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1.
Nat Struct Mol Biol ; 26(7): 567-570, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31270470

RESUMO

The cyclic enzymatic removal and ligation of the C-terminal tyrosine of α-tubulin generates heterogeneous microtubules and affects their functions. Here we describe the crystal and solution structure of the tubulin carboxypeptidase complex between vasohibin (VASH1) and small vasohibin-binding protein (SVBP), which folds in a long helix, which stabilizes the VASH1 catalytic domain. This structure, combined with molecular docking and mutagenesis experiments, reveals which residues are responsible for recognition and cleavage of the tubulin C-terminal tyrosine.

2.
J Virol ; 93(13)2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30996093

RESUMO

Vaccinia virus is a promising viral vaccine and gene delivery candidate and has historically been used as a model to study poxvirus-host cell interactions. We employed a genome-wide insertional mutagenesis approach in human haploid cells to identify host factors crucial for vaccinia virus infection. A library of mutagenized HAP1 cells was exposed to modified vaccinia virus Ankara (MVA). Deep-sequencing analysis of virus-resistant cells identified host factors involved in heparan sulfate synthesis, Golgi organization, and vesicular protein trafficking. We validated EXT1, TM9SF2, and TMED10 (TMP21/p23/p24δ) as important host factors for vaccinia virus infection. The critical roles of EXT1 in heparan sulfate synthesis and vaccinia virus infection were confirmed. TM9SF2 was validated as a player mediating heparan sulfate expression, explaining its contribution to vaccinia virus infection. In addition, TMED10 was found to be crucial for virus-induced plasma membrane blebbing and phosphatidylserine-induced macropinocytosis, presumably by regulating the cell surface expression of the TAM receptor Axl.IMPORTANCE Poxviruses are large DNA viruses that can infect a wide range of host species. A number of these viruses are clinically important to humans, including variola virus (smallpox) and vaccinia virus. Since the eradication of smallpox, zoonotic infections with monkeypox virus and cowpox virus are emerging. Additionally, poxviruses can be engineered to specifically target cancer cells and are used as a vaccine vector against tuberculosis, influenza, and coronaviruses. Poxviruses rely on host factors for most stages of their life cycle, including attachment to the cell and entry. These host factors are crucial for virus infectivity and host cell tropism. We used a genome-wide knockout library of host cells to identify host factors necessary for vaccinia virus infection. We confirm a dominant role for heparin sulfate in mediating virus attachment. Additionally, we show that TMED10, previously not implicated in virus infections, facilitates virus uptake by modulating the cellular response to phosphatidylserine.

3.
Nat Med ; 25(4): 612-619, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833751

RESUMO

Cancer cells can evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of programmed death ligand 1 in tumor microenvironments is a major immune checkpoint for tumor-specific T cell responses as it binds to programmed cell death protein-1 on activated and dysfunctional T cells1. The activity of myeloid cells such as macrophages and neutrophils is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein creates a 'don't eat me' signal on tumor cells by binding to SIRPα expressed on myeloid cells2-5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like protein (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated killing of tumor cells in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway and thereby augment antibody therapy of cancer.


Assuntos
Aminoaciltransferases/metabolismo , Antígenos de Diferenciação/metabolismo , Antígeno CD47/metabolismo , Imunoterapia , Neoplasias/imunologia , Neoplasias/terapia , Receptores Imunológicos/metabolismo , Aminoaciltransferases/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Humanos , Camundongos Transgênicos , Neoplasias/patologia , Proteínas Opsonizantes/metabolismo , Ácido Pirrolidonocarboxílico/metabolismo
4.
PLoS One ; 14(2): e0212053, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30753225

RESUMO

Experimental and clinical observations have highlighted the role of cytotoxic T cells in human tumor control. However, the parameters that control tumor cell sensitivity to T cell attack remain incompletely understood. To identify modulators of tumor cell sensitivity to T cell effector mechanisms, we performed a whole genome haploid screen in HAP1 cells. Selection of tumor cells by exposure to tumor-specific T cells identified components of the interferon-γ (IFN-γ) receptor (IFNGR) signaling pathway, and tumor cell killing by cytotoxic T cells was shown to be in large part mediated by the pro-apoptotic effects of IFN-γ. Notably, we identified schlafen 11 (SLFN11), a known modulator of DNA damage toxicity, as a regulator of tumor cell sensitivity to T cell-secreted IFN-γ. SLFN11 does not influence IFNGR signaling, but couples IFNGR signaling to the induction of the DNA damage response (DDR) in a context dependent fashion. In line with this role of SLFN11, loss of SLFN11 can reduce IFN-γ mediated toxicity. Collectively, our data indicate that SLFN11 can couple IFN-γ exposure of tumor cells to DDR and cellular apoptosis. Future work should reveal the mechanistic basis for the link between IFNGR signaling and DNA damage response, and identify tumor cell types in which SLFN11 contributes to the anti-tumor activity of T cells.

5.
Nat Commun ; 10(1): 100, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30626869

RESUMO

Loss of BRCA2 affects genome stability and is deleterious for cellular survival. Using a genome-wide genetic screen in near-haploid KBM-7 cells, we show that tumor necrosis factor-alpha (TNFα) signaling is a determinant of cell survival upon BRCA2 inactivation. Specifically, inactivation of the TNF receptor (TNFR1) or its downstream effector SAM68 rescues cell death induced by BRCA2 inactivation. BRCA2 inactivation leads to pro-inflammatory cytokine production, including TNFα, and increases sensitivity to TNFα. Enhanced TNFα sensitivity is not restricted to BRCA2 inactivation, as BRCA1 or FANCD2 inactivation, or hydroxyurea treatment also sensitizes cells to TNFα. Mechanistically, BRCA2 inactivation leads to cGAS-positive micronuclei and results in a cell-intrinsic interferon response, as assessed by quantitative mass-spectrometry and gene expression profiling, and requires ASK1 and JNK signaling. Combined, our data reveals that micronuclei induced by loss of BRCA2 instigate a cGAS/STING-mediated interferon response, which encompasses re-wired TNFα signaling and enhances TNFα sensitivity.


Assuntos
Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Sobrevivência Celular/fisiologia , Inflamação/metabolismo , Nucleotidiltransferases/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Linhagem Celular , Deleção de Genes , Humanos , Nucleotidiltransferases/genética , Transdução de Sinais
6.
Nature ; 563(7732): 559-563, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30464266

RESUMO

The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

7.
Science ; 362(6419): 1171-1177, 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30442766

RESUMO

In genetic screens aimed at understanding drug resistance mechanisms in chronic myeloid leukemia cells, inactivation of the cullin 3 adapter protein-encoding leucine zipper-like transcription regulator 1 (LZTR1) gene led to enhanced mitogen-activated protein kinase (MAPK) pathway activity and reduced sensitivity to tyrosine kinase inhibitors. Knockdown of the Drosophila LZTR1 ortholog CG3711 resulted in a Ras-dependent gain-of-function phenotype. Endogenous human LZTR1 associates with the main RAS isoforms. Inactivation of LZTR1 led to decreased ubiquitination and enhanced plasma membrane localization of endogenous KRAS (V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog). We propose that LZTR1 acts as a conserved regulator of RAS ubiquitination and MAPK pathway activation. Because LZTR1 disease mutations failed to revert loss-of-function phenotypes, our findings provide a molecular rationale for LZTR1 involvement in a variety of inherited and acquired human disorders.

8.
Trends Cell Biol ; 2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-30213517

RESUMO

Microtubules are subjected to a variety of post-translational modifications (PTMs). The combination of different α- and ß-tubulin isoforms and PTMs are referred to as the tubulin code. PTMs are generated by a suite of enzymes thought to affect tubulin-interacting proteins. One PTM is the cyclic removal and ligation of the C-terminal tyrosine of α-tubulin. This has been implicated in cellular processes such as mitosis, cardiomyocyte contraction, and neuronal function. Recently, vasohibins (VASHs) were identified as the first tubulin-detyrosinating enzymes, A cell-autonomous role for VASHs in regulating the cytoskeleton was unexpected due to their previous association with angiogenesis. This review discusses the functionality of the tubulin detyrosination cycle, the biology of VASHs, and highlights the emerging questions accompanying this link.

9.
Cancer Res ; 78(23): 6621-6631, 2018 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-30213788

RESUMO

: Neuroblastoma is the second most common tumor in children. The cause of neuroblastoma is thought to lie in aberrant development of embryonic neural crest cells and is accompanied by low MHC-1 expression and suppression of the NF-κB transcription factor, thereby gearing cells toward escape from immunosurveillance. Here, we assess regulation of the MHC-1 gene in neuroblastoma to enhance its immunogenic potential for therapeutic T-cell targeting. A genome-wide CRISPR screen identified N4BP1 and TNIP1 as inhibitory factors of NF-κB-mediated MHC-1 expression in neuroblastoma. Patients with advanced stage neuroblastoma who expressed high levels of TNIP1 and N4BP1 exhibited worse overall survival. Depletion of N4BP1 or TNIP1 increased NF-κB and MHC-1 expression and stimulated recognition by antigen-specific CD8+ T cells. We confirmed that TNIP1 inhibited canonical NF-κB member RelA by preventing activation of the RelA/p50 NF-κB dimer. Furthermore, N4BP1 inhibited both canonical and noncanonical NF-κB through binding of deubiquitinating enzyme CEZANNE, resulting in stabilization of TRAF3 and degradation of NF-κB-inducing kinase NIK. These data suggest that N4BP1/CEZANNE or TNIP1 may be candidate targets for immunotherapy in neuroblastoma tumors and should lift NF-κB suppression, thereby triggering increased peptide/MHC1-mediated tumor reactivity to enhance therapeutic T-cell targeting. SIGNIFICANCE: Aberrant regulation of NF-κB and MHC-1 in neuroblastoma tumors provides new targets for immunotherapeutic approaches against neuroblastoma.

10.
Curr Biol ; 28(17): 2685-2696.e4, 2018 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-30122526

RESUMO

The spindle assembly checkpoint (SAC) halts anaphase progression until all kinetochores have obtained bipolar, stable attachments to the mitotic spindle. Upon initial attachment, chromosomes undergo oscillatory movements to reach metaphase. Once a chromosome is correctly attached and positioned, these oscillatory movements are reduced by the motor protein Kif18A, and loss of Kif18A results in chromosome hyper-oscillations. By using a haploid genetic approach, we found that loss of Kif18A is lethal in wild-type human HAP1 cells, but not in SAC-deficient HAP1 cells. Unexpectedly, we found that the hyper-oscillations after Kif18A loss are not associated with chromosome missegregations. Rather, we found that loss of Kif18A results in a loss of tension across a subset of kinetochores accompanying SAC activation. Strikingly, the SAC-active kinetochores appear to have established fully functional kinetochore-microtubule (k-Mt) attachments, allowing proper chromosome segregation. These findings shed new light on the role of Kif18A in chromosome segregation and demonstrate that the SAC can be activated at kinetochores that are occupied by fully functional k-Mts that lack tension.

