RESUMEN
OBJECTIVES: To describe the current state of research and future research hotspots through a metrological analysis of the literature in the field of forensic anthropological remains identification research. METHODS: The data retrieved and extracted from the Web of Science Core Collection (WoSCC), the core database of the Web of Science information service platform (hereinafter referred to as "WoS"), was used to analyze the trends and topic changes in research on forensic identification of human remains from 1991 to 2022. Network visualisation of publication trends, countries (regions), institutions, authors and topics related to the identification of remains in forensic anthropology was analysed using python 3.9.2 and Gephi 0.10. RESULTS: A total of 873 papers written in English in the field of forensic anthropological remains identification research were obtained. The journal with the largest number of publications was Forensic Science International (164 articles). The country (region) with the largest number of published papers was China (90 articles). Katholieke Univ Leuven (Netherlands, 21 articles) was the institution with the largest number of publications. Topic analysis revealed that the focus of forensic anthropological remains identification research was sex estimation and age estimation, and the most commonly studied remains were teeth. CONCLUSIONS: The volume of publications in the field of forensic anthropological remains identification research has a distinct phasing. However, the scope of both international and domestic collaborations remains limited. Traditionally, human remains identification has primarily relied on key areas such as the pelvis, skull, and teeth. Looking ahead, future research will likely focus on the more accurate and efficient identification of multiple skeletal remains through the use of machine learning and deep learning techniques.
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Bibliometría , Restos Mortales , Antropología Forense , Humanos , Antropología Forense/métodos , Publicaciones/estadística & datos numéricosRESUMEN
Heterobifunctional proteolysis-targeting chimeras (PROTACs) offer a promising cancer treatment avenue by efficiently degrading unwanted cellular proteins. A recent study from Zhang et al. demonstrated the successful utilization of the N-end rule in PROTAC design, allowing for a modular degradation rate tailored to the oncogenic driver BCR-ABL.
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Proteínas , Ubiquitina-Proteína Ligasas , Proteolisis , Proteínas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Protein-protein interactions (PPIs) play important roles in biological processes of life, and predicting PPIs becomes a critical scientific issue of concern. Most PPIs occur through small domains or motifs (fragments), which are challenging and laborious to map by standard biochemical approaches because they generally require the cloning of several truncation mutants. Here, we present a computational method, named as PPI-Miner, to fish potential protein interacting partners utilizing protein motifs as queries. In brief, this work first developed a motif-matching algorithm designed to identify the proteins that contain sequential or structural similar motifs with the given query motif. Being aligned to the query motif, the binding mode of the discovered motif and its receptor protein will be initially determined to be used to build PPI complexes accordingly. Eventually, a PPI complex structure could be built and optimized with a designed automatic protocol. Besides discovering PPIs, PPI-Miner can also be applied to other areas, i.e., the rational design of molecular glues and protein vaccines. In this work, PPI-Miner was employed to mine the potential cereblon (CRBN) substrates from human proteome. As a result, 1,739 candidates were predicted, and 16 of them have been experimentally validated in previous studies. The source code of PPI-Miner can be obtained from the GitHub repository (https://github.com/Wang-Lin-boop/PPI-Miner), the webserver is freely available for users (https://bailab.siais.shanghaitech.edu.cn/services/ppi-miner), and the database of predicted CRBN substrates is accessible at https://bailab.siais.shanghaitech.edu.cn/services/crbn-subslib.
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Mapeo de Interacción de Proteínas , Programas Informáticos , Humanos , Animales , Mapeo de Interacción de Proteínas/métodos , Algoritmos , Secuencias de Aminoácidos , Proteoma , Bases de Datos de ProteínasRESUMEN
Aberrant overexpression of nicotinamide phosphoribosyltransferase (NAMPT) has been reported in a variety of tumor cells and is a poor prognosis factor for patient survival. It plays an important role in tumor cell proliferation, acting concurrently as an nicotinamide adenine dinucleotide (NAD+) synthase and, unexpectedly, as an extracellular signaling molecule for several tumor-promoting pathways. Although previous efforts to modulate NAMPT activity were limited to enzymatic inhibitors with low success in clinical studies, protein degradation offers the possibility to simultaneously disrupt NAMPT's enzyme activity and ligand capabilities. Here we report the development of two highly selective proteolysis-targeting chimeras (PROTACs) that promote NAMPT degradation in a cereblon-dependent manner. Both PROTAC degraders outperform a clinical candidate, FK866, in killing effect on hematological tumor cells. These results emphasize the importance and feasibility of applying PROTACs as a superior strategy for targeting proteins with multiple tumor-promoting functions like NAMPT, which is not easily achieved by conventional enzymatic inhibitors.
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Neoplasias , Nicotinamida Fosforribosiltransferasa , Humanos , Proliferación Celular , NAD , Neoplasias/tratamiento farmacológico , ProteolisisRESUMEN
Programmed cell death protein 1 (PD-1) checkpoint blockade therapy requires the CD28 co-stimulatory receptor for CD8+ T cell expansion and cytotoxicity. However, CD28 expression is frequently lost in exhausted T cells and during immune senescence, limiting the clinical benefits of PD-1 immunotherapy in individuals with cancer. Here, using a cereblon knockin mouse model that regains in vivo T cell response to lenalidomide, an immunomodulatory imide drug, we show that lenalidomide reinstates the anti-tumor activity of CD28-deficient CD8+ T cells after PD-1 blockade. Lenalidomide redirects the CRL4Crbn ubiquitin ligase to degrade Ikzf1 and Ikzf3 in T cells and unleashes paracrine interleukin-2 (IL-2) and intracellular Notch signaling, which collectively bypass the CD28 requirement for activation of intratumoral CD8+ T cells and inhibition of tumor growth by PD-1 blockade. Our results suggest that PD-1 immunotherapy can benefit from a lenalidomide combination when treating solid tumors infiltrated with abundant CD28- T cells.
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Antígenos CD28 , Receptor de Muerte Celular Programada 1 , Animales , Linfocitos T CD8-positivos , Factores Inmunológicos , Inmunoterapia/métodos , Lenalidomida/farmacología , RatonesRESUMEN
Cancer cells acquire genetic heterogeneity to escape from immune surveillance during tumor evolution, but a systematic approach to distinguish driver from passenger mutations is lacking. Here we investigate the impact of different immune pressure on tumor clonal dynamics and immune evasion mechanism, by combining massive parallel sequencing of immune edited tumors and CRISPR library screens in syngeneic mouse tumor model and co-culture system. We find that the core microRNA (miRNA) biogenesis and targeting machinery maintains the sensitivity of cancer cells to PD-1-independent T cell-mediated cytotoxicity. Genetic inactivation of the machinery or re-introduction of ANKRD52 frequent patient mutations dampens the JAK-STAT-interferon-γ signaling and antigen presentation in cancer cells, largely by abolishing miR-155-targeted silencing of suppressor of cytokine signaling 1 (SOCS1). Expression of each miRNA machinery component strongly correlates with intratumoral T cell infiltration in nearly all human cancer types. Our data indicate that the evolutionarily conserved miRNA pathway can be exploited by cancer cells to escape from T cell-mediated elimination and immunotherapy.
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Evasión Inmune , MicroARNs/metabolismo , Neoplasias , Animales , Línea Celular Tumoral , Quimiocinas/metabolismo , Heterogeneidad Genética , Humanos , Inmunoterapia , Interferón gamma , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Neoplasias/genética , Fosfoproteínas Fosfatasas , Receptor de Muerte Celular Programada 1 , Transducción de Señal , Proteína 1 Supresora de la Señalización de Citocinas , Linfocitos TRESUMEN
Immunomodulatory drugs (IMiDs) have markedly improved patient outcome in multiple myeloma (MM); however, resistance to IMiDs commonly underlies relapse of disease. Here, we identify that tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) knockdown (KD)/knockout (KO) in MM cells mediates IMiD resistance via activation of noncanonical nuclear factor κB (NF-κB) and extracellular signal-regulated kinase (ERK) signaling. Within MM bone marrow (BM) stromal cell supernatants, TNF-α induces proteasomal degradation of TRAF2, noncanonical NF-κB, and downstream ERK signaling in MM cells, whereas interleukin-6 directly triggers ERK activation. RNA sequencing of MM patient samples shows nearly universal ERK pathway activation at relapse on lenalidomide maintenance therapy, confirming its clinical relevance. Combination MEK inhibitor treatment restores IMiD sensitivity of TRAF2 KO cells both in vitro and in vivo. Our studies provide the framework for clinical trials of MEK inhibitors to overcome IMiD resistance in the BM microenvironment and improve patient outcome in MM.
