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1.
Cell ; 180(6): 1245-1261.e21, 2020 03 19.
Artículo en Inglés | MEDLINE | ID: mdl-32142654

RESUMEN

In response to transcription-blocking DNA damage, cells orchestrate a multi-pronged reaction, involving transcription-coupled DNA repair, degradation of RNA polymerase II (RNAPII), and genome-wide transcription shutdown. Here, we provide insight into how these responses are connected by the finding that ubiquitylation of RNAPII itself, at a single lysine (RPB1 K1268), is the focal point for DNA-damage-response coordination. K1268 ubiquitylation affects DNA repair and signals RNAPII degradation, essential for surviving genotoxic insult. RNAPII degradation results in a shutdown of transcriptional initiation, in the absence of which cells display dramatic transcriptome alterations. Additionally, regulation of RNAPII stability is central to transcription recovery-persistent RNAPII depletion underlies the failure of this process in Cockayne syndrome B cells. These data expose regulation of global RNAPII levels as integral to the cellular DNA-damage response and open the intriguing possibility that RNAPII pool size generally affects cell-specific transcription programs in genome instability disorders and even normal cells.


Asunto(s)
Daño del ADN , ARN Polimerasa II/metabolismo , Reparación del ADN , Células HEK293 , Humanos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Ubiquitinación , Rayos Ultravioleta
2.
Cell ; 167(7): 1750-1761.e16, 2016 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-27984725

RESUMEN

S phase and mitotic onset are brought about by the action of multiple different cyclin-CDK complexes. However, it has been suggested that changes in the total level of CDK kinase activity, rather than substrate specificity, drive the temporal ordering of S phase and mitosis. Here, we present a phosphoproteomics-based systems analysis of CDK substrates in fission yeast and demonstrate that the phosphorylation of different CDK substrates can be temporally ordered during the cell cycle by a single cyclin-CDK. This is achieved by rising CDK activity and the differential sensitivity of substrates to CDK activity over a wide dynamic range. This is combined with rapid phosphorylation turnover to generate clearly resolved substrate-specific activity thresholds, which in turn ensures the appropriate ordering of downstream cell-cycle events. Comparative analysis with wild-type cells expressing multiple cyclin-CDK complexes reveals how cyclin-substrate specificity works alongside activity thresholds to fine-tune the patterns of substrate phosphorylation.


Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Ciclinas/metabolismo , Mitosis , Fosforilación
3.
Nature ; 627(8004): 671-679, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38448585

RESUMEN

DNA and histone modifications combine into characteristic patterns that demarcate functional regions of the genome1,2. While many 'readers' of individual modifications have been described3-5, how chromatin states comprising composite modification signatures, histone variants and internucleosomal linker DNA are interpreted is a major open question. Here we use a multidimensional proteomics strategy to systematically examine the interaction of around 2,000 nuclear proteins with over 80 modified dinucleosomes representing promoter, enhancer and heterochromatin states. By deconvoluting complex nucleosome-binding profiles into networks of co-regulated proteins and distinct nucleosomal features driving protein recruitment or exclusion, we show comprehensively how chromatin states are decoded by chromatin readers. We find highly distinctive binding responses to different features, many factors that recognize multiple features, and that nucleosomal modifications and linker DNA operate largely independently in regulating protein binding to chromatin. Our online resource, the Modification Atlas of Regulation by Chromatin States (MARCS), provides in-depth analysis tools to engage with our results and advance the discovery of fundamental principles of genome regulation by chromatin states.


Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina , Proteínas Nucleares , Nucleosomas , Proteómica , Humanos , Sitios de Unión , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , ADN/genética , ADN/metabolismo , Elementos de Facilitación Genéticos , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/metabolismo , Nucleosomas/química , Nucleosomas/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Proteómica/métodos
4.
Mol Cell ; 81(13): 2808-2822.e10, 2021 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-34111399