11.
J Cell Sci ; 131(15)2018 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-30076240

RESUMO

In order to replicate, most pathogens need to enter their target cells. Many viruses enter the host cell through an endocytic pathway and hijack endosomes for their journey towards sites of replication. For delivery of their genome to the host cell cytoplasm and to avoid degradation, viruses have to escape this endosomal compartment without host detection. Viruses have developed complex mechanisms to penetrate the endosomal membrane and have evolved to co-opt several host factors to facilitate endosomal escape. Conversely, there is an extensive variety of cellular mechanisms to counteract or impede viral replication. At the level of cell entry, there are cellular defense mechanisms that recognize endosomal membrane damage caused by virus-induced membrane fusion and pore formation, as well as restriction factors that block these processes. In this Cell Science at a Glance article and accompanying poster, we describe the different mechanisms that viruses have evolved to escape the endosomal compartment, as well as the counteracting cellular protection mechanisms. We provide examples for enveloped and non-enveloped viruses, for which we discuss some unique and unexpected cellular responses to virus-entry-induced membrane damage.

12.
Cell Host Microbe ; 23(5): 636-643.e5, 2018 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-29681460

RESUMO

Human type A Enteroviruses (EV-As) cause diseases ranging from hand-foot-and-mouth disease to poliomyelitis-like disease. Although cellular receptors are identified for some EV-As, they remain elusive for the majority of EV-As. We identify the cell surface molecule KREMEN1 as an entry receptor for coxsackievirus A10 (CV-A10). Whereas loss of KREMEN1 renders cells resistant to CV-A10 infection, KREMEN1 overexpression enhances CV-A10 binding to the cell surface and increases susceptibility to infection, indicating that KREMEN1 is a rate-limiting factor for CV-A10 infection. Furthermore, the extracellular domain of KREMEN1 binds CV-A10 and functions as a neutralizing agent during infection. Kremen-deficient mice are resistant to CV-A10-induced lethal paralysis, emphasizing the relevance of Kremen for infection in vivo. KREMEN1 is also essential for infection by a phylogenetic and pathogenic related group of EV-As. Collectively these findings highlight the importance of KREMEN1 for these emerging pathogens and its potential as an antiviral therapeutic target.


Assuntos
Enterovirus Humano A/metabolismo , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/metabolismo , Proteínas de Membrana/metabolismo , Internalização do Vírus , Animais , Antígenos de Superfície , Linhagem Celular , Linhagem Celular Tumoral , Enterovirus/patogenicidade , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Feminino , Técnicas de Inativação de Genes , Células HCT116 , Células HEK293 , Doença de Mão, Pé e Boca/virologia , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Mutagênese , Filogenia , Domínios Proteicos
13.
Mol Oncol ; 12(6): 953-971, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29689640

RESUMO

The absence of biomarkers to accurately predict anticancer therapy response remains a major obstacle in clinical oncology. We applied a genome-wide loss-of-function screening approach in human haploid cells to characterize genetic vulnerabilities to classical microtubule-targeting agents. Using docetaxel and vinorelbine, two well-established chemotherapeutic agents, we sought to identify genetic alterations sensitizing human HAP1 cells to these drugs. Despite the fact that both drugs act on microtubules, a set of distinct genes were identified whose disruption affects drug sensitivity. For docetaxel, this included a number of genes with a function in mitosis, while for vinorelbine we identified inactivation of FBXW7, RB1, and NF2, three frequently mutated tumor suppressor genes, as sensitizing factors. We validated these genes using independent knockout clones and confirmed FBXW7 as an important regulator of the mitotic spindle assembly. Upon FBXW7 depletion, vinorelbine treatment led to decreased survival of cells due to defective mitotic progression and subsequent mitotic catastrophe. We show that haploid insertional mutagenesis screens are a useful tool to study genetic vulnerabilities to classical chemotherapeutic drugs by identifying thus far unknown sensitivity factors. These results provide a rationale for investigating patient response to vinca alkaloid-based anticancer treatment in relation to the mutational status of these three tumor suppressor genes, and could in the future lead to the establishment of novel predictive biomarkers or suggest new drug combinations based on molecular mechanisms of drug sensitivity.

14.
Cell Rep ; 22(6): 1424-1438, 2018 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-29425499

RESUMO

The spindle assembly checkpoint (SAC) ensures faithful segregation of chromosomes. Although most mammalian cell types depend on the SAC for viability, we found that human HAP1 cells can grow SAC independently. We generated MAD1- and MAD2-deficient cells and mutagenized them to identify synthetic lethal interactions, revealing that chromosome congression factors become essential upon SAC deficiency. Besides expected hits, we also found that BUB1 becomes essential in SAC-deficient cells. We found that the BUB1 C terminus regulates alignment as well as recruitment of CENPF. Second, we found that BUBR1 was not essential in SAC-deficient HAP1 cells. We confirmed that BUBR1 does not regulate chromosome alignment in HAP1 cells and that BUB1 does not regulate chromosome alignment through BUBR1. Taken together, our data resolve some long-standing questions about the interplay between BUB1 and BUBR1 and their respective roles in the SAC and chromosome alignment.

15.
Genome Med ; 9(1): 118, 2017 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-29273094

RESUMO

BACKGROUND: The phenotypic severity of congenital muscular dystrophy-dystroglycanopathy (MDDG) syndromes associated with aberrant glycosylation of α-dystroglycan ranges from the severe Walker-Warburg syndrome or muscle-eye-brain disease to mild, late-onset, isolated limb-girdle muscular dystrophy without neural involvement. However, muscular dystrophy is invariably found across the spectrum of MDDG patients. METHODS: Using linkage mapping and whole-exome sequencing in two families with an unexplained neurodevelopmental disorder, we have identified homozygous and compound heterozygous mutations in B3GALNT2. RESULTS: The first family comprises two brothers of Dutch non-consanguineous parents presenting with mild ID and behavioral problems. Immunohistochemical analysis of muscle biopsy revealed no significant aberrations, in line with the absence of a muscular phenotype in the affected siblings. The second family includes five affected individuals from an Iranian consanguineous kindred with mild-to-moderate intellectual disability (ID) and epilepsy without any notable neuroimaging, muscle, or eye abnormalities. Complementation assays of the compound heterozygous mutations identified in the two brothers had a comparable effect on the O-glycosylation of α-dystroglycan as previously reported mutations that are associated with severe muscular phenotypes. CONCLUSIONS: In conclusion, we show that mutations in B3GALNT2 can give rise to a novel MDDG syndrome presentation, characterized by ID associated variably with seizure, but without any apparent muscular involvement. Importantly, B3GALNT2 activity does not fully correlate with the severity of the phenotype as assessed by the complementation assay.


Assuntos
Deficiência Intelectual/genética , Mutação , N-Acetilgalactosaminiltransferases/genética , Fenótipo , Síndrome de Walker-Warburg/genética , Adolescente , Adulto , Linhagem Celular , Criança , Feminino , Genes Recessivos , Genótipo , Humanos , Deficiência Intelectual/patologia , Masculino , N-Acetilgalactosaminiltransferases/metabolismo , Linhagem , Síndrome de Walker-Warburg/patologia
16.
Cell Host Microbe ; 22(5): 688-696.e5, 2017 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-29120745

RESUMO

Arenaviruses cause fatal hemorrhagic disease in humans. Old World arenavirus glycoproteins (GPs) mainly engage α-dystroglycan as a cell-surface receptor, while New World arenaviruses hijack transferrin receptor. However, the Lujo virus (LUJV) GP does not cluster with New or Old World arenaviruses. Using a recombinant vesicular stomatitis virus containing LUJV GP as its sole attachment and fusion protein (VSV-LUJV), we demonstrate that infection is independent of known arenavirus receptor genes. A genome-wide haploid genetic screen identified the transmembrane protein neuropilin 2 (NRP2) and tetraspanin CD63 as factors for LUJV GP-mediated infection. LUJV GP binds the N-terminal domain of NRP2, while CD63 stimulates pH-activated LUJV GP-mediated membrane fusion. Overexpression of NRP2 or its N-terminal domain enhances VSV-LUJV infection, and cells lacking NRP2 are deficient in wild-type LUJV infection. These findings uncover this distinct set of host cell entry factors in LUJV infection and are attractive focus points for therapeutic intervention.