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Agentes Inmunomoduladores , Mieloma Múltiple , Médula Ósea/patología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/uso terapéutico , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , FN-kappa B/metabolismo , Recurrencia Local de Neoplasia , Factor 2 Asociado a Receptor de TNF/metabolismo , Microambiente TumoralAsunto(s)
División Celular , Proteínas de Unión al ADN/fisiología , Fase G2 , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Precursoras de Linfocitos B/citología , Animales , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células Precursoras de Linfocitos B/metabolismoRESUMEN
About 257 million people with chronic infection of hepatitis B virus (HBV) worldwide are at high risk of developing terminal liver diseases. Reactivation of virus replication has been frequently reported in those patient populations receiving imatinib (an Abl kinase inhibitor) or bortezomib (a proteasome inhibitor) to treat concurrent diseases, but the underlying mechanism for this reactivation is unknown. We report that the HBV polymerase protein is recruited by Cdt2 to the cullin-RING ligase 4 (CRL4) for ubiquitination and proteasome degradation and that this process is stimulated by the c-Abl nonreceptor tyrosine kinase. Genetic ablation of the Abl-CRL4Cdt2 axis or pharmaceutical inhibition of this process stabilizes HBV polymerase protein and increases viral loads in HBV-infected liver cancer cell lines. Our study reveals a kinase-dependent activation of CRL4 ubiquitin ligase that can be targeted for blocking HBV replication.
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Productos del Gen pol/metabolismo , Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Hepatitis B/virología , Interacciones Huésped-Patógeno , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Replicación Viral , Línea Celular Tumoral , Estabilidad de Enzimas , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Unión Proteica , Proteolisis , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Immunomodulatory drugs (IMiDs) including lenalidomide and pomalidomide bind cereblon (CRBN) and activate the CRL4CRBN ubiquitin ligase to trigger proteasomal degradation of the essential transcription factors IKZF1 and IKZF3 and multiple myeloma (MM) cytotoxicity. We have shown that CRBN is also targeted for degradation by SCFFbxo7 ubiquitin ligase. In the current study, we explored the mechanisms underlying sensitivity of MM cells to IMiDs using genome-wide CRISPR-Cas9 screening. We validate that CSN9 signalosome complex, a deactivator of Cullin-RING ubiquitin ligase, inhibits SCFFbxo7 E3 ligase-mediated CRBN degradation, thereby conferring sensitivity to IMiDs; conversely, loss of function of CSN9 signalosome activates SCFFbxo7 complex, thereby enhancing degradation of CRBN and conferring IMiD resistance. Finally, we show that pretreatment with either proteasome inhibitors or NEDD8 activating enzyme (NAE) inhibitors can abrogate degradation and maintain levels of CRBN, thereby enhancing sensitivity to IMiDs. These studies therefore demonstrate that CSN9 signalosome complex regulates sensitivity to IMiDs by modulating CRBN expression.
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Complejo del Señalosoma COP9/metabolismo , Sistemas CRISPR-Cas , Factor de Transcripción Ikaros/metabolismo , Factores Inmunológicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Péptido Hidrolasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Antineoplásicos/farmacología , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Bortezomib/farmacología , Complejo del Señalosoma COP9/antagonistas & inhibidores , Complejo del Señalosoma COP9/genética , Ciclopentanos/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Factor de Transcripción Ikaros/genética , Mieloma Múltiple/genética , Mieloma Múltiple/patología , Péptido Hidrolasas/genética , Pronóstico , Proteolisis , Pirimidinas/farmacología , Células Tumorales Cultivadas , Ubiquitina-Proteína Ligasas , UbiquitinaciónRESUMEN
Adipocytes attached to the extracellular matrix (ECM) mainly consist of collagen in adipose tissues, while the degradation of ECM by collagenase induces the apoptosis of adipocytes, leading to a decrease in local subcutaneous adipose. To achieve this goal, we are developing a mutant collagenase H (ColH) to remove local subcutaneous fat such as submental fat (SMF). Three vectors were constructed for expressing rColH(FM, mutant for fat melting, with 6xHis tag), rColH(WT, wild-type, with 6xHis tag), and rColH(E451D, E451D mutant, without 6xHis tag) in Escherichia coli. rColH(FM) & rColH(WT) were purified by Ni Sepharose on a laboratory scale, while rColH(E451D) was purified by five chromatography purification steps on a large scale. Then, the stability of rColH(FM) and rColH(WT) was tested by SDS-PAGE to investigate the influence of the E451D mutation on stability. Afterwards, the enzyme kinetics of ColH (mutant or wild-type, with or without His tag) were investigated and compared. Finally, the adipolysis of rColH(E451D) at various doses was tested in vitro and in vivo. The ultrasound results in minipigs suggested that effective adipolysis was induced by rColH(E451D) compared with the negative control, and the histological results suggest dose-dependent fibrosis, necrosis, inflammation and cholesterol cleft formation. These findings indicate the possibility of rColH(E451D) becoming a new injectable drug to safely remove subcutaneous adipose.
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Adipocitos/efectos de los fármacos , Proteínas Bacterianas/farmacología , Colágeno/metabolismo , Colagenasas/farmacología , Lipólisis/efectos de los fármacos , Grasa Subcutánea/efectos de los fármacos , Adipocitos/patología , Animales , Proteínas Bacterianas/aislamiento & purificación , Colagenasas/aislamiento & purificación , Escherichia coli/genética , Ratones , Ratones Obesos , Obesidad/tratamiento farmacológico , Grasa Subcutánea/patología , Porcinos , Porcinos EnanosRESUMEN
Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBN mutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7 ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN-associated ID.
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Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Aprendizaje/fisiología , Memoria/fisiología , Proteínas del Tejido Nervioso/metabolismo , Proteolisis , Proteínas Ligasas SKP Cullina F-box/antagonistas & inhibidores , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Células Cultivadas , Femenino , Células HEK293 , Humanos , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Ligasas SKP Cullina F-box/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/genética , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Transcription from chromosomes is regulated by posttranslational modifications to histones, such as methylation and ubiquitination. Monoubiquitination of histones H2A and H2B influences H3 methylation to reinforce the activation or repression of gene expression. Here, we provide evidence that H3 polyubiquitination represses transcription of fetal and cell-cycle genes in postnatal mouse liver by crosstalk with H3K9 methylation. We found that the CRL4 ubiquitin ligase targets H3 for polyubiquitination at K79 via the DCAF8 substrate receptor in hepatocytes. Genetic inactivation of DCAF8 and overexpression of an H3K79 mutant in cells or inducible deletion of CRL4 in mouse liver abrogates H3 ubiquitination, reactivates the expression of fetal liver and cell-cycle genes by interfering with methylated H3K9 occupancy, and leads to cell senescence. Restoring CRL4DCAF8 expression in cells with decreased H3 ubiquitination reinstates the epigenetic gene silencing. Our results suggest that progressive H3 ubiquitination plays an important role in postnatal liver maturation.
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Histonas/metabolismo , Hígado/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Ciclo Celular/fisiología , Senescencia Celular/fisiología , Epigénesis Genética , Silenciador del Gen/fisiología , Hepatocitos/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Metilación , Ratones , Ratones Transgénicos , Ubiquitina/metabolismo , Ubiquitinación/fisiologíaRESUMEN
Cullin-RING ligase 4 (CRL4), a complex of Cul4 and DDB1, regulates the cell cycle, DNA damage repair, and chromatin replication by targeting a variety of substrates for ubiquitination. CRL4 is also hijacked by viral proteins or thalidomide-derived compounds to degrade host restriction factors. Here we report that the c-Abl non-receptor kinase phosphorylates DDB1 at residue Tyr-316 to recruit a small regulatory protein, DDA1, leading to increased substrate ubiquitination. Pharmacological inhibition or genetic ablation of the Abl-DDB1-DDA1 axis decreases the ubiquitination of CRL4 substrates, including IKZF1 and IKZF3, in lenalidomide-treated multiple myeloma cells. Importantly, panobinostat, a recently approved anti-myeloma drug, and dexamethasone enhance lenalidomide-induced substrate degradation and cytotoxicity by activating c-Abl, therefore providing a mechanism underlying their combination with lenalidomide to treat multiple myeloma.
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Proteínas de Unión al ADN/metabolismo , Proteínas Proto-Oncogénicas c-abl/metabolismo , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligasas/metabolismo , Inhibidores de la Angiogénesis/farmacología , Línea Celular Tumoral , Supervivencia Celular , Dexametasona/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Lenalidomida , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/metabolismo , Panobinostat , Unión Proteica , Proteolisis , Talidomida/farmacología , Tirosina/química , UbiquitinaciónRESUMEN
Interleukin 6 (IL6), tumor necrosis factor α (TNFα) and TNF receptor-1(TNFR1) have been shown to involve in oval cell proliferation and hepatocellular carcinoma (HCC) development. However, their role in these processes is still unclear. In the present study, by using hepatocytes-specific DDB1 deletion mouse models, we explored the role and mechanism of IL6, TNFα and TNFR1 in oval cell proliferation and HCC development in the context of inflammation, which is the common features of HCC pathogenesis in humans. Our results showed that IL6 promotes oval cell proliferation and liver regeneration, while TNFα/TNFR1 does not affect this process. Deletion of IL6 accelerates HCC development and increases tumor burden. The number of natural killer(NK) cells is significantly decreased in tumors without IL6, implying that IL6 suppresses HCC by NK cells. In contrast to IL6, TNFR1-mediated signaling pathway promotes HCC development, and deletion of TNFR1 reduced tumor incidence. Increased apoptosis, compensatory proliferation and activation of MAPK/MEK/ERK cascade contribute to the oncogenic function of TNFR1-mediated signaling pathway. Intriguingly, deletion of TNFα accelerates tumor development, which shows divergent roles of TNFα and TNFR1 in hepatocarcinogenesis.
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Transformación Celular Neoplásica/genética , Inflamación/genética , Interleucina-6/genética , Regeneración Hepática/genética , Hígado/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proliferación Celular/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Interleucina-6/deficiencia , Hígado/patología , Hígado/fisiopatología , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Ratones Noqueados , Ratones Transgénicos , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.
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Encefalopatías Metabólicas Innatas/tratamiento farmacológico , Cardiomiopatías/tratamiento farmacológico , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Mutación/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Encefalopatías Metabólicas Innatas/genética , Modelos Animales de Enfermedad , Isocitrato Deshidrogenasa/genética , Ratones , Mutación/genéticaRESUMEN
The most common congenital disorder of glycosylation (CDG), phosphomannomutase 2 (PMM2)-CDG, is caused by mutations in PMM2 that limit availability of mannose precursors required for protein N-glycosylation. The disorder has no therapy and there are no models to test new treatments. We generated compound heterozygous mice with the R137H and F115L mutations in Pmm2 that correspond to the most prevalent alleles found in patients with PMM2-CDG. Many Pmm2R137H/F115L mice died prenatally, while survivors had significantly stunted growth. These animals and cells derived from them showed protein glycosylation deficiencies similar to those found in patients with PMM2-CDG. Growth-related glycoproteins insulin-like growth factor (IGF) 1, IGF binding protein-3 and acid-labile subunit, along with antithrombin III, were all deficient in Pmm2R137H/F115L mice, but their levels in heterozygous mice were comparable to wild-type (WT) littermates. These imbalances, resulting from defective glycosylation, are likely the cause of the stunted growth seen both in our model and in PMM2-CDG patients. Both Pmm2R137H/F115L mouse and PMM2-CDG patient-derived fibroblasts displayed reductions in PMM activity, guanosine diphosphate mannose, lipid-linked oligosaccharide precursor and total cellular protein glycosylation, along with hypoglycosylation of a new endogenous biomarker, glycoprotein 130 (gp130). Over-expression of WT-PMM2 in patient-derived fibroblasts rescued all these defects, showing that restoration of mutant PMM2 activity is a viable therapeutic strategy. This functional mouse model of PMM2-CDG, in vitro assays and identification of the novel gp130 biomarker all shed light on the human disease, and moreover, provide the essential tools to test potential therapeutics for this untreatable disease.
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Biomarcadores , Trastornos Congénitos de Glicosilación/genética , Receptor gp130 de Citocinas/genética , Fosfotransferasas (Fosfomutasas)/genética , Animales , Trastornos Congénitos de Glicosilación/metabolismo , Trastornos Congénitos de Glicosilación/patología , Receptor gp130 de Citocinas/biosíntesis , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Genotipo , Glicosilación , Humanos , Manosa/genética , Manosa/metabolismo , Ratones , MutaciónRESUMEN
Sorafenib is a multikinase inhibitor approved as the first line treatment for late stage hepatocellular carcinoma (HCC). Due to its significant variation in clinical benefits among patients, defining prognostic biomarkers for sorafenib sensitivity in HCC would allow targeted treatment. Phosphorylated extracellular signaling-regulated kinase (pERK) was proposed to predict the response to sorafenib in HCC, but clinical supports are mixed or even contradictory. Here we found that pERK expression levels are variable in different nodules from individual patient liver. Xenografts derived from resected tumors are resistant to sorafenib inhibition when expressing low levels of pERK. This correlation of low pERK levels and sorafenib resistance is corroborated by histological characterization of chemical-induced and genetic mouse models for pERK-positive and pERK-negative HCC respectively, as well as computed tomography (CT) imaging of patient tumors with validated pERK expression. Mouse and human HCC samples expressing low pERK show strong inflammatory infiltrating cells and significant enrichment of intratumoral CD8+ cytotoxic T lymphocytes that express programmed death receptor-1 (PD-1). These pERK-PD-1+ patients have poorer overall and disease-free survival than pERK+PD-1- patients. In conclusion, our data suggest that anti-PD-1 immunotherapy might complement sorafenib in treating HCC patients by targeting sorafenib-resistant cancer cells, and the dual pERK and PD-1 biomarkers would help HCC patient selection to achieve optimal clinical benefits.
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Carcinoma Hepatocelular/tratamiento farmacológico , Resistencia a Antineoplásicos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Niacinamida/análogos & derivados , Compuestos de Fenilurea/uso terapéutico , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Puntos de Control del Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Femenino , Células Hep G2 , Humanos , Células K562 , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Niacinamida/uso terapéutico , Fosforilación/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , SorafenibRESUMEN
Protein palmitoylation regulates many aspects of cell function and is carried out by acyl transferases that contain zf-DHHC motifs. The in vivo physiological function of protein palmitoylation is largely unknown. Here we generated mice deficient in the acyl transferase Aph2 (Ablphilin 2 or zf-DHHC16) and demonstrated an essential role for Aph2 in embryonic/postnatal survival, eye development, and heart development. Aph2(-/-) embryos and pups showed cardiomyopathy and cardiac defects including bradycardia. We identified phospholamban, a protein often associated with human cardiomyopathy, as an interacting partner and a substrate of Aph2. Aph2-mediated palmitoylation of phospholamban on cysteine 36 differentially alters its interaction with PKA and protein phosphatase 1 α, augmenting serine 16 phosphorylation, and regulates phospholamban pentamer formation. Aph2 deficiency results in phospholamban hypophosphorylation, a hyperinhibitory form. Ablation of phospholamban in Aph2(-/-) mice histologically and functionally alleviated the heart defects. These findings establish Aph2 as a critical in vivo regulator of cardiac function and reveal roles for protein palmitoylation in the development of other organs including eyes.
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Aciltransferasas/fisiología , Cardiomiopatías/etiología , Proteínas Portadoras/fisiología , Animales , Células COS , Proteínas de Unión al Calcio/metabolismo , Chlorocebus aethiops , Ecocardiografía , Ojo/embriología , Lipoilación , Ratones , FosforilaciónRESUMEN
Little is known on post-transcriptional regulation of adult and embryonic stem cell maintenance and differentiation. Here we characterize the role of Ddb1, a component of the CUL4-DDB1 ubiquitin ligase complex. Ddb1 is highly expressed in multipotent hematopoietic progenitors and its deletion leads to abrogation of both adult and fetal hematopoiesis, targeting specifically transiently amplifying progenitor subsets. However, Ddb1 deletion in non-dividing lymphocytes has no discernible phenotypes. Ddb1 silencing activates Trp53 pathway and leads to significant effects on cell cycle progression and rapid apoptosis. The abrogation of hematopoietic progenitor cells can be partially rescued by simultaneous deletion of Trp53. Conversely, depletion of DDB1 in embryonic stem cell (ESC) leads to differentiation albeit negative effects on cell cycle and apoptosis. Mass spectrometry reveals differing protein interactions between DDB1 and distinct DCAFs, the substrate recognizing components of the E3 complex, between cell types. Our studies identify CUL4-DDB1 complex as a novel post-translational regulator of stem and progenitor maintenance and differentiation.