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1.
Brain ; 146(8): 3373-3391, 2023 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-36825461

RESUMEN

GGC repeat expansion in the 5' untranslated region (UTR) of NOTCH2NLC is associated with a broad spectrum of neurological disorders, especially neuronal intranuclear inclusion disease (NIID). Studies have found that GGC repeat expansion in NOTCH2NLC induces the formation of polyglycine (polyG)-containing protein, which is involved in the formation of neuronal intranuclear inclusions. However, the mechanism of neurotoxicity induced by NOTCH2NLC GGC repeats is unclear. Here, we used NIID patient-specific induced pluripotent stem cell (iPSC)-derived 3D cerebral organoids (3DCOs) and cellular models to investigate the pathophysiological mechanisms of NOTCH2NLC GGC repeat expansion. IPSC-derived 3DCOs and cellular models showed the deposition of polyG-containing intranuclear inclusions. The NOTCH2NLC GGC repeats could induce the upregulation of autophagic flux, enhance integrated stress response and activate EIF2α phosphorylation. Bulk RNA sequencing for iPSC-derived neurons and single-cell RNA sequencing (scRNA-seq) for iPSC-derived 3DCOs revealed that NOTCH2NLC GGC repeats may be associated with dysfunctions in ribosome biogenesis and translation. Moreover, NOTCH2NLC GGC repeats could induce the NPM1 nucleoplasm translocation, increase nucleolar stress, impair ribosome biogenesis and induce ribosomal RNA sequestration, suggesting dysfunction of membraneless organelles in the NIID cellular model. Dysfunctions in ribosome biogenesis and phosphorylated EIF2α and the resulting increase in the formation of G3BP1-positive stress granules may together lead to whole-cell translational inhibition, which may eventually cause cell death. Interestingly, scRNA-seq revealed that NOTCH2NLC GGC repeats may be associated with a significantly decreased proportion of immature neurons while 3DCOs were developing. Together, our results underscore the value of patient-specific iPSC-derived 3DCOs in investigating the mechanisms of polyG diseases, especially those caused by repeats in human-specific genes.


Asunto(s)
ADN Helicasas , ARN Helicasas , Humanos , Proteínas de Unión a Poli-ADP-Ribosa , Proteínas con Motivos de Reconocimiento de ARN , Regiones no Traducidas 5' , Cuerpos de Inclusión Intranucleares , Ribosomas , Expansión de Repetición de Trinucleótido/genética
2.
Nature ; 461(7260): 114-9, 2009 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-19675569

RESUMEN

TRAF6 is a ubiquitin ligase that is essential for the activation of NF-kappaB and MAP kinases in several signalling pathways, including those emanating from the interleukin 1 and Toll-like receptors. TRAF6 functions together with a ubiquitin-conjugating enzyme complex consisting of UBC13 (also known as UBE2N) and UEV1A (UBE2V1) to catalyse Lys 63-linked polyubiquitination, which activates the TAK1 (also known as MAP3K7) kinase complex. TAK1 in turn phosphorylates and activates IkappaB kinase (IKK), leading to the activation of NF-kappaB. Although several proteins are known to be polyubiquitinated in the IL1R and Toll-like receptor pathways, it is not clear whether ubiquitination of any of these proteins is important for TAK1 or IKK activation. By reconstituting TAK1 activation in vitro using purified proteins, here we show that free Lys 63 polyubiquitin chains, which are not conjugated to any target protein, directly activate TAK1 by binding to the ubiquitin receptor TAB2 (also known as MAP3K7IP2). This binding leads to autophosphorylation and activation of TAK1. Furthermore, we found that unanchored polyubiquitin chains synthesized by TRAF6 and UBCH5C (also known as UBE2D3) activate the IKK complex. Disassembly of the polyubiquitin chains by deubiquitination enzymes prevented TAK1 and IKK activation. These results indicate that unanchored polyubiquitin chains directly activate TAK1 and IKK, suggesting a new mechanism of protein kinase regulation.


Asunto(s)
Quinasa I-kappa B/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Poliubiquitina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Línea Celular , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , Enzima Desubiquitinante CYLD , Activación Enzimática/efectos de los fármacos , Células HeLa , Humanos , Interleucina-1beta/farmacología , Lisina/metabolismo , Fosforilación , Poliubiquitina/biosíntesis , Receptores Inmunológicos , Factor 6 Asociado a Receptor de TNF/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Enzimas Ubiquitina-Conjugadoras , Ubiquitinación
3.
Sci Rep ; 11(1): 3857, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33594198

RESUMEN

Accurate evaluation of liver steatosis is required from brain-dead donors (BDDs) with nonalcoholic fatty liver disease (NAFLD). Our purposes were to investigate expression and regulation of connective tissue growth factor (CTGF) expression in livers from human and rat after brain death, and further evaluate its potential application. NAFLD and brain death models were established in rats. LX2 cells were cultured under hypoxia/reoxygenation. CTGF protein and mRNA levels were measured in liver samples from BDDs of human and rat by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction. YAP-regulated CTGF expression was investigated in LX2 cells via YAP small interfering RNA and Verteporfin treatment. Blood CTGF level from BDDs was measured by enzyme-linked immunosorbent assay. After brain death, CTGF, transforming growth factor-ß and YAP were overexpressed in non-alcoholic steatotic liver, whereas CTGF was downregulated in non-steatotic liver. Time-series analysis revealed that CTGF and YAP expression was comparable, as confirmed by inhibited YAP expression in LX2 cells. CTGF level and NAFLD activity were linearly correlated. CTGF expression and regulation differ between non-steatosis and nonalcoholic steatosis livers from BDDs. CTGF may be an important factor to evaluate graft quality from BDDs with NAFLD.


Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Animales , Biomarcadores/metabolismo , Muerte Encefálica , Humanos , Masculino , Ratas Endogámicas Lew , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Señalizadoras YAP/metabolismo
4.
Sci Adv ; 7(41): eabh1756, 2021 Oct 08.
Artículo en Inglés | MEDLINE | ID: mdl-34613781

RESUMEN

RIP1 has emerged as a master regulator in TNFα signaling that controls two distinct cellular fates: cell survival versus programmed cell death. Because the default response of most cells to TNFα is NF-κB­mediated inflammation and survival, a specific mechanism must exist to control the divergence of signaling outcome. Here, we identify HSPA13 as a transcription-independent checkpoint to modulate the role of RIP1 in TNFα signaling. Through specific binding to TNFR1 and RIP1, HSPA13 enhances TNFα-induced recruitment of RIP1 to TNFR1, and consequently promotes downstream NF-κB transcriptional responses. Meanwhile, HSPA13 attenuates the participation of RIP1 in cytosolic complex II and prevents cells from programmed death. Loss of HSPA13 shifts the transition of RIP1 from complex I to complex II and promotes both apoptosis and necroptosis. Thus, our study provides compelling evidence for the cellular protective function of HSPA13 in fine-tuning TNFα responses.

5.
Sci STKE ; 2005(272): pe7, 2005 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-15728425

RESUMEN

Ubiquitination is best known for its role in targeting proteins for degradation by the proteasome, but evidence of the nonproteolytic functions of ubiquitin is also rapidly accumulating. One example of the regulatory, rather than proteolytic, function of ubiquitin is provided by study of the tumor necrosis factor (TNF) receptor-associated factor (TRAF) proteins, which function as ubiquitin ligases to synthesize lysine 63 (K(63))-linked polyubiquitin chains to mediate protein kinase activation through a proteasome-independent mechanism. Some TRAF proteins, such as TRAF2 and TRAF3, have recently been shown to have a positive role in the canonical pathway that activates nuclear factor kappaB (NF-kappaB) through IkappaB kinase beta (IKKbeta), but a negative role in the noncanonical pathway that activates NF-kappaB through IKKalpha. These opposing roles of TRAF proteins may be linked to their ability to synthesize distinct forms of polyubiquitin chains. Indeed, the TRAF2-interacting protein RIP can mediate IKK activation when it is modified by K(63) polyubiquitin chains, but is targeted to degradation by the proteasome when it is K(48)-polyubiquitinted by the NF-kappaB inhibitor A20. Thus, ubiquitin chains are dynamic switches that can influence signaling outputs in dramatically different ways.


Asunto(s)
Transducción de Señal/fisiología , Factor 2 Asociado a Receptor de TNF/fisiología , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Citocinas/fisiología , Regulación de la Expresión Génica/fisiología , Humanos , Quinasa I-kappa B/fisiología , Macrófagos/fisiología , Modelos Biológicos , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Factor 6 Asociado a Receptor de TNF/fisiología , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/fisiología , Ubiquitina/metabolismo , Enzimas Activadoras de Ubiquitina/fisiología
6.
Mol Cell ; 22(2): 245-57, 2006 Apr 21.
Artículo en Inglés | MEDLINE | ID: mdl-16603398

RESUMEN

The receptor interacting protein kinase 1 (RIP1) is essential for the activation of nuclear factor kappaB (NF-kappaB) by tumor necrosis factor alpha (TNFalpha). Here, we present evidence that TNFalpha induces the polyubiquitination of RIP1 at Lys-377 and that this polyubiquitination is required for the activation of IkappaB kinase (IKK) and NF-kappaB. A point mutation of RIP1 at Lys-377 (K377R) abolishes its polyubiquitination as well as its ability to restore IKK activation in a RIP1-deficient cell line. The K377R mutation of RIP1 also prevents the recruitment of TAK1 and IKK complexes to TNF receptor. Interestingly, polyubiquitinated RIP1 recruits IKK through the binding between the polyubiquitin chains and NEMO, a regulatory subunit of the IKK complex. Mutations of NEMO that disrupt its polyubiquitin binding also abolish IKK activation. These results reveal the biochemical mechanism underlying the essential signaling function of NEMO and provide direct evidence that signal-induced site-specific ubiquitination of RIP1 is required for IKK activation.


Asunto(s)
Proteínas Portadoras/metabolismo , Quinasa I-kappa B/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Poliubiquitina/metabolismo , Proteínas de Unión al ARN/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Línea Celular , Activación Enzimática , Humanos , Células Jurkat , Lisina/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas de Complejo Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/deficiencia , Proteínas de Complejo Poro Nuclear/genética , Mutación Puntual , Estructura Terciaria de Proteína , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Homología de Secuencia de Aminoácido
7.
Proc Natl Acad Sci U S A ; 99(26): 16672-7, 2002 Dec 24.
Artículo en Inglés | MEDLINE | ID: mdl-12482938

RESUMEN

We report the isolation and characterization of a new line of mutant Chinese hamster ovary cells, designated SRD-5, that are resistant to 25HC, a potent suppressor of cleavage of sterol regulatory element-binding proteins (SREBPs) in mammalian cells. In SRD-5 cells, SREBPs are cleaved constitutively, generating transcriptionally active nuclear SREBP even in the presence of sterols. Sequence analysis of SREBP cleavage-activating protein (SCAP) transcripts from SRD-5 cells revealed the presence of a mutation in one SCAP allele that results in substitution of a conserved Leu by Phe at amino acid 315 within the sterol-sensing domain. Sterols fail to inhibit the packaging of SREBPSCAP(L315F) complexes into budding vesicles in vitro. Sterols also fail to induce binding of SCAP(L315F) to insig-1 or insig-2, two proteins that function in the sterol-mediated retention of SREBPSCAP complexes in the endoplasmic reticulum. Similar findings were observed for SCAP(D443N) and SCAP(Y298C), both of which cause a sterol-resistant phenotype. Thus, three different point mutations, each within the sterol-sensing domain of SCAP, prevent sterol-induced binding of SCAP to insig proteins and abolish feedback regulation of SREBP processing by sterols.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Esteroles/farmacología , Factores de Transcripción , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Ratones , Datos de Secuencia Molecular , Mutación , Proteína 1 de Unión a los Elementos Reguladores de Esteroles , Relación Estructura-Actividad
8.
Mol Cell ; 14(3): 289-301, 2004 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-15125833

RESUMEN

The CARD domain protein BCL10 and paracaspase MALT1 are essential for the activation of IkappaB kinase (IKK) and NF-kappaB in response to T cell receptor (TCR) stimulation. Here we present evidence that TRAF6 ubiquitin ligase and TAK1 protein kinase mediate IKK activation by BCL10 and MALT1. RNAi-mediated silencing of MALT1, TAK1, TRAF6, and TRAF2 suppressed TCR-dependent IKK activation and interleukin-2 production in T cells. Furthermore, we have reconstituted the pathway from BCL10 to IKK activation in vitro with purified proteins of MALT1, TRAF6, TAK1, and ubiquitination enzymes including Ubc13/Uev1A. We find that a small fraction of BCL10 and MALT1 proteins form high molecular weight oligomers. Strikingly, only these oligomeric forms of BCL10 and MALT1 can activate IKK in vitro. The MALT1 oligomers bind to TRAF6, induce TRAF6 oligomerization, and activate the ligase activity of TRAF6 to polyubiquitinate NEMO. These results reveal an oligomerization --> ubiquitination --> phosphorylation cascade that culminates in NF-kappaB activation in T lymphocytes.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/metabolismo , Linfoma de Células B de la Zona Marginal , Quinasas Quinasa Quinasa PAM/metabolismo , Proteínas de Neoplasias , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas/metabolismo , Linfocitos T/enzimología , Proteína 10 de la LLC-Linfoma de Células B , Caspasas , Células HeLa , Humanos , Quinasa I-kappa B , Inmunidad Innata/fisiología , Interleucina-2/metabolismo , Sustancias Macromoleculares , Proteína 1 de la Translocación del Linfoma del Tejido Linfático Asociado a Mucosas , FN-kappa B/metabolismo , Fosforilación , Unión Proteica/fisiología , Interferencia de ARN/fisiología , Transducción de Señal/fisiología , Linfocitos T/inmunología , Factor 2 Asociado a Receptor de TNF , Factor 6 Asociado a Receptor de TNF , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo
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