RESUMEN

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway senses cytosolic DNA and induces interferon-stimulated genes (ISGs) to activate the innate immune system. Here, we report the unexpected discovery that cGAS also senses dysfunctional protein production. Purified ribosomes interact directly with cGAS and stimulate its DNA-dependent activity in vitro. Disruption of the ribosome-associated protein quality control (RQC) pathway, which detects and resolves ribosome collision during translation, results in cGAS-dependent ISG expression and causes re-localization of cGAS from the nucleus to the cytosol. Indeed, cGAS preferentially binds collided ribosomes in vitro, and orthogonal perturbations that result in elevated levels of collided ribosomes and RQC activation cause sub-cellular re-localization of cGAS and ribosome binding in vivo as well. Thus, translation stress potently increases DNA-dependent cGAS activation. These findings have implications for the inflammatory response to viral infection and tumorigenesis, both of which substantially reprogram cellular protein synthesis.


Asunto(s)
Núcleo Celular , Nucleotidiltransferasas , Biosíntesis de Proteínas , Ribosomas , Transducción de Señal , Estrés Fisiológico , Transporte Activo de Núcleo Celular , Núcleo Celular/química , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Ribosomas/química , Ribosomas/genética , Ribosomas/metabolismo
5.
Mol Cell ; 81(14): 2944-2959.e10, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34166609

RESUMEN

A number of regulatory factors are recruited to chromatin by specialized RNAs. Whether RNA has a more general role in regulating the interaction of proteins with chromatin has not been determined. We used proteomics methods to measure the global impact of nascent RNA on chromatin in embryonic stem cells. Surprisingly, we found that nascent RNA primarily antagonized the interaction of chromatin modifiers and transcriptional regulators with chromatin. Transcriptional inhibition and RNA degradation induced recruitment of a set of transcriptional regulators, chromatin modifiers, nucleosome remodelers, and regulators of higher-order structure. RNA directly bound to factors, including BAF, NuRD, EHMT1, and INO80 and inhibited their interaction with nucleosomes. The transcriptional elongation factor P-TEFb directly bound pre-mRNA, and its recruitment to chromatin upon Pol II inhibition was regulated by the 7SK ribonucleoprotein complex. We postulate that by antagonizing the interaction of regulatory proteins with chromatin, nascent RNA links transcriptional output with chromatin composition.


Asunto(s)
Cromatina/metabolismo , ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/fisiología , Células HEK293 , Humanos , Masculino , Ratones , Nucleosomas/metabolismo , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica/fisiología , Proteómica/métodos , ARN Polimerasa II/metabolismo , Transcripción Genética/fisiología , Factores de Elongación Transcripcional/metabolismo
6.
Mol Cell ; 79(2): 332-341.e7, 2020 07 16.
Artículo en Inglés | MEDLINE | ID: mdl-32521225

RESUMEN

The Ddi1/DDI2 proteins are ubiquitin shuttling factors, implicated in a variety of cellular functions. In addition to ubiquitin-binding and ubiquitin-like domains, they contain a conserved region with similarity to retroviral proteases, but whether and how DDI2 functions as a protease has remained unknown. Here, we show that DDI2 knockout cells are sensitive to proteasome inhibition and accumulate high-molecular weight, ubiquitylated proteins that are poorly degraded by the proteasome. These proteins are targets for the protease activity of purified DDI2. No evidence for DDI2 acting as a de-ubiquitylating enzyme was uncovered, which could suggest that it cleaves the ubiquitylated protein itself. In support of this idea, cleavage of transcription factor NRF1 is known to require DDI2 activity in vivo. We show that DDI2 is indeed capable of cleaving NRF1 in vitro but only when NRF1 protein is highly poly-ubiquitylated. Together, these data suggest that DDI2 is a ubiquitin-directed endoprotease.


Asunto(s)
Proteasas de Ácido Aspártico/metabolismo , Factor Nuclear 1 de Respiración/metabolismo , Ubiquitina/metabolismo , Proteasas de Ácido Aspártico/genética , Sitios de Unión , Sistemas CRISPR-Cas , Línea Celular , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Biosíntesis de Proteínas , Proteolisis
7.
Mol Cell ; 79(6): 917-933.e9, 2020 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-32755595

RESUMEN

Despite key roles in sister chromatid cohesion and chromosome organization, the mechanism by which cohesin rings are loaded onto DNA is still unknown. Here we combine biochemical approaches and cryoelectron microscopy (cryo-EM) to visualize a cohesin loading intermediate in which DNA is locked between two gates that lead into the cohesin ring. Building on this structural framework, we design experiments to establish the order of events during cohesin loading. In an initial step, DNA traverses an N-terminal kleisin gate that is first opened upon ATP binding and then closed as the cohesin loader locks the DNA against the ATPase gate. ATP hydrolysis will lead to ATPase gate opening to complete DNA entry. Whether DNA loading is successful or results in loop extrusion might be dictated by a conserved kleisin N-terminal tail that guides the DNA through the kleisin gate. Our results establish the molecular basis for cohesin loading onto DNA.


Asunto(s)
Proteínas de Ciclo Celular/ultraestructura , Cromátides/ultraestructura , Proteínas Cromosómicas no Histona/ultraestructura , ADN/ultraestructura , Intercambio de Cromátides Hermanas/genética , Adenosina Trifosfatasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica/genética , Microscopía por Crioelectrón , ADN/genética , Conformación de Ácido Nucleico , Conformación Proteica , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/ultraestructura , Cohesinas
8.
Nature ; 589(7840): 103-109, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33239783

RESUMEN

Mammalian telomeres protect chromosome ends from aberrant DNA repair1. TRF2, a component of the telomere-specific shelterin protein complex, facilitates end protection through sequestration of the terminal telomere repeat sequence within a lariat T-loop structure2,3. Deleting TRF2 (also known as TERF2) in somatic cells abolishes T-loop formation, which coincides with telomere deprotection, chromosome end-to-end fusions and inviability3-9. Here we establish that, by contrast, TRF2 is largely dispensable for telomere protection in mouse pluripotent embryonic stem (ES) and epiblast stem cells. ES cell telomeres devoid of TRF2 instead activate an attenuated telomeric DNA damage response that lacks accompanying telomere fusions, and propagate for multiple generations. The induction of telomere dysfunction in ES cells, consistent with somatic deletion of Trf2 (also known as Terf2), occurs only following the removal of the entire shelterin complex. Consistent with TRF2 being largely dispensable for telomere protection specifically during early embryonic development, cells exiting pluripotency rapidly switch to TRF2-dependent end protection. In addition, Trf2-null embryos arrest before implantation, with evidence of strong DNA damage response signalling and apoptosis specifically in the non-pluripotent compartment. Finally, we show that ES cells form T-loops independently of TRF2, which reveals why TRF2 is dispensable for end protection during pluripotency. Collectively, these data establish that telomere protection is solved by distinct mechanisms in pluripotent and somatic tissues.


Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Pluripotentes/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/deficiencia , Animales , Blastocisto/citología , Blastocisto/metabolismo , Supervivencia Celular , Cromosomas de los Mamíferos/genética , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Telómero/genética , Proteína 2 de Unión a Repeticiones Teloméricas/genética , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo
9.
Immunity ; 47(6): 1083-1099.e6, 2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-29246442

RESUMEN

The immunosuppressive protein PD-L1 is upregulated in many cancers and contributes to evasion of the host immune system. The relative importance of the tumor microenvironment and cancer cell-intrinsic signaling in the regulation of PD-L1 expression remains unclear. We report that oncogenic RAS signaling can upregulate tumor cell PD-L1 expression through a mechanism involving increases in PD-L1 mRNA stability via modulation of the AU-rich element-binding protein tristetraprolin (TTP). TTP negatively regulates PD-L1 expression through AU-rich elements in the 3' UTR of PD-L1 mRNA. MEK signaling downstream of RAS leads to phosphorylation and inhibition of TTP by the kinase MK2. In human lung and colorectal tumors, RAS pathway activation is associated with elevated PD-L1 expression. In vivo, restoration of TTP expression enhances anti-tumor immunity dependent on degradation of PD-L1 mRNA. We demonstrate that RAS can drive cell-intrinsic PD-L1 expression, thus presenting therapeutic opportunities to reverse the innately immunoresistant phenotype of RAS mutant cancers.


Asunto(s)
Antígeno B7-H1/inmunología , Neoplasias Colorrectales/inmunología , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/inmunología , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Tristetraprolina/inmunología , Escape del Tumor , Animales , Antígeno B7-H1/genética , Línea Celular Tumoral , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Células Epiteliales/inmunología , Células Epiteliales/patología , Femenino , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Trasplante de Neoplasias , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Proteínas Proto-Oncogénicas p21(ras)/genética , División del ARN , Estabilidad del ARN , ARN Mensajero/genética , ARN Mensajero/inmunología , Transducción de Señal , Tristetraprolina/genética
10.
Mol Cell ; 72(6): 942-954.e7, 2018 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-30576656

RESUMEN

Many active eukaryotic gene promoters exhibit divergent noncoding transcription, but the mechanisms restricting expression of these transcripts are not well understood. Here, we demonstrate how a sequence-specific transcription factor represses divergent noncoding transcription at highly expressed genes in yeast. We find that depletion of the transcription factor Rap1 induces noncoding transcription in a large fraction of Rap1-regulated gene promoters. Specifically, Rap1 prevents transcription initiation at cryptic promoters near its binding sites, which is uncoupled from transcription regulation in the protein-coding direction. We further provide evidence that Rap1 acts independently of previously described chromatin-based mechanisms to repress cryptic or divergent transcription. Finally, we show that divergent transcription in the absence of Rap1 is elicited by the RSC chromatin remodeler. We propose that a sequence-specific transcription factor limits access of basal transcription machinery to regulatory elements and adjacent sequences that act as divergent cryptic promoters, thereby providing directionality toward productive transcription.


Asunto(s)
Regulación Fúngica de la Expresión Génica , ARN de Hongos/metabolismo , ARN no Traducido/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética , Sitios de Unión , Ensamble y Desensamble de Cromatina , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , ARN de Hongos/genética , ARN no Traducido/genética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genética , Factores de Transcripción/genética
11.
Mol Cell ; 70(4): 707-721.e7, 2018 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-29754823

RESUMEN

DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.


Asunto(s)
Carcinogénesis/patología , ADN Polimerasa II/química , ADN Polimerasa II/fisiología , Replicación del ADN , Discapacidades del Desarrollo/etiología , Trastornos del Crecimiento/etiología , Leucopenia/etiología , Animales , Carcinogénesis/genética , Células Cultivadas , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Femenino , Humanos , Recién Nacido , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Proteína p53 Supresora de Tumor/fisiología
12.
EMBO J ; 40(10): e106188, 2021 05 17.
Artículo en Inglés | MEDLINE | ID: mdl-33881780

RESUMEN

Tumour progression locus 2 (TPL-2) kinase mediates Toll-like receptor (TLR) activation of ERK1/2 and p38α MAP kinases in myeloid cells to modulate expression of key cytokines in innate immunity. This study identified a novel MAP kinase-independent regulatory function for TPL-2 in phagosome maturation, an essential process for killing of phagocytosed microbes. TPL-2 catalytic activity was demonstrated to induce phagosome acidification and proteolysis in primary mouse and human macrophages following uptake of latex beads. Quantitative proteomics revealed that blocking TPL-2 catalytic activity significantly altered the protein composition of phagosomes, particularly reducing the abundance of V-ATPase proton pump subunits. Furthermore, TPL-2 stimulated the phosphorylation of DMXL1, a regulator of V-ATPases, to induce V-ATPase assembly and phagosome acidification. Consistent with these results, TPL-2 catalytic activity was required for phagosome acidification and the efficient killing of Staphylococcus aureus and Citrobacter rodentium following phagocytic uptake by macrophages. TPL-2 therefore controls innate immune responses of macrophages to bacteria via V-ATPase induction of phagosome maturation.


Asunto(s)
Macrófagos/metabolismo , Fagosomas/metabolismo , Animales , Humanos , Quinasas Quinasa Quinasa PAM/metabolismo , Fosforilación/fisiología , Proteínas/metabolismo , Transducción de Señal/fisiología , Staphylococcus aureus/metabolismo
13.
EMBO J ; 40(11): e107226, 2021 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-33932049

RESUMEN

Malaria parasite egress from host erythrocytes (RBCs) is regulated by discharge of a parasite serine protease called SUB1 into the parasitophorous vacuole (PV). There, SUB1 activates a PV-resident cysteine protease called SERA6, enabling host RBC rupture through SERA6-mediated degradation of the RBC cytoskeleton protein ß-spectrin. Here, we show that the activation of Plasmodium falciparum SERA6 involves a second, autocatalytic step that is triggered by SUB1 cleavage. Unexpectedly, autoproteolytic maturation of SERA6 requires interaction in multimolecular complexes with a distinct PV-located protein cofactor, MSA180, that is itself a SUB1 substrate. Genetic ablation of MSA180 mimics SERA6 disruption, producing a fatal block in ß-spectrin cleavage and RBC rupture. Drug-like inhibitors of SERA6 autoprocessing similarly prevent ß-spectrin cleavage and egress in both P. falciparum and the emerging zoonotic pathogen P. knowlesi. Our results elucidate the egress pathway and identify SERA6 as a target for a new class of antimalarial drugs designed to prevent disease progression.


Asunto(s)
Antimaláricos/farmacología , Proteasas de Cisteína/metabolismo , Plasmodium falciparum/metabolismo , Inhibidores de Proteasas/farmacología , Proteínas Protozoarias/metabolismo , Células Cultivadas , Eritrocitos/metabolismo , Eritrocitos/parasitología , Humanos , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/patogenicidad , Proteolisis , Proteínas Protozoarias/antagonistas & inhibidores , Serina Proteasas/metabolismo , Espectrina/metabolismo
14.
EMBO J ; 40(14): e105985, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-34121209

RESUMEN

Autophagy is a process through which intracellular cargoes are catabolised inside lysosomes. It involves the formation of autophagosomes initiated by the serine/threonine kinase ULK and class III PI3 kinase VPS34 complexes. Here, unbiased phosphoproteomics screens in mouse embryonic fibroblasts deleted for Ulk1/2 reveal that ULK loss significantly alters the phosphoproteome, with novel high confidence substrates identified including VPS34 complex member VPS15 and AMPK complex subunit PRKAG2. We identify six ULK-dependent phosphorylation sites on VPS15, mutation of which reduces autophagosome formation in cells and VPS34 activity in vitro. Mutation of serine 861, the major VPS15 phosphosite, decreases both autophagy initiation and autophagic flux. Analysis of VPS15 knockout cells reveals two novel ULK-dependent phenotypes downstream of VPS15 removal that can be partially recapitulated by chronic VPS34 inhibition, starvation-independent accumulation of ULK substrates and kinase activity-regulated recruitment of autophagy proteins to ubiquitin-positive structures.


Asunto(s)
Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Autofagia/fisiología , Fosfatidilinositol 3-Quinasas Clase III/metabolismo , Proteína de Clasificación Vacuolar VPS15/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Ratones , Proteómica/métodos
15.
PLoS Pathog ; 19(6): e1011449, 2023 06.
Artículo en Inglés | MEDLINE | ID: mdl-37352369

RESUMEN

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles of LCAT-null parasites showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites.


Asunto(s)
Malaria Falciparum , Malaria , Parásitos , Animales , Parásitos/metabolismo , Fosfolipasas , Perforina , Proteómica , Eritrocitos/parasitología , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Malaria Falciparum/parasitología
16.
Nature ; 575(7783): 523-527, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31723267

RESUMEN

The protection of telomere ends by the shelterin complex prevents DNA damage signalling and promiscuous repair at chromosome ends. Evidence suggests that the 3' single-stranded telomere end can assemble into a lasso-like t-loop configuration1,2, which has been proposed to safeguard chromosome ends from being recognized as DNA double-strand breaks2. Mechanisms must also exist to transiently disassemble t-loops to allow accurate telomere replication and to permit telomerase access to the 3' end to solve the end-replication problem. However, the regulation and physiological importance of t-loops in the protection of telomere ends remains unknown. Here we identify a CDK phosphorylation site in the shelterin subunit at Ser365 of TRF2, whose dephosphorylation in S phase by the PP6R3 phosphatase provides a narrow window during which the RTEL1 helicase can transiently access and unwind t-loops to facilitate telomere replication. Re-phosphorylation of TRF2 at Ser365 outside of S phase is required to release RTEL1 from telomeres, which not only protects t-loops from promiscuous unwinding and inappropriate activation of ATM, but also counteracts replication conflicts at DNA secondary structures that arise within telomeres and across the genome. Hence, a phospho-switch in TRF2 coordinates the assembly and disassembly of t-loops during the cell cycle, which protects telomeres from replication stress and an unscheduled DNA damage response.


Asunto(s)
Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Fosfoserina/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/química , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , ADN/biosíntesis , ADN/química , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , Fibroblastos , Genoma/genética , Células HEK293 , Humanos , Ratones , Mutación , Fenotipo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Fase S , Complejo Shelterina , Telomerasa/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/química , Proteínas de Unión a Telómeros/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/genética
18.
Mol Cell ; 65(3): 393-402.e3, 2017 Feb 02.
Artículo en Inglés | MEDLINE | ID: mdl-28132839

RESUMEN

In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2ACdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Factores de Transcripción/metabolismo , Ciclo Celular , Quinasas Ciclina-Dependientes/metabolismo , Fosforilación , Serina/metabolismo , Transducción de Señal
19.
PLoS Biol ; 19(10): e3001408, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34695132

RESUMEN

We have combined chemical biology and genetic modification approaches to investigate the importance of protein myristoylation in the human malaria parasite, Plasmodium falciparum. Parasite treatment during schizogony in the last 10 to 15 hours of the erythrocytic cycle with IMP-1002, an inhibitor of N-myristoyl transferase (NMT), led to a significant blockade in parasite egress from the infected erythrocyte. Two rhoptry proteins were mislocalized in the cell, suggesting that rhoptry function is disrupted. We identified 16 NMT substrates for which myristoylation was significantly reduced by NMT inhibitor (NMTi) treatment, and, of these, 6 proteins were substantially reduced in abundance. In a viability screen, we showed that for 4 of these proteins replacement of the N-terminal glycine with alanine to prevent myristoylation had a substantial effect on parasite fitness. In detailed studies of one NMT substrate, glideosome-associated protein 45 (GAP45), loss of myristoylation had no impact on protein location or glideosome assembly, in contrast to the disruption caused by GAP45 gene deletion, but GAP45 myristoylation was essential for erythrocyte invasion. Therefore, there are at least 3 mechanisms by which inhibition of NMT can disrupt parasite development and growth: early in parasite development, leading to the inhibition of schizogony and formation of "pseudoschizonts," which has been described previously; at the end of schizogony, with disruption of rhoptry formation, merozoite development and egress from the infected erythrocyte; and at invasion, when impairment of motor complex function prevents invasion of new erythrocytes. These results underline the importance of P. falciparum NMT as a drug target because of the pleiotropic effect of its inhibition.


Asunto(s)
Eritrocitos/parasitología , Ácido Mirístico/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Aciltransferasas/antagonistas & inhibidores , Aciltransferasas/metabolismo , Animales , Sistemas CRISPR-Cas/genética , Supervivencia Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Lipoilación/efectos de los fármacos , Merozoítos/efectos de los fármacos , Merozoítos/metabolismo , Parásitos/efectos de los fármacos , Parásitos/crecimiento & desarrollo , Plasmodium falciparum/efectos de los fármacos , Plasmodium falciparum/enzimología , Plasmodium falciparum/ultraestructura , Solubilidad , Especificidad por Sustrato/efectos de los fármacos
20.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-33883278

RESUMEN

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.


Asunto(s)
Neoplasias/tratamiento farmacológico , Estrés Fisiológico/efectos de los fármacos , Antineoplásicos/farmacología , Autofagia/fisiología , Línea Celular Tumoral , Humanos , Metaboloma/genética , Mitocondrias/metabolismo , Mieloma Múltiple/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatología , Inhibidores de Proteasoma/farmacología , Proteolisis , Proteoma/genética , Análisis de Sistemas , Transcriptoma/genética
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