FAM208A binds to endogenous retroviruses (ERVs) and long interspersed element-1s (LINE-1s or L1s), it is mainly required to repress evolutionarily young L1s (mouse-specific lineages <5 million years old). TRIM28, in contrast, is necessary to repress both ERVs and young L1s. Genes co-repressed by TRIM28 and FAM208A are evolutionarily young, or exhibit tissue-specific expression, are enriched in young L1s, and display evidence for regulation through LTR promoters. Finally, we demonstrate that the HUSH complex is also required to repress L1 elements in human cells. Overall, these data indicate that the HUSH complex and TRIM28 co-repress young retrotransposons and new genes rewired by retrotransposon noncoding DNA.


Assuntos
Genoma Humano , Proteínas Nucleares/genética , Retroelementos/genética , Proteína 28 com Motivo Tripartido/genética , Animais , Metilação de DNA/genética , Retrovirus Endógenos/genética , Heterocromatina/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos/genética , Camundongos , Regiões Promotoras Genéticas
10.
Nat Commun ; 9(1): 651, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29440755

RESUMO

Missense mutations in MORC2 cause neuropathies including spinal muscular atrophy and Charcot-Marie-Tooth disease. We recently identified MORC2 as an effector of epigenetic silencing by the human silencing hub (HUSH). Here we report the biochemical and cellular activities of MORC2 variants, alongside crystal structures of wild-type and neuropathic forms of a human MORC2 fragment comprising the GHKL-type ATPase module and CW-type zinc finger. This fragment dimerizes upon binding ATP and contains a hinged, functionally critical coiled-coil insertion absent in other GHKL ATPases. We find that dimerization and DNA binding of the MORC2 ATPase module transduce HUSH-dependent silencing. Disease mutations change the dynamics of dimerization by distinct structural mechanisms: destabilizing the ATPase-CW module, trapping the ATP lid, or perturbing the dimer interface. These defects lead to the modulation of HUSH function, thus providing a molecular basis for understanding MORC2-associated neuropathies.


Assuntos
Adenosina Trifosfatases/metabolismo , Epigênese Genética , Inativação Gênica , Mutação de Sentido Incorreto , Doenças do Sistema Nervoso/genética , Fatores de Transcrição/genética , Trifosfato de Adenosina/metabolismo , Animais , Doença de Charcot-Marie-Tooth/genética , Doença de Charcot-Marie-Tooth/patologia , Cristalografia por Raios X , DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Atrofia Muscular Espinal , Doenças do Sistema Nervoso/patologia , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Células Sf9 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Dedos de Zinco
11.
Mol Cell ; 69(1): 5-7, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29304334

RESUMO

Post-translational modifications of histones are widely used to discriminate between different types of chromatin. In a recent issue of Molecular Cell, Becker et al. (2017) delineate chromatin states based on physical properties, thereby expanding our understanding of chromatin function.


Assuntos
Cromatina , Código das Histonas , Genômica , Histonas/genética , Processamento de Proteína Pós-Traducional , Proteômica
12.
Nat Genet ; 49(7): 1035-1044, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28581500

RESUMO

Dominant mutations in the MORC2 gene have recently been shown to cause axonal Charcot-Marie-Tooth (CMT) disease, but the cellular function of MORC2 is poorly understood. Here, through a genome-wide CRISPR-Cas9-mediated forward genetic screen, we identified MORC2 as an essential gene required for epigenetic silencing by the HUSH complex. HUSH recruits MORC2 to target sites in heterochromatin. We exploited a new method, differential viral accessibility (DIVA), to show that loss of MORC2 results in chromatin decompaction at these target loci, which is concomitant with a loss of H3K9me3 deposition and transcriptional derepression. The ATPase activity of MORC2 is critical for HUSH-mediated silencing, and the most common alteration affecting the ATPase domain in CMT patients (p.Arg252Trp) hyperactivates HUSH-mediated repression in neuronal cells. These data define a critical role for MORC2 in epigenetic silencing by the HUSH complex and provide a mechanistic basis underpinning the role of MORC2 mutations in CMT disease.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Montagem e Desmontagem da Cromatina/genética , Repressão Epigenética/genética , Inativação Gênica , Heterocromatina/genética , Fatores de Transcrição/genética , Adenosina Trifosfatases/metabolismo , Sistemas CRISPR-Cas , Doença de Charcot-Marie-Tooth/metabolismo , Células HeLa , Heterocromatina/metabolismo , Código das Histonas , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Lisina/química , Metilação , Complexos Multiproteicos , Mutação de Sentido Incorreto , Neurônios/metabolismo , Domínios Proteicos , Mapeamento de Interação de Proteínas , Proteínas Metiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Fatores de Transcrição/fisiologia , Transgenes
13.
Cell Rep ; 17(3): 653-659, 2016 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-27732843

RESUMO

The histone methyltransferase SETDB1 plays a central role in repressive chromatin processes, but the functional requirement for its binding partner ATF7IP has remained enigmatic. Here, we show that ATF7IP is essential for SETDB1 stability: nuclear SETDB1 protein is degraded by the proteasome upon ablation of ATF7IP. As a result, ATF7IP is critical for repression that requires H3K9 trimethylation by SETDB1, including transgene silencing by the HUSH complex. Furthermore, we show that loss of ATF7IP phenocopies loss of SETDB1 in genome-wide assays. ATF7IP and SETDB1 knockout cells exhibit near-identical defects in the global deposition of H3K9me3, which results in similar dysregulation of the transcriptome. Overall, these data identify a critical functional role for ATF7IP in heterochromatin formation by regulating SETDB1 abundance in the nucleus.


