RESUMO
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.
Assuntos
Proteínas , Ubiquitina-Proteína Ligases , Proteólise , Proteínas/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
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.
Assuntos
Bibliometria , Restos Mortais , Antropologia Forense , Humanos , Antropologia Forense/métodos , Publicações/estatística & dados numéricosRESUMO
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.
Assuntos
Mapeamento de Interação de Proteínas , Software , Humanos , Animais , Mapeamento de Interação de Proteínas/métodos , Algoritmos , Motivos de Aminoácidos , Proteoma , Bases de Dados de ProteínasRESUMO
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.
Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Proteínas Ligases SKP Culina F-Box/antagonistas & inibidores , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Deficiência Intelectual/genética , Deficiência Intelectual/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Ligases SKP Culina F-Box/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Talidomida/análogos & derivados , Ubiquitina-Proteína Ligases/metabolismo , Inibidores da Angiogênese/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Dexametasona/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Lenalidomida , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Panobinostat , Ligação Proteica , Proteólise , Talidomida/farmacologia , Tirosina/química , UbiquitinaçãoRESUMO
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.
Assuntos
Biomarcadores , Defeitos Congênitos da Glicosilação/genética , Receptor gp130 de Citocina/genética , Fosfotransferases (Fosfomutases)/genética , Animais , Defeitos Congênitos da Glicosilação/metabolismo , Defeitos Congênitos da Glicosilação/patologia , Receptor gp130 de Citocina/biossíntese , Modelos Animais de Doenças , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genótipo , Glicosilação , Humanos , Manose/genética , Manose/metabolismo , Camundongos , MutaçãoRESUMO
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.
Assuntos
Aciltransferases/fisiologia , Cardiomiopatias/etiologia , Proteínas de Transporte/fisiologia , Animais , Células COS , Proteínas de Ligação ao Cálcio/metabolismo , Chlorocebus aethiops , Ecocardiografia , Olho/embriologia , Lipoilação , Camundongos , FosforilaçãoRESUMO
Mitochondrial aldehyde dehydrogenase 2 (ALDH2) in the liver removes toxic aldehydes including acetaldehyde, an intermediate of ethanol metabolism. Nearly 40% of East Asians inherit an inactive ALDH2*2 variant, which has a lysine-for-glutamate substitution at position 487 (E487K), and show a characteristic alcohol flush reaction after drinking and a higher risk for gastrointestinal cancers. Here we report the characterization of knockin mice in which the ALDH2(E487K) mutation is inserted into the endogenous murine Aldh2 locus. These mutants recapitulate essentially all human phenotypes including impaired clearance of acetaldehyde, increased sensitivity to acute or chronic alcohol-induced toxicity, and reduced ALDH2 expression due to a dominant-negative effect of the mutation. When treated with a chemical carcinogen, these mutants exhibit increased DNA damage response in hepatocytes, pronounced liver injury, and accelerated development of hepatocellular carcinoma (HCC). Importantly, ALDH2 protein levels are also significantly lower in patient HCC than in peritumor or normal liver tissues. Our results reveal that ALDH2 functions as a tumor suppressor by maintaining genomic stability in the liver, and the common human ALDH2 variant would present a significant risk factor for hepatocarcinogenesis. Our study suggests that the ALDH2*2 allele-alcohol interaction may be an even greater human public health hazard than previously appreciated.
Assuntos
Aldeído Desidrogenase/genética , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Mutação/genética , Intoxicação Alcoólica/enzimologia , Intoxicação Alcoólica/patologia , Aldeído-Desidrogenase Mitocondrial , Substituição de Aminoácidos , Animais , Sequência de Bases , Carcinogênese/patologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Etanol/efeitos adversos , Técnicas de Introdução de Genes , Técnicas de Genotipagem , Hepatócitos/enzimologia , Hepatócitos/patologia , Humanos , Hiperpigmentação/patologia , Imuno-Histoquímica , Fígado/enzimologia , Fígado/patologia , Neoplasias Hepáticas/patologia , Camundongos Endogâmicos C57BL , Proteínas Mutantes/metabolismo , Polimorfismo Genético , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Pele/patologia , Análise de SobrevidaRESUMO
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.
Assuntos
Encefalopatias Metabólicas Congênitas/tratamento farmacológico , Cardiomiopatias/tratamento farmacológico , