RESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a common cause of familial and sporadic Parkinson's disease (PD). Elevated LRRK2 kinase activity and neurodegeneration are linked, but the phosphosubstrate that connects LRRK2 kinase activity to neurodegeneration is not known. Here, we show that ribosomal protein s15 is a key pathogenic LRRK2 substrate in Drosophila and human neuron PD models. Phosphodeficient s15 carrying a threonine 136 to alanine substitution rescues dopamine neuron degeneration and age-related locomotor deficits in G2019S LRRK2 transgenic Drosophila and substantially reduces G2019S LRRK2-mediated neurite loss and cell death in human dopamine and cortical neurons. Remarkably, pathogenic LRRK2 stimulates both cap-dependent and cap-independent mRNA translation and induces a bulk increase in protein synthesis in Drosophila, which can be prevented by phosphodeficient T136A s15. These results reveal a novel mechanism of PD pathogenesis linked to elevated LRRK2 kinase activity and aberrant protein synthesis in vivo.
Asunto(s)
Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Ribosómicas/metabolismo , Secuencia de Aminoácidos , Animales , Drosophila melanogaster , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Datos de Secuencia Molecular , Neuronas/patología , Enfermedad de Parkinson/patología , Proteínas Ribosómicas/químicaRESUMEN
The NLRP3 inflammasome plays a key role in responding to pathogens, and endogenous damage and mitochondria are intensively involved in inflammasome activation. The NLRP3 inflammasome forms multiprotein complexes and its sequential assembly is important for its activation. Here, we show that NLRP3 is ubiquitinated by the mitochondria-associated E3 ligase, MARCH5. Myeloid cell-specific March5 conditional knockout (March5 cKO) mice failed to secrete IL-1ß and IL-18 and exhibited an attenuated mortality rate upon LPS or Pseudomonas aeruginosa challenge. Macrophages derived from March5 cKO mice also did not produce IL-1ß and IL-18 after microbial infection. Mechanistically, MARCH5 interacts with the NACHT domain of NLRP3 and promotes K27-linked polyubiquitination on K324 and K430 residues of NLRP3. Ubiquitination-defective NLRP3 mutants on K324 and K430 residues are not able to bind to NEK7, nor form NLRP3 oligomers leading to abortive ASC speck formation and diminished IL-1ß production. Thus, MARCH5-dependent NLRP3 ubiquitination on the mitochondria is required for NLRP3-NEK7 complex formation and NLRP3 oligomerization. We propose that the E3 ligase MARCH5 is a regulator of NLRP3 inflammasome activation on the mitochondria.
Asunto(s)
Inflamasomas , Proteína con Dominio Pirina 3 de la Familia NLR , Animales , Ratones , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Interleucina-18/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Caspasa 1/metabolismoRESUMEN
Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/metabolismo , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismo , Mitofagia , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Dinaminas , Metabolismo Energético , GTP Fosfohidrolasas/genética , Células HEK293 , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial , Proteínas Asociadas a Microtúbulos/genética , Mitocondrias/genética , Mitocondrias/patología , Proteínas Mitocondriales/genética , Mutación , Neuronas/metabolismo , Neuronas/patología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas Sprague-Dawley , Transducción de Señal , Factores de Tiempo , Zinc/metabolismoRESUMEN
The synaptic insertion or removal of AMPA receptors (AMPAR) plays critical roles in the regulation of synaptic activity reflected in the expression of long-term potentiation (LTP) and long-term depression (LTD). The cellular events underlying this important process in learning and memory are still being revealed. Here we describe and characterize the AAA+ ATPase Thorase, which regulates the expression of surface AMPAR. In an ATPase-dependent manner Thorase mediates the internalization of AMPAR by disassembling the AMPAR-GRIP1 complex. Following genetic deletion of Thorase, the internalization of AMPAR is substantially reduced, leading to increased amplitudes of miniature excitatory postsynaptic currents, enhancement of LTP, and elimination of LTD. These molecular events are expressed as deficits in learning and memory in Thorase null mice. This study identifies an AAA+ ATPase that plays a critical role in regulating the surface expression of AMPAR and thereby regulates synaptic plasticity and learning and memory.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Plasticidad Neuronal , Receptores AMPA/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Células Cultivadas , Femenino , Perfilación de la Expresión Génica , Humanos , Aprendizaje , Masculino , Memoria , Ratones , Datos de Secuencia Molecular , Ratas , Alineación de Secuencia , SinapsisRESUMEN
Receptor-interacting protein kinase-3 (RIP3 or RIPK3) is a central protein in necroptosis, but posttranslational processes that regulate RIP3 activity and stability remain poorly understood. Here, we identify pellino E3 ubiquitin protein ligase 1 (PELI1) as an E3 ligase that targets RIP3 for proteasome-dependent degradation. Phosphorylation of RIP3 on T182 leads to interaction with the forkhead-associated (FHA) domain of PELI1 and PELI1-mediated K48-linked polyubiquitylation of RIP3 on K363. This same phosphorylation event is also important for RIP3 kinase activity; thus, PELI1 preferentially targets kinase-active RIP3 for degradation. PELI1-mediated RIP3 degradation effectively prevents cell death triggered by RIP3 hyperactivation. Importantly, upregulated RIP3 expression in keratinocytes from toxic epidermal necrolysis (TEN) patients is correlated with low expression of PELI1, suggesting that loss of PELI1 may play a role in the pathogenesis of TEN. We propose that PELI1 may function to control inadvertent activation of RIP3, thus preventing aberrant cell death and maintaining cellular homeostasis.
Asunto(s)
Queratinocitos/enzimología , Proteínas Nucleares/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Síndrome de Stevens-Johnson/enzimología , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Muerte Celular , Fibroblastos/enzimología , Fibroblastos/patología , Células HEK293 , Células HT29 , Células HeLa , Humanos , Queratinocitos/patología , Ratones , Proteínas Nucleares/genética , Fosforilación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Transducción de Señal , Síndrome de Stevens-Johnson/genética , Síndrome de Stevens-Johnson/patología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Exploring the connection between ubiquitin-like modifiers (ULMs) and the DNA damage response (DDR), we employed several advanced DNA damage and repair assay techniques and identified a crucial role for LC3B. Notably, its RNA recognition motif (RRM) plays a pivotal role in the context of transcription-associated homologous recombination (HR) repair (TA-HRR), a particular subset of HRR pathways. Surprisingly, independent of autophagy flux, LC3B interacts directly with R-loops at DNA lesions within transcriptionally active sites via its RRM, promoting TA-HRR. Using native RNA immunoprecipitation (nRIP) coupled with high-throughput sequencing (nRIP-seq), we discovered that LC3B also directly interacts with the 3'UTR AU-rich elements (AREs) of BRCA1 via its RRM, influencing its stability. This suggests that LC3B regulates TA-HRR both proximal to and distal from DNA lesions. Data from our LC3B depletion experiments showed that LC3B knockdown disrupts end-resection for TA-HRR, redirecting it towards the non-homologous end joining (NHEJ) pathway and leading to chromosomal instability, as evidenced by alterations in sister chromatid exchange (SCE) and interchromosomal fusion (ICF). Thus, our findings unveil autophagy-independent functions of LC3B in DNA damage and repair pathways, highlighting its importance. This could reshape our understanding of TA-HRR and the interaction between autophagy and DDR.
Asunto(s)
Proteína BRCA1 , Proteínas Asociadas a Microtúbulos , Estructuras R-Loop , Reparación del ADN por Recombinación , Transcripción Genética , Humanos , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Daño del ADN , Reparación del ADN por Unión de Extremidades , Regiones no Traducidas 3' , Recombinación Homóloga , Línea Celular Tumoral , Intercambio de Cromátides HermanasRESUMEN
PARP inhibitors (PARPi) show selective efficacy in tumors with homologous recombination repair (HRR)-defects but the activation mechanism of HRR pathway in PARPi-treated cells remains enigmatic. To unveil it, we searched for the mediator bridging PARP1 to ATM pathways by screening 211 human ubiquitin-related proteins. We discovered TRIM44 as a crucial mediator that recruits the MRN complex to damaged chromatin, independent of PARP1 activity. TRIM44 binds PARP1 and regulates the ubiquitination-PARylation balance of PARP1, which facilitates timely recruitment of the MRN complex for DSB repair. Upon exposure to PARPi, TRIM44 shifts its binding from PARP1 to the MRN complex via its ZnF UBP domain. Knockdown of TRIM44 in cells significantly enhances the sensitivity to olaparib and overcomes the resistance to olaparib induced by 53BP1 deficiency. These observations emphasize the central role of TRIM44 in tethering PARP1 to the ATM-mediated repair pathway. Suppression of TRIM44 may enhance PARPi effectiveness and broaden their use even to HR-proficient tumors.