Assuntos
Lujo virus/fisiologia , Neuropilina-2/metabolismo , Tetraspanina 30/metabolismo , Proteínas Virais de Fusão/metabolismo , Proteínas Virais/metabolismo , Internalização do Vírus , Proteínas de Transporte , Linhagem Celular , Interações Hospedeiro-Patógeno/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Lujo virus/genética , Lujo virus/patogenicidade , Domínios e Motivos de Interação entre Proteínas , Receptores de Superfície Celular/metabolismo , Receptores da Transferrina , Proteínas Virais de Fusão/genética , Proteínas Virais/genética
17.
Science ; 358(6369): 1453-1456, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29146869

RESUMO

Tubulin is subjected to a number of posttranslational modifications to generate heterogeneous microtubules. The modifications include removal and ligation of the C-terminal tyrosine of ⍺-tubulin. The enzymes responsible for detyrosination, an activity first observed 40 years ago, have remained elusive. We applied a genetic screen in haploid human cells to find regulators of tubulin detyrosination. We identified SVBP, a peptide that regulates the abundance of vasohibins (VASH1 and VASH2). Vasohibins, but not SVBP alone, increased detyrosination of ⍺-tubulin, and purified vasohibins removed the C-terminal tyrosine of ⍺-tubulin. We found that vasohibins play a cell type-dependent role in detyrosination, although cells also contain an additional detyrosinating activity. Thus, vasohibins, hitherto studied as secreted angiogenesis regulators, constitute a long-sought missing link in the tubulin tyrosination cycle.


Assuntos
Proteínas Angiogênicas/metabolismo , Carboxipeptidases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina/metabolismo , Proteínas Angiogênicas/genética , Biocatálise , Carboxipeptidases/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Haploidia , Humanos , Neovascularização Fisiológica
18.
Cell Host Microbe ; 22(4): 460-470.e5, 2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29024641

RESUMO

Respiratory and arthropod-borne viral infections are a global threat due to the lack of effective antivirals and vaccines. A potential strategy is to target host proteins required for viruses but non-essential for the host. To identify such proteins, we performed a genome-wide knockout screen in human haploid cells and identified the calcium pump SPCA1. SPCA1 is required by viruses from the Paramyxoviridae, Flaviviridae, and Togaviridae families, including measles, dengue, West Nile, Zika, and chikungunya viruses. Calcium transport activity is required for SPCA1 to promote virus spread. SPCA1 regulates proteases within the trans-Golgi network that require calcium for their activity and are critical for virus glycoprotein maturation. Consistent with these findings, viral glycoproteins fail to mature in SPCA1-deficient cells preventing viral spread, which is evident even in cells with partial loss of SPCA1. Thus, SPCA1 is an attractive antiviral host target for a broad spectrum of established and emerging viral infections.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Flaviviridae/fisiologia , Interações Hospedeiro-Patógeno , Paramyxoviridae/fisiologia , Togaviridae/fisiologia , Proteínas Virais/metabolismo , Células A549 , Animais , ATPases Transportadoras de Cálcio/genética , Cercopithecus aethiops , Feminino , Técnicas de Inativação de Genes , Estudo de Associação Genômica Ampla , Haploidia , Células HeLa , Humanos , Masculino , Células Vero , Proteínas Virais/genética , Rede trans-Golgi/enzimologia
19.
Nature ; 549(7670): 106-110, 2017 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-28813410

RESUMO

The clinical benefit for patients with diverse types of metastatic cancers that has been observed upon blockade of the interaction between PD-1 and PD-L1 has highlighted the importance of this inhibitory axis in the suppression of tumour-specific T-cell responses. Notwithstanding the key role of PD-L1 expression by cells within the tumour micro-environment, our understanding of the regulation of the PD-L1 protein is limited. Here we identify, using a haploid genetic screen, CMTM6, a type-3 transmembrane protein of previously unknown function, as a regulator of the PD-L1 protein. Interference with CMTM6 expression results in impaired PD-L1 protein expression in all human tumour cell types tested and in primary human dendritic cells. Furthermore, through both a haploid genetic modifier screen in CMTM6-deficient cells and genetic complementation experiments, we demonstrate that this function is shared by its closest family member, CMTM4, but not by any of the other CMTM members tested. Notably, CMTM6 increases the PD-L1 protein pool without affecting PD-L1 (also known as CD274) transcription levels. Rather, we demonstrate that CMTM6 is present at the cell surface, associates with the PD-L1 protein, reduces its ubiquitination and increases PD-L1 protein half-life. Consistent with its role in PD-L1 protein regulation, CMTM6 enhances the ability of PD-L1-expressing tumour cells to inhibit T cells. Collectively, our data reveal that PD-L1 relies on CMTM6/4 to efficiently carry out its inhibitory function, and suggest potential new avenues to block this pathway.


Assuntos
Antígeno B7-H1/metabolismo , Proteínas com Domínio MARVEL/metabolismo , Antígeno B7-H1/biossíntese , Antígeno B7-H1/química , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Células Dendríticas/metabolismo , Teste de Complementação Genética , Haploidia , Humanos , Proteínas com Domínio MARVEL/genética , Melanoma/genética , Melanoma/metabolismo , Ligação Proteica , Estabilidade Proteica , Ubiquitinação
20.
Nature ; 546(7657): 307-311, 2017 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-28562590

RESUMO

As key executers of biological functions, the activity and abundance of proteins are subjected to extensive regulation. Deciphering the genetic architecture underlying this regulation is critical for understanding cellular signalling events and responses to environmental cues. Using random mutagenesis in haploid human cells, we apply a sensitive approach to directly couple genomic mutations to protein measurements in individual cells. Here we use this to examine a suite of cellular processes, such as transcriptional induction, regulation of protein abundance and splicing, signalling cascades (mitogen-activated protein kinase (MAPK), G-protein-coupled receptor (GPCR), protein kinase B (AKT), interferon, and Wingless and Int-related protein (WNT) pathways) and epigenetic modifications (histone crotonylation and methylation). This scalable, sequencing-based procedure elucidates the genetic landscapes that control protein states, identifying genes that cause very narrow phenotypic effects and genes that lead to broad phenotypic consequences. The resulting genetic wiring map identifies the E3-ligase substrate adaptor KCTD5 (ref. 1) as a negative regulator of the AKT pathway, a key signalling cascade frequently deregulated in cancer. KCTD5-deficient cells show elevated levels of phospho-AKT at S473 that could not be attributed to effects on canonical pathway components. To reveal the genetic requirements for this phenotype, we iteratively analysed the regulatory network linked to AKT activity in the knockout background. This genetic modifier screen exposes suppressors of the KCTD5 phenotype and mechanistically demonstrates that KCTD5 acts as an off-switch for GPCR signalling by triggering proteolysis of Gßγ heterodimers dissociated from the Gα subunit. Although biological networks have previously been constructed on the basis of gene expression, protein-protein associations, or genetic interaction profiles, we foresee that the approach described here will enable the generation of a comprehensive genetic wiring map for human cells on the basis of quantitative protein states.


Assuntos
Canais de Potássio/metabolismo , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/genética , Análise de Célula Única/métodos , Células Cultivadas , Haploidia , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Histonas/química , Histonas/metabolismo , Humanos , Interferons/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutagênese , Fenótipo , Fosforilação/genética , Canais de Potássio/deficiência , Canais de Potássio/genética , Proteólise , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Via de Sinalização Wnt
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