Assuntos
Heterocromatina/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Metiltransferases/metabolismo , Fatores de Transcrição/metabolismo , Núcleo Celular/metabolismo , Estabilidade Enzimática , Epigênese Genética , Deleção de Genes , Inativação Gênica , Células HeLa , Histona-Lisina N-Metiltransferase , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteólise , Proteínas Repressoras , Transcriptoma/genética
14.
Nat Commun ; 7: 11786, 2016 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-27283361

RESUMO

The application of forward genetic screens to cultured human cells represents a powerful method to study gene function. The repurposing of the bacterial CRISPR/Cas9 system provides an effective method to disrupt gene function in mammalian cells, and has been applied to genome-wide screens. Here, we compare the efficacy of genome-wide CRISPR/Cas9-mediated forward genetic screens versus gene-trap mutagenesis screens in haploid human cells, which represent the existing 'gold standard' method. This head-to-head comparison aimed to identify genes required for the endoplasmic reticulum-associated degradation (ERAD) of MHC class I molecules. The two approaches show high concordance (>70%), successfully identifying the majority of the known components of the canonical glycoprotein ERAD pathway. Both screens also identify a role for the uncharacterized gene TXNDC11, which we show encodes an EDEM2/3-associated disulphide reductase. Genome-wide CRISPR/Cas9-mediated screens together with haploid genetic screens provide a powerful addition to the forward genetic toolbox.


Assuntos
Sistemas CRISPR-Cas/genética , Degradação Associada com o Retículo Endoplasmático/genética , Testes Genéticos , Haploidia , Mamíferos/genética , Animais , Corantes Fluorescentes/metabolismo , Genes Reporter , Glicoproteínas/metabolismo , Células HEK293 , Células HeLa , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Oxirredução , Ligação Proteica , Domínios Proteicos , Tiorredoxinas/química , Tiorredoxinas/metabolismo , alfa-Glucosidases/metabolismo
15.
Bioessays ; 38(4): 333-43, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26853531

RESUMO

Much of what we understand about heterochromatin formation in mammals has been extrapolated from forward genetic screens for modifiers of position-effect variegation (PEV) in the fruit fly Drosophila melanogaster. The recent identification of the HUSH (Human Silencing Hub) complex suggests that more recent evolutionary developments contribute to the mechanisms underlying PEV in human cells. Although HUSH-mediated repression also involves heterochromatin spreading through the reading and writing of the repressive H3K9me3 histone modification, clear orthologues of HUSH subunits are not found in Drosophila but are conserved in vertebrates. Here we compare the insights into the mechanisms of PEV derived from genetic screens in the fly, the mouse and in human cells, review what is currently known about the HUSH complex and discuss the implications of HUSH-mediated silencing for viral latency. Future studies will provide mechanistic insight into HUSH complex function and reveal the relationship between HUSH and other epigenetic silencing complexes.


Assuntos
Antígenos de Neoplasias/genética , Efeitos da Posição Cromossômica , Inativação Gênica , Heterocromatina/metabolismo , Histonas/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Animais , Antígenos de Neoplasias/metabolismo , Evolução Biológica , Linhagem Celular Tumoral , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Testes Genéticos , HIV-1/genética , HIV-1/metabolismo , Haploidia , Heterocromatina/química , Histonas/metabolismo , Humanos , Camundongos , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Latência Viral
16.
Science ; 348(6242): 1481-1485, 2015 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-26022416

RESUMO

Forward genetic screens in Drosophila melanogaster for modifiers of position-effect variegation have revealed the basis of much of our understanding of heterochromatin. We took an analogous approach to identify genes required for epigenetic repression in human cells. A nonlethal forward genetic screen in near-haploid KBM7 cells identified the HUSH (human silencing hub) complex, comprising three poorly characterized proteins, TASOR, MPP8, and periphilin; this complex is absent from Drosophila but is conserved from fish to humans. Loss of HUSH components resulted in decreased H3K9me3 both at endogenous genomic loci and at retroviruses integrated into heterochromatin. Our results suggest that the HUSH complex is recruited to genomic loci rich in H3K9me3, where subsequent recruitment of the methyltransferase SETDB1 is required for further H3K9me3 deposition to maintain transcriptional silencing.


Assuntos
Antígenos de Neoplasias/metabolismo , Efeitos da Posição Cromossômica , Inativação Gênica , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Animais , Antígenos de Neoplasias/genética , Sequência Conservada , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Evolução Molecular , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde/genética , Células HeLa , Heterocromatina/metabolismo , Histona-Lisina N-Metiltransferase , Humanos , Imunoprecipitação , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Fosfoproteínas/genética , Proteínas Metiltransferases/metabolismo
17.
PLoS Pathog ; 9(11): e1003772, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24278019

RESUMO

The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2), a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.


Assuntos
Membrana Celular/metabolismo , Regulação para Baixo , Herpesvirus Humano 8/enzimologia , Proteínas Imediatamente Precoces/biossíntese , Proteínas com Domínio MARVEL/biossíntese , Proteolipídeos/biossíntese , Ubiquitina-Proteína Ligases/biossíntese , Proteínas Virais/biossíntese , Membrana Celular/genética , Membrana Celular/imunologia , Testes Genéticos , Células HeLa , Células Hep G2 , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas com Domínio MARVEL/genética , Proteínas com Domínio MARVEL/imunologia , Proteolipídeos/genética , Proteolipídeos/imunologia , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/imunologia , Sarcoma de Kaposi/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia
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