RESUMEN
Tight control of the type I interferon (IFN) signaling pathway is critical for maintaining host innate immune responses, and the ubiquitination and deubiquitination of signaling molecules are essential for signal transduction. Deubiquitinase ubiquitin-specific protein 19 (USP19) is known to be involved in deubiquitinating Beclin1, TRAF3, and TRIF for downregulation of the type I IFN signaling. Here, we show that SIAH1, a cellular E3 ubiquitin ligase that is involved in multicellular pathway, is a potent positive regulator of virus-mediated type I IFN signaling that maintains homeostasis within the antiviral immune response by targeting USP19. In the early stages of virus infection, stabilized SIAH1 directly interacts with the USP19 and simultaneously mediates K27-linked ubiquitination of 489, 490, and 610 residues of USP19 for proteasomal degradation. Additionally, we found that USP19 specifically interacts with MAVS and deubiquitinates K63-linked ubiquitinated MAVS for negative regulation of type I IFN signaling. Ultimately, we identified that SIAH1-mediated degradation of USP19 reversed USP19-mediated deubiquitination of MAVS, Beclin1, TRAF3, and TRIF, resulting in the activation of antiviral immune responses. Taken together, these findings provide new insights into the molecular mechanism of USP19 and SIAH1, and suggest a critical role of SIAH1 in antiviral immune response and homeostasis.
Asunto(s)
Interferón Tipo I , Ubiquitina , Humanos , Ubiquitina/metabolismo , Factor 3 Asociado a Receptor de TNF/genética , Beclina-1 , Ubiquitinación , Inmunidad Innata , Interferón Tipo I/metabolismo , Enzimas Desubicuitinizantes/genética , Enzimas Desubicuitinizantes/metabolismo , Proteínas Adaptadoras del Transporte Vesicular , Endopeptidasas/genética , Endopeptidasas/metabolismoRESUMEN
Systemic lupus erythematosus (SLE) is a chronic inflammatory disease caused by autoantibodies. Serum samples from patients with SLE (n = 10) were compared with those from normal controls (NCs, n = 5) using 21K protein chip analysis to identify a biomarker for SLE, revealing 63 SLE-specific autoantibodies. The anti-chaperonin-containing t-complex polypeptide-1 (TCP1) antibody exhibited higher expression in patients with SLE than in NCs. To validate the specificity of the anti-TCP1 antibody in SLE, dot blot analysis was conducted using sera from patients with SLE (n = 100), rheumatoid arthritis (RA; n = 25), Behçet's disease (BD; n = 28), and systemic sclerosis (SSc; n = 30) and NCs (n = 50). The results confirmed the detection of anti-TCP1 antibodies in 79 of 100 patients with SLE, with substantially elevated expression compared to both NCs and patients with other autoimmune diseases. We performed an enzyme-linked immunosorbent assay to determine the relative amounts of anti-TCP1 antibodies; markedly elevated anti-TCP1 antibody levels were detected in the sera of patients with SLE (50.1 ± 17.3 arbitrary unit (AU), n = 251) compared to those in NCs (33.9 ± 9.3 AU), RA (35 ± 8.7 AU), BD (37.5 ± 11.6 AU), and SSc (43 ± 11.9 AU). These data suggest that the anti-TCP1 antibody is a potential diagnostic biomarker for SLE.
Asunto(s)
Autoanticuerpos , Biomarcadores , Lupus Eritematoso Sistémico , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/sangre , Biomarcadores/sangre , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Femenino , Masculino , Adulto , Persona de Mediana Edad , Ensayo de Inmunoadsorción Enzimática/métodos , Estudios de Casos y ControlesRESUMEN
BACKGROUND: Anti-heat shock protein (HSP) autoantibodies are detected in autoimmune diseases. We sought to ascertain whether anti-HSP10 IgG is present in patients with CSU and to elucidate the role of HSP10 in CSU pathogenesis. METHOD: Using a human proteome microarray, six potential autoantibodies had higher expression in 10 CSU samples compared with 10 normal controls (NCs). Among them, HSP10 IgG autoantibody was quantified by immune dot-blot assay in sera from 86 CSU patients and 44 NCs. The serum levels of HSP10 and microRNA-101-5p were measured in CSU patients and NCs. The effects of HSP10 and miR-101-5p on mast cell degranulation in response to IgE, compound 48/80, and platelet-activating factor (PAF) were investigated. RESULTS: CSU patients had higher IgG positivity to HSP10 (40.7% vs. 11.4%, p = .001), lower serum HSP10 levels (5.8 ± 3.6 vs. 12.2 ± 6.6 pg/mL, p < .001) than in NCs, and their urticaria severity was associated with anti-HSP10 IgG positivity, while HSP10 levels were related to urticaria control status. MiR-101-5p was increased in CSU patients. PAF enhanced IL4 production in PBMCs from CSU patients. IL-4 upregulated miR-101-5p and reduced HSP10 expression in keratinocytes. Transfection of miR-101-5p reduced HSP10 expression in keratinocytes. MiR-101-5p promoted PAF-induced mast cell degranulation, while HSP10 specifically prevented it. CONCLUSION: A new autoantibody, anti-HSP10 IgG was detected in CSU patients, which showed a significant correlation with UAS7 scores. A decreased serum HSP10 level was associated with upregulation of miR-101-5p due to increased IL-4 and PAF in CSU patients. Modulation of miR-101-5p and HSP10 may be a novel therapeutic approach for CSU.
Asunto(s)
Urticaria Crónica , MicroARNs , Urticaria , Humanos , MicroARNs/genética , Factor de Activación Plaquetaria , Interleucina-4 , Enfermedad Crónica , Autoanticuerpos , Inmunoglobulina GRESUMEN
DNA lesions impact on local transcription and the damage-induced transcriptional repression facilitates efficient DNA repair. However, how chromatin dynamics cooperates with these two events remained largely unknown. We here show that histone H2A acetylation at K118 is enriched in transcriptionally active regions. Under DNA damage, the RSF1 chromatin remodeling factor recruits HDAC1 to DSB sites. The RSF1-HDAC1 complex induces the deacetylation of H2A(X)-K118 and its deacetylation is indispensable for the ubiquitination of histone H2A at K119. Accordingly, the acetylation mimetic H2A-K118Q suppressed the H2A-K119ub level, perturbing the transcriptional repression at DNA lesions. Intriguingly, deacetylation of H2AX at K118 also licenses the propagation of γH2AX and recruitment of MDC1. Consequently, the H2AX-K118Q limits DNA repair. Together, the RSF1-HDAC1 complex controls the traffic of the DNA damage response and transcription simultaneously in transcriptionally active chromatins. The interplay between chromatin remodelers and histone modifiers highlights the importance of chromatin versatility in the maintenance of genome integrity.
Asunto(s)
Ensamble y Desensamble de Cromatina , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , Epigénesis Genética , Regulación de la Expresión Génica , Proteínas Nucleares/genética , Transactivadores/genética , Acetilación , Animales , Línea Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Células HEK293 , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Ratones , Microscopía Confocal , Células 3T3 NIH , Proteínas Nucleares/metabolismo , Unión Proteica , Interferencia de ARN , Transactivadores/metabolismo , UbiquitinaciónRESUMEN
Mutual crosstalk among poly(ADP-ribose) (PAR), activated PAR polymerase 1 (PARP1) metabolites, and DNA repair machinery has emerged as a key regulatory mechanism of the DNA damage response (DDR). However, there is no conclusive evidence of how PAR precisely controls DDR. Herein, six deubiquitinating enzymes (DUBs) associated with PAR-coupled DDR were identified, and the role of USP39, an inactive DUB involved in spliceosome assembly, was characterized. USP39 rapidly localizes to DNA lesions in a PAR-dependent manner, where it regulates non-homologous end-joining (NHEJ) via a tripartite RG motif located in the N-terminus comprising 46 amino acids (N46). Furthermore, USP39 acts as a molecular trigger for liquid demixing in a PAR-coupled N46-dependent manner, thereby directly interacting with the XRCC4/LIG4 complex during NHEJ. In parallel, the USP39-associated spliceosome complex controls homologous recombination repair in a PAR-independent manner. These findings provide mechanistic insights into how PAR chains precisely control DNA repair processes in the DDR.
Asunto(s)
Reparación del ADN por Unión de Extremidades , ADN Ligasa (ATP)/genética , Proteínas de Unión al ADN/genética , ADN/genética , Poli(ADP-Ribosa) Polimerasas/genética , Proteasas Ubiquitina-Específicas/genética , Secuencias de Aminoácidos , Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , ADN/metabolismo , Roturas del ADN de Doble Cadena , ADN Ligasa (ATP)/metabolismo , Proteínas de Unión al ADN/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Osteoblastos/citología , Osteoblastos/metabolismo , Poli Adenosina Difosfato Ribosa/metabolismo , Poli(ADP-Ribosa) Polimerasas/metabolismo , Reparación del ADN por Recombinación , Transducción de Señal , Empalmosomas , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Proteasas Ubiquitina-Específicas/metabolismoRESUMEN
Aberrant activation of the non-receptor kinase c-Abl is implicated in the development of pathogenic hallmarks of Parkinson's disease, such as α-synuclein aggregation and progressive neuronal loss. c-Abl-mediated phosphorylation and inhibition of parkin ligase function lead to accumulation of parkin interacting substrate (PARIS) that mediates α-synuclein pathology-initiated dopaminergic neurodegeneration. Here we show that, in addition to PARIS accumulation, c-Abl phosphorylation of PARIS is required for PARIS-induced cytotoxicity. c-Abl-mediated phosphorylation of PARIS at Y137 (within the Krüppel-associated box domain) drives its association with KAP1 and the repression of genes with diverse functions in pathways such as chromatin remodelling and p53-dependent cell death. One phosphorylation-dependent PARIS target, MDM4 (a p53 inhibitor that associates with MDM2; also known as MDMX), is transcriptionally repressed in a histone deacetylase-dependent manner via PARIS binding to insulin response sequence motifs within the MDM4 promoter. Virally induced PARIS transgenic mice develop c-Abl activity-dependent Parkinson's disease features such as motor deficits, dopaminergic neuron loss and neuroinflammation. PARIS expression in the midbrain resulted in c-Abl activation, PARIS phosphorylation, MDM4 repression and p53 activation, all of which are blocked by the c-Abl inhibitor nilotinib. Importantly, we also observed aberrant c-Abl activation and PARIS phosphorylation along with PARIS accumulation in the midbrain of adult parkin knockout mice, implicating c-Abl in recessive Parkinson's disease. Inhibition of c-Abl or PARIS phosphorylation by nilotinib or Y137F-PARIS expression in adult parkin knockout mice blocked MDM4 repression and p53 activation, preventing motor deficits and dopaminergic neurodegeneration. Finally, we found correlative increases in PARIS phosphorylation, MDM4 repression and p53 activation in post-mortem Parkinson's disease brains, pointing to clinical relevance of the c-Abl-PARIS-MDM4-p53 pathway. Taken together, our results describe a novel mechanism of epigenetic regulation of dopaminergic degeneration downstream of pathological c-Abl activation in Parkinson's disease. Since c-Abl activation has been shown in sporadic Parkinson's disease, PARIS phosphorylation might serve as both a useful biomarker and a potential therapeutic target to regulate neuronal loss in Parkinson's disease.
Asunto(s)
Neuronas Dopaminérgicas/patología , Degeneración Nerviosa/patología , Trastornos Parkinsonianos/patología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Proteínas Represoras/metabolismo , Animales , Neuronas Dopaminérgicas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Degeneración Nerviosa/metabolismo , Trastornos Parkinsonianos/metabolismo , FosforilaciónRESUMEN
PSMD14, a subunit of the 19S regulatory particles of the 26S proteasome, was recently identified as a potential prognostic marker and therapeutic target in diverse human cancers. Here, we show that the silencing and pharmacological blockade of PSMD14 in MDA-MB 435S breast cancer cells induce paraptosis, a non-apoptotic cell death mode characterized by extensive vacuolation derived from the endoplasmic reticulum (ER) and mitochondria. The PSMD14 inhibitor, capzimin (CZM), inhibits proteasome activity but differs from the 20S proteasome subunit-inhibiting bortezomib (Bz) in that it does not induce aggresome formation or Nrf1 upregulation, which underlie Bz resistance in cancer cells. In addition to proteasome inhibition, the release of Ca2+ from the ER into the cytosol critically contributes to CZM-induced paraptosis. Induction of paraptosis by targeting PSMD14 may provide an attractive therapeutic strategy against cancer cells resistant to proteasome inhibitors or pro-apoptotic drugs.
