RESUMEN
Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Estabilidad del ARN , ARN Mensajero/metabolismo , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Ribosomas/metabolismoRESUMEN
Certain proteins and organelles can be selectively degraded by autophagy. Typical substrates and receptors of selective autophagy have LC3-interacting regions (LIRs) that bind to autophagosomal LC3 and GABARAP family proteins. Here, we performed a differential interactome screen using wild-type LC3B and a LIR recognition-deficient mutant and identified TEX264 as a receptor for autophagic degradation of the endoplasmic reticulum (ER-phagy). TEX264 is an ER protein with a single transmembrane domain and a LIR motif. TEX264 interacts with LC3 and GABARAP family proteins more efficiently and is expressed more ubiquitously than previously known ER-phagy receptors. ER-phagy is profoundly blocked by deletion of TEX264 alone and almost completely by additional deletion of FAM134B and CCPG1. A long intrinsically disordered region of TEX264 is required for its ER-phagy receptor function to bridge the gap between the ER and autophagosomal membranes independently of its amino acid sequence. These results suggest that TEX264 is a major ER-phagy receptor.
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Proteínas Relacionadas con la Autofagia/genética , Autofagia/genética , Retículo Endoplásmico/genética , Proteínas Intrínsecamente Desordenadas/genética , Secuencia de Aminoácidos/genética , Proteínas Relacionadas con la Autofagia/química , Proteínas de Ciclo Celular/genética , Retículo Endoplásmico/química , Estrés del Retículo Endoplásmico/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteínas Intrínsecamente Desordenadas/química , Proteínas de la Membrana , Proteínas Asociadas a Microtúbulos/genética , Proteínas de Neoplasias/genética , ProteolisisRESUMEN
Mbtd1 (mbt domain containing 1) encodes a nuclear protein containing a zinc finger domain and four malignant brain tumor (MBT) repeats. We previously generated Mbtd1-deficient mice and found that MBTD1 is highly expressed in fetal hematopoietic stem cells (HSCs) and sustains the number and function of fetal HSCs. However, since Mbtd1-deficient mice die soon after birth possibly due to skeletal abnormalities, its role in adult hematopoiesis remains unclear. To address this issue, we generated Mbtd1 conditional knockout mice and analyzed adult hematopoietic tissues deficient in Mbtd1. We observed that the numbers of HSCs and progenitors increased and Mbtd1-deficient HSCs exhibited hyperactive cell cycle, resulting in a defective response to exogenous stresses. Mechanistically, we found that MBTD1 directly binds to the promoter region of FoxO3a, encoding a forkhead protein essential for HSC quiescence, and interacts with components of TIP60 chromatin remodeling complex and other proteins involved in HSC and other stem cell functions. Restoration of FOXO3a activity in Mbtd1-deficient HSCs in vivo rescued cell cycle and pool size abnormalities. These findings indicate that MBTD1 is a critical regulator for HSC pool size and function, mainly through the maintenance of cell cycle quiescence by FOXO3a.
Asunto(s)
Médula Ósea , Células Madre Hematopoyéticas , Animales , Ratones , Ciclo Celular/genética , Hematopoyesis/genética , Células Madre Hematopoyéticas/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Factores de Transcripción/metabolismoRESUMEN
Nuclear stress bodies (nSBs) are nuclear membraneless organelles formed around stress-inducible HSATIII architectural long noncoding RNAs (lncRNAs). nSBs repress splicing of hundreds of introns during thermal stress recovery, which are partly regulated by CLK1 kinase phosphorylation of temperature-dependent Ser/Arg-rich splicing factors (SRSFs). Here, we report a distinct mechanism for this splicing repression through protein sequestration by nSBs. Comprehensive identification of RNA-binding proteins revealed HSATIII association with proteins related to N6 -methyladenosine (m6 A) RNA modification. 11% of the first adenosine in the repetitive HSATIII sequence were m6 A-modified. nSBs sequester the m6 A writer complex to methylate HSATIII, leading to subsequent sequestration of the nuclear m6 A reader, YTHDC1. Sequestration of these factors from the nucleoplasm represses m6 A modification of pre-mRNAs, leading to repression of m6 A-dependent splicing during stress recovery phase. Thus, nSBs serve as a common platform for regulation of temperature-dependent splicing through dual mechanisms employing two distinct ribonucleoprotein modules with partially m6 A-modified architectural lncRNAs.
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Proteínas del Tejido Nervioso/genética , Factores de Empalme de ARN/genética , Empalme del ARN , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Núcleo Celular/genética , Células HeLa , Humanos , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Factores de Empalme de ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetitivas de Ácidos Nucleicos , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismo , TemperaturaRESUMEN
Although the herpes simplex virus type 1 (HSV-1) genome was thought to contain approximately 80 different protein coding sequences (CDSs), recent multi-omics analyses reported HSV-1 encodes more than 200 potential CDSs. However, few of the newly identified CDSs were confirmed to be expressed at the peptide or protein level in HSV-1-infected cells. Furthermore, the impact of the proteins they encode on HSV-1 infection is largely unknown. This study focused on a newly identified CDS, UL31.6. Re-analyzation of our previous chemical proteomics data verified that UL31.6 was expressed at the peptide level in HSV-1-infected cells. Antisera raised against a viral protein encoded by UL31.6 (pUL31.6) reacted with a protein with an approximate molecular mass of 37 kDa in lysates of Vero cells infected with each of three HSV-1 strains. pUL31.6 was efficiently dissociated from virions in high-salt solution. A UL31.6-null mutation had a minimal effect on HSV-1 gene expression, replication, cell-to-cell spread, and morphogenesis in Vero cells; in contrast, it significantly reduced HSV-1 cell-to-cell spread in three neural cells but not in four non-neural cells including Vero cells. The UL31.6-null mutation also significantly reduced the mortality and viral replication in the brains of mice after intracranial infection, but had minimal effects on pathogenic manifestations in and around the eyes, and viral replication detected in the tear films of mice after ocular infection. These results indicated that pUL31.6 was a tegument protein and specifically acted as a neurovirulence factor by potentially promoting viral transmission between neuronal cells in the central nervous system.IMPORTANCERecent multi-omics analyses reported the herpes simplex virus type 1 (HSV-1) genome encodes an additional number of potential coding sequences (CDSs). However, the expressions of these CDSs at the peptide or protein levels and the biological effects of these CDSs on HSV-1 infection remain largely unknown. This study annotated a cryptic orphan CDS, termed UL31.6, an HSV-1 gene that encodes a tegument protein with an approximate molecular mass of 37 kDa, which specifically acts as a neurovirulence factor. Our study indicates that HSV-1 proteins important for viral pathogenesis remain to be identified and a comprehensive understanding of the pathogenesis of HSV-1 will require not only the identification of cryptic orphan CDSs using emerging technologies but also step-by-step and in-depth analyses of each of the cryptic orphan CDSs.
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Herpesvirus Humano 1 , Proteínas Virales , Replicación Viral , Animales , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/patogenicidad , Herpesvirus Humano 1/fisiología , Chlorocebus aethiops , Células Vero , Ratones , Proteínas Virales/genética , Proteínas Virales/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Herpes Simple/virología , Virulencia , Femenino , HumanosRESUMEN
A number of long noncoding RNAs (lncRNAs) are induced in response to specific stresses to construct membrane-less nuclear bodies; however, their function remains poorly understood. Here, we report the role of nuclear stress bodies (nSBs) formed on highly repetitive satellite III (HSATIII) lncRNAs derived from primate-specific satellite III repeats upon thermal stress exposure. A transcriptomic analysis revealed that depletion of HSATIII lncRNAs, resulting in elimination of nSBs, promoted splicing of 533 retained introns during thermal stress recovery. A HSATIII-Comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS) analysis identified multiple splicing factors in nSBs, including serine and arginine-rich pre-mRNA splicing factors (SRSFs), the phosphorylation states of which affect splicing patterns. SRSFs are rapidly de-phosphorylated upon thermal stress exposure. During stress recovery, CDC like kinase 1 (CLK1) was recruited to nSBs and accelerated the re-phosphorylation of SRSF9, thereby promoting target intron retention. Our findings suggest that HSATIII-dependent nSBs serve as a conditional platform for phosphorylation of SRSFs by CLK1 to promote the rapid adaptation of gene expression through intron retention following thermal stress exposure.
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Núcleo Celular/metabolismo , Respuesta al Choque Térmico , Repeticiones de Microsatélite , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , ARN Largo no Codificante/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Animales , Células CHO , Cricetulus , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Intrones , Fosforilación , Factores de Empalme de ARN/metabolismo , Secuenciación del ExomaRESUMEN
The 26S proteasome is an enzymatic complex that degrades ubiquitinated proteins in eukaryotic cells. It is composed of the 20S core particle (CP) and the 19S regulatory particle (RP). The latter is further divided into the lid and base subcomplexes. While the mechanism involved in the assembly of the CP is well investigated, that of the RP is poorly understood. Here, we show that the formation of the mammalian base subcomplex involves three distinct modules, where specific pairs of ATPase subunits are associated with the distinct chaperones p28, S5b, or p27. The process of base formation starts from association of the p28-Rpt3-Rpt6-Rpn14 complex with the S5b-Rpt1-Rpt2-Rpn1 complex, followed by incorporation of the p27-Rpt5-Rpt4 complex and Rpn2, where p28, S5b, and p27 regulate the associations between the modules. These chaperones dissociate before completion of 26S proteasome formation. Our results demonstrate that base assembly is facilitated by multiple proteasome-dedicated chaperones, like CP assembly.
Asunto(s)
Complejo de la Endopetidasa Proteasomal/metabolismo , Línea Celular , Técnicas de Silenciamiento del Gen , Humanos , Chaperonas Moleculares/metabolismoRESUMEN
The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwi-dependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Silenciador del Gen , ARN Interferente Pequeño/metabolismo , Animales , Proteínas Argonautas/metabolismo , Femenino , Regulación de la Expresión Génica , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Transcripción GenéticaRESUMEN
This study developed a system consisting of two rounds of screening cellular proteins involved in the nuclear egress of herpes simplex virus 1 (HSV-1). Using this system, we first screened cellular proteins that interacted with the HSV-1 nuclear egress complex (NEC) consisting of UL34 and UL31 in HSV-1-infected cells, which are critical for the nuclear egress of HSV-1, by tandem affinity purification coupled with mass spectrometry-based proteomics technology. Next, we performed CRISPR/Cas9-based screening of live HSV-1-infected reporter cells under fluorescence microscopy using single guide RNAs targeting the cellular proteins identified in the first proteomic screening to detect the mislocalization of the lamin-associated protein emerin, which is a phenotype for defects in HSV-1 nuclear egress. This study focused on a cellular orphan transporter SLC35E1, one of the cellular proteins identified by the screening system. Knockout of SLC35E1 reduced HSV-1 replication and induced membranous invaginations containing perinuclear enveloped virions (PEVs) adjacent to the nuclear membrane (NM), aberrant accumulation of PEVs in the perinuclear space between the inner and outer NMs and the invagination structures, and mislocalization of the NEC. These effects were similar to those of previously reported mutation(s) in HSV-1 proteins and depletion of cellular proteins that are important for HSV-1 de-envelopment, one of the steps required for HSV-1 nuclear egress. Our newly established screening system enabled us to identify a novel cellular protein required for efficient HSV-1 de-envelopment. IMPORTANCE The identification of cellular protein(s) that interact with viral effector proteins and function in important viral procedures is necessary for enhancing our understanding of the mechanics of various viral processes. In this study, we established a new system consisting of interactome screening for the herpes simplex virus 1 (HSV-1) nuclear egress complex (NEC), followed by loss-of-function screening to target the identified putative NEC-interacting cellular proteins to detect a defect in HSV-1 nuclear egress. This newly established system identified SLC35E1, an orphan transporter, as a novel cellular protein required for efficient HSV-1 de-envelopment, providing an insight into the mechanisms involved in this viral procedure.
Asunto(s)
Herpesvirus Humano 1 , Proteínas de Transporte de Membrana , Liberación del Virus , Animales , Sistemas CRISPR-Cas , Chlorocebus aethiops , Técnicas de Inactivación de Genes , Células HEK293 , Células HeLa , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/fisiología , Humanos , Proteínas de Transporte de Membrana/metabolismo , Membrana Nuclear/metabolismo , Proteínas Nucleares , Proteómica , Células Vero , Proteínas Virales/metabolismoRESUMEN
Viral RNA in the cytoplasm of mammalian host cells is recognized by retinoic acid-inducible protein-I-like receptors (RLRs), which localize to cytoplasmic stress granules (SGs). Activated RLRs associate with the mitochondrial adaptor protein IPS-1, which activates antiviral host defense mechanisms, including type I IFN induction. It has remained unclear, however, how RLRs in SGs and IPS-1 in the mitochondrial outer membrane associate physically and engage in information transfer. In this study, we show that NUDT21, an RNA-binding protein that regulates alternative transcript polyadenylation, physically associates with IPS-1 and mediates its localization to SGs in response to transfection with polyinosinic-polycytidylic acid [poly(I:C)], a mimic of viral dsRNA. We found that despite its well-established function in the nucleus, a fraction of NUDT21 localizes to mitochondria in resting cells and becomes localized to SGs in response to poly(I:C) transfection. NUDT21 was also found to be required for efficient type I IFN induction in response to viral infection in both human HeLa cells and mouse macrophage cell line RAW264.7 cells. Our results together indicate that NUDT21 links RLRs in SGs to mitochondrial IPS-1 and thereby activates host defense responses to viral infection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Infecciones por Cardiovirus/metabolismo , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteína 58 DEAD Box/metabolismo , Virus de la Encefalomiocarditis/fisiología , Mitocondrias/metabolismo , Enfermedad de Newcastle/metabolismo , Virus de la Enfermedad de Newcastle/fisiología , Receptores Inmunológicos/metabolismo , Vesículas Secretoras/metabolismo , Animales , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Ratones , Poli I-C/inmunología , Transporte de Proteínas , Células RAW 264.7 , ARN Interferente Pequeño/genética , ARN Viral/inmunología , Estrés FisiológicoRESUMEN
Unlimited organ availability would represent a paradigm shift in transplantation. Long-term in vivo engraftment and function of scaled-up bioengineered liver grafts have not been previously reported. In this study, we describe a human-scale transplantable liver graft engineered on a porcine liver-derived scaffold. We repopulated the scaffold parenchyma with primary hepatocytes and the vascular system with endothelial cells. For in vivo functional testing, we performed auxiliary transplantation of the repopulated scaffold in pigs with induced liver failure. It was observed that the auxiliary bioengineered liver graft improved liver function for 28 days and exhibited upregulation of liver-specific genes. This study is the first of its kind to present 28 days of posttransplant evaluation of a bioengineered liver graft using a preclinical large animal model. Furthermore, it provides definitive evidence for the feasibility of engineering human-scale transplantable liver grafts for clinical applications.
Asunto(s)
Fallo Hepático , Trasplante de Hígado , Animales , Células Endoteliales , Hepatocitos/trasplante , Hígado/irrigación sanguínea , Porcinos , Ingeniería de Tejidos , Andamios del TejidoRESUMEN
The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.
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Glicoproteínas de Membrana/agonistas , ARN Nuclear Pequeño/inmunología , Receptor Toll-Like 7/agonistas , Adulto , Alarminas/química , Animales , Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/inmunología , Secuencia de Bases , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunosupresores/síntesis química , Inmunosupresores/farmacología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/inmunología , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Persona de Mediana Edad , ARN/inmunología , ARN/metabolismo , Ribonucleoproteínas Nucleares Pequeñas/química , Ribonucleoproteínas Nucleares Pequeñas/inmunología , Análisis de Secuencia de ARN , Receptor Toll-Like 7/deficiencia , Adulto JovenRESUMEN
Codon bias has been implicated as one of the major factors contributing to mRNA stability in several model organisms. However, the molecular mechanisms of codon bias on mRNA stability remain unclear in humans. Here, we show that human cells possess a mechanism to modulate RNA stability through a unique codon bias. Bioinformatics analysis showed that codons could be clustered into two distinct groups-codons with G or C at the third base position (GC3) and codons with either A or T at the third base position (AT3): the former stabilizing while the latter destabilizing mRNA. Quantification of codon bias showed that increased GC3-content entails proportionately higher GC-content. Through bioinformatics, ribosome profiling, and in vitro analysis, we show that decoupling the effects of codon bias reveals two modes of mRNA regulation, one GC3- and one GC-content dependent. Employing an immunoprecipitation-based strategy, we identify ILF2 and ILF3 as RNA-binding proteins that differentially regulate global mRNA abundances based on codon bias. Our results demonstrate that codon bias is a two-pronged system that governs mRNA abundance.
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Uso de Codones , Codón , ARN Mensajero/genética , Biología Computacional/métodos , Guanilato Ciclasa/genética , Humanos , Proteína del Factor Nuclear 45/metabolismo , Estabilidad del ARN , Ribosomas/genética , Ribosomas/metabolismo , Transcripción GenéticaRESUMEN
Although mechanisms for protein homeostasis in the cytosol have been studied extensively, those in the nucleus remain largely unknown. Here, we identified that a protein complex mediates export of polyubiquitinated proteins from the nucleus to the cytosol. UBIN, a ubiquitin-associated (UBA) domain-containing protein, shuttled between the nucleus and the cytosol in a CRM1-dependent manner, despite the lack of intrinsic nuclear export signal (NES). Instead, the UBIN binding protein polyubiquitinated substrate transporter (POST) harboring an NES shuttled UBIN through nuclear pores. UBIN bound to polyubiquitin chain through its UBA domain, and the UBIN-POST complex exported them from the nucleus to the cytosol. Ubiquitinated proteins accumulated in the cytosol in response to proteasome inhibition, whereas cotreatment with CRM1 inhibitor led to their accumulation in the nucleus. Our results suggest that ubiquitinated proteins are exported from the nucleus to the cytosol in the UBIN-POST complex-dependent manner for the maintenance of nuclear protein homeostasis.
Asunto(s)
Proteínas Portadoras/metabolismo , Núcleo Celular/metabolismo , Citosol/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Ubiquitinadas/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Animales , Proteínas Portadoras/genética , Núcleo Celular/genética , Células HEK293 , Células HeLa , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Proteínas de la Membrana/genética , Ratones , Células 3T3 NIH , Proteínas Nucleares/genética , Proteínas Transportadoras de Solutos , Proteínas Ubiquitinadas/genéticaRESUMEN
Metabolic reprogramming, including the Warburg effect, is a hallmark of cancer. Indeed, the diversity of cancer metabolism leads to cancer heterogeneity, but accurate assessment of metabolic properties in tumors has not yet been undertaken. Here, we performed absolute quantification of the expression levels of 113 proteins related to carbohydrate metabolism and antioxidant pathways, in stage III colorectal cancer surgical specimens from 70 patients. The Warburg effect appeared in absolute protein levels between tumor and normal mucosa specimens demonstrated. Notably, the levels of proteins associated with the tricarboxylic citric acid cycle were remarkably reduced in the malignant tumors which had relapsed after surgery and treatment with 5-fluorouracil-based adjuvant therapy. In addition, the efficacy of 5-fluorouracil also decreased in the cultured cancer cell lines with promotion of the Warburg effect. We further identified nine and eight important proteins, which are closely related to the Warburg effect, for relapse risk and 5-fluorouracil benefit, respectively, using a biomarker exploration procedure. These results provide us a clue for bridging between metabolic protein expression profiles and benefit from 5-fluorouracil adjuvant chemotherapy.
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Antioxidantes/metabolismo , Metabolismo de los Hidratos de Carbono , Neoplasias Colorrectales/tratamiento farmacológico , Fluorouracilo/administración & dosificación , Adulto , Anciano , Quimioterapia Adyuvante , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Femenino , Fluorouracilo/farmacología , Fluorouracilo/uso terapéutico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Transducción de Señal/efectos de los fármacos , Resultado del TratamientoRESUMEN
Maintaining protein homeostasis is central to cell survival. The ubiquitin-proteasome system and autophagy play pivotal roles in protein quality control through protein degradation. Activities of these degradative pathways are carefully orchestrated, and autophagy is up-regulated during proteasome dysfunction for cellular homeostasis. However, the mechanism by which proteasome impairment induces compensatory autophagy has remained largely elusive. Here, we show that FAM48A mediates autophagy induction during proteasome inhibition. FAM48A is degraded by the proteasome and accumulates in cells by proteasome inhibition. Knockdown of FAM48A led to defective induction of autophagy during proteasome inhibition and accompanied by defective localization of Atg9 on recycling endosomes. Our results indicate that FAM48A is a kind of sensor that is required for compensatory autophagy induction upon proteasome impairment.
Asunto(s)
Autofagia , Complejo de la Endopetidasa Proteasomal/metabolismo , Factores de Transcripción/genética , Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Humanos , Inmunohistoquímica , Especificidad por Sustrato , Factores de Transcripción/metabolismoRESUMEN
Targeted proteomics approaches are of value for deep and accurate quantification of protein abundance. Extending such methods to quantify large numbers of proteins requires the construction of predefined targeted assays. We developed a targeted proteomics platform-in vitro proteome-assisted multiple reaction monitoring (MRM) for protein absolute quantification (iMPAQT)-by using >18,000 human recombinant proteins, thus enabling protein absolute quantification on a genome-wide scale. Our platform comprises experimentally confirmed MRM assays of mass tag (mTRAQ)-labeled peptides to allow for rapid and straightforward measurement of the absolute abundance of predefined sets of proteins by mass spectrometry. We applied iMPAQT to delineate the quantitative metabolic landscape of normal and transformed human fibroblasts. Oncogenic transformation gave rise to relatively small but global changes in metabolic pathways resulting in aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. iMPAQT should facilitate quantitative biology studies based on protein abundance measurements.
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Genoma Humano/genética , Espectrometría de Masas/métodos , Proteoma/análisis , Proteómica/métodos , Línea Celular Transformada , Fibroblastos/metabolismo , Glucólisis/fisiología , Humanos , Biblioteca de Péptidos , Proteínas Recombinantes/análisisRESUMEN
Reactive oxygen species (ROS)-induced activation of Apoptosis signal-regulating kinase 1 (ASK1) plays crucial roles in oxidative stress-mediated cell death through the activation of the JNK and p38 MAPK pathways. However, the regulatory mechanism of ASK1 in the oxidative stress response remains to be elucidated. Here, we identified the kelch repeat protein, Slim, as an activator of ASK1 through a Drosophila misexpression screen. We also performed a proteomics screen and revealed that Kelch domain containing 10 (KLHDC10), a mammalian ortholog of Slim, interacted with Protein phosphatase 5 (PP5), which has been shown to inactivate ASK1 in response to ROS. KLHDC10 bound to the phosphatase domain of PP5 and suppressed its phosphatase activity. Moreover, KLHDC10 was required for H(2)O(2)-induced sustained activation of ASK1 and cell death in Neuro2A cells. These findings suggest that Slim/KLHDC10 is an activator of ASK1, contributing to oxidative stress-induced cell death through the suppression of PP5.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas Nucleares/metabolismo , Estrés Oxidativo , Fosfoproteínas Fosfatasas/metabolismo , Animales , Proteínas Portadoras/genética , Muerte Celular , Línea Celular Tumoral , Clonación Molecular , Proteínas de Drosophila/genética , Drosophila melanogaster/efectos de los fármacos , Drosophila melanogaster/genética , Activación Enzimática , Regulación de la Expresión Génica , Células HEK293 , Humanos , Peróxido de Hidrógeno/farmacología , MAP Quinasa Quinasa Quinasa 5/genética , Quinasas Quinasa Quinasa PAM/genética , Melaninas/metabolismo , Ratones , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/genética , Oxidantes/farmacología , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Fosfoproteínas Fosfatasas/genética , Mutación Puntual , Dominios y Motivos de Interacción de Proteínas , Mapeo de Interacción de Proteínas , Proteómica , Interferencia de ARN , Transducción de Señal , Factores de Tiempo , Transfección , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Transcription factor access to regulatory elements is prevented by the nucleosome. Heat shock factor 1 (HSF1) is a winged helix transcription factor that plays roles in control and stressed conditions by gaining access to target elements, but mechanisms of HSF1 access are not well known in mammalian cells. Here, we show the physical interaction between the wing motif of human HSF1 and replication protein A (RPA), which is involved in DNA metabolism. Depletion of RPA1 abolishes HSF1 access to the promoter of HSP70 in unstressed condition and delays its rapid activation in response to heat shock. The HSF1-RPA complex leads to preloading of RNA polymerase II and opens the chromatin structure by recruiting a histone chaperone, FACT. Furthermore, this interaction is required for melanoma cell proliferation. These results provide a mechanism of constitutive HSF1 access to nucleosomal DNA, which is important for both basal and inducible gene expression.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad , Elementos Reguladores de la Transcripción , Proteína de Replicación A/metabolismo , Factores de Transcripción/metabolismo , Factores de Elongación Transcripcional , Secuencia de Aminoácidos , Secuencia de Bases , Cromatina/genética , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/genética , Células HEK293 , Factores de Transcripción del Choque Térmico , Proteínas del Grupo de Alta Movilidad/genética , Proteínas del Grupo de Alta Movilidad/metabolismo , Humanos , Datos de Secuencia Molecular , Nucleosomas/genética , Regiones Promotoras Genéticas , Unión Proteica , Dominios y Motivos de Interacción de Proteínas/genética , ARN Polimerasa II/metabolismo , Factores de Transcripción/genética , Factores de Elongación Transcripcional/genética , Factores de Elongación Transcripcional/metabolismoRESUMEN
Transcripts of alpha-fetoprotein (Afp), H19, and insulin-like growth factor 2 (Igf2) genes are highly expressed in mouse fetal liver, but decrease drastically during maturation. While transcriptional regulation of these genes has been well studied, the post-transcriptional regulation of their developmental decrease is poorly understood. Here, we show that shortening of poly(A) tails and subsequent RNA decay are largely responsible for the postnatal decrease of Afp, H19, and Igf2 transcripts in mouse liver. IGF2 mRNA binding protein 1 (IMP1), which regulates stability and translation efficiency of target mRNAs, binds to these fetal liver transcripts. When IMP1 is exogenously expressed in mouse adult liver, fetal liver transcripts show higher expression and possess longer poly(A) tails, suggesting that IMP1 stabilizes them. IMP1 declines concomitantly with fetal liver transcripts as liver matures. Instead, RNA-binding proteins (RBPs) that promote RNA decay, such as cold shock domain containing protein E1 (CSDE1), K-homology domain splicing regulatory protein (KSRP), and CUG-BP1 and ETR3-like factors 1 (CELF1), bind to 3' regions of fetal liver transcripts. These data suggest that transitions among RBPs associated with fetal liver transcripts shift regulation from stabilization to decay, leading to a postnatal decrease in those fetal transcripts.