Asunto(s)
Neoplasias de la Mama , Calcio/metabolismo , Complejo de la Endopetidasa Proteasomal , Apoptosis , Bortezomib/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Inhibidores de Proteasoma/farmacología , TransactivadoresRESUMEN
BACKGROUND: Necroptosis is emerging as a new target for cancer immunotherapy as it is now recognized as a form of cell death that increases tumor immunogenicity, which would be especially helpful in treating immune-desert tumors. De novo synthesis of inflammatory proteins during necroptosis appears especially important in facilitating increased anti-tumor immune responses. While late-stage transcription mediated by NF-κB during cell death is believed to play a role in this process, it is otherwise unclear what cell signaling events initiate this transactivation of inflammatory genes. METHODS: We employed tandem-affinity purification linked to mass spectrometry (TAP-MS), in combination with the analysis of RNA-sequencing (RNA-Seq) datasets to identify the Tripartite Motif Protein 28 (TRIM28) as a candidate co-repressor. Comprehensive biochemical and molecular biology techniques were used to characterize the role of TRIM28 in RIPK3 activation-induced transcriptional and immunomodulatory events. The cell composition estimation module was used to evaluate the correlation between RIPK3/TRIM28 levels and CD8+ T cells or dendritic cells (DC) in all TCGA tumors. RESULTS: We identified TRIM28 as a co-repressor that regulates transcriptional activity during necroptosis. Activated RIPK3 phosphorylates TRIM28 on serine 473, inhibiting its chromatin binding activity, thereby contributing to the transactivation of NF-κB and other transcription factors, such as SOX9. This leads to elevated cytokine expression, which then potentiates immunoregulatory processes, such as DC maturation. The expression of RIPK3 has a significant positive association with the tumor-infiltrating immune cells populations in various tumor type, thereby activating anti-cancer responses. CONCLUSION: Our data suggest that RIPK3 activation-dependent derepression of TRIM28 in cancer cells leads to increased immunostimulatory cytokine production in the tumor microenvironment, which then contributes to robust cytotoxic anti-tumor immunity.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteína 28 que Contiene Motivos Tripartito/genética , Microambiente Tumoral/genética , Animales , Sitios de Unión , Muerte Celular , Línea Celular , Citocinas/metabolismo , Humanos , Ratones , Modelos Biológicos , FN-kappa B/metabolismo , Necroptosis , Neoplasias/genética , Neoplasias/metabolismo , Unión Proteica , Transducción de SeñalRESUMEN
OBJECTIVES: Recently, necroptosis has attracted increasing attention in arthritis research; however, it remains unclear whether its regulation is involved in osteoarthritis (OA) pathogenesis. Since receptor-interacting protein kinase-3 (RIP3) plays a pivotal role in necroptosis and its dysregulation is involved in various pathological processes, we investigated the role of the RIP3 axis in OA pathogenesis. METHODS: Experimental OA was induced in wild-type or Rip3 knockout mice by surgery to destabilise the medial meniscus (DMM) or the intra-articular injection of adenovirus carrying a target gene (Ad-Rip3 and Ad-Trim24 shRNA). RIP3 expression was examined in OA cartilage from human patients; Trim24, a negative regulator of RIP3, was identified by microarray and in silico analysis. Connectivity map (CMap) and in silico binding approaches were used to identify RIP3 inhibitors and to examine their direct regulation of RIP3 activation in OA pathogenesis. RESULTS: RIP3 expression was markedly higher in damaged cartilage from patients with OA than in undamaged cartilage. In the mouse model, adenoviral RIP3 overexpression accelerated cartilage disruption, whereas Rip3 depletion reduced DMM-induced OA pathogenesis. Additionally, TRIM24 knockdown upregulated RIP3 expression; its downregulation promoted OA pathogenesis in knee joint tissues. The CMap approach and in silico binding assay identified AZ-628 as a potent RIP3 inhibitor and demonstrated that it abolished RIP3-mediated OA pathogenesis by inhibiting RIP3 kinase activity. CONCLUSIONS: TRIM24-RIP3 axis perturbation promotes OA chronicity by activating RIP3 kinase, suggesting that the therapeutic manipulation of this pathway could provide new avenues for treating OA.
Asunto(s)
Proteínas Portadoras/metabolismo , Osteoartritis/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Femenino , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Necroptosis/fisiología , Proteínas Nucleares/metabolismo , Osteoartritis/patología , Transducción de Señal/fisiología , Factores de Transcripción/metabolismoRESUMEN
During hematopoiesis, red blood cells originate from the hematopoietic stem cell reservoir. Although the regulation of erythropoiesis and globin expression has been intensively investigated, the underlining mechanisms are not fully understood, including the interplay between transcription factors and epigenetic factors. Here, we uncover that the Mbd2-free NuRD chromatin remodeling complex potentiates erythroid differentiation of proerythroblasts via managing functions of the CP2c complexes. We found that both Mbd2 and Mbd3 expression is downregulated during differentiation of MEL cells in vitro and in normal erythropoiesis in mouse bone marrow, and Mbd2 downregulation is crucial for erythropoiesis. In uninduced MEL cells, the Mbd2-NuRD complex is recruited to the promoter via Gata1/Fog1, and, via direct binding through p66α, it acts as a transcriptional inhibitor of the CP2c complexes, preventing their DNA binding and promoting degradation of the CP2c family proteins to suppress globin gene expression. Conversely, during erythropoiesis in vitro and in vivo, the Mbd2-free NuRD does not dissociate from the chromatin and acts as a transcriptional coactivator aiding the recruitment of the CP2c complexes to chromatin, and thereby leading to the induction of the active hemoglobin synthesis and erythroid differentiation. Our study highlights the regulation of erythroid differentiation by the Mbd2-CP2c loop.
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Proteínas de Unión al ADN/metabolismo , Eritropoyesis/fisiología , Globinas/genética , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Diferenciación Celular , Ensamble y Desensamble de Cromatina , Proteínas de Unión al ADN/genética , Células Eritroides/citología , Eritropoyesis/genética , Factor de Transcripción GATA1/metabolismo , Regulación de la Expresión Génica , Hemoglobinas/biosíntesis , Hemoglobinas/genética , Humanos , Masculino , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Ratones Endogámicos BALB C , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genéticaRESUMEN
Mutations in parkin lead to early-onset autosomal recessive Parkinson's disease (PD) and inactivation of parkin is thought to contribute to sporadic PD. Adult knockout of parkin in the ventral midbrain of mice leads to an age-dependent loss of dopamine neurons that is dependent on the accumulation of parkin interacting substrate (PARIS), zinc finger protein 746 (ZNF746), and its transcriptional repression of PGC-1α. Here we show that adult knockout of parkin in mouse ventral midbrain leads to decreases in mitochondrial size, number, and protein markers consistent with a defect in mitochondrial biogenesis. This decrease in mitochondrial mass is prevented by short hairpin RNA knockdown of PARIS. PARIS overexpression in mouse ventral midbrain leads to decreases in mitochondrial number and protein markers and PGC-1α-dependent deficits in mitochondrial respiration. Taken together, these results suggest that parkin loss impairs mitochondrial biogenesis, leading to declining function of the mitochondrial pool and cell death.
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Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Animales , Encéfalo/embriología , Encéfalo/metabolismo , Muerte Celular , Línea Celular Tumoral , Dopamina/metabolismo , Neuronas Dopaminérgicas/metabolismo , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Consumo de Oxígeno , Enfermedad de Parkinson/metabolismo , Proteínas Represoras/fisiología , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.
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Antineoplásicos/metabolismo , Cetuximab/metabolismo , Receptores ErbB/antagonistas & inhibidores , Escherichia coli/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Antineoplásicos/química , Línea Celular Tumoral , Cetuximab/química , Cetuximab/genética , Cristalografía por Rayos X , Escherichia coli/genética , Humanos , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Espectrometría de Masas , Unión Proteica , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially S-nitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO's actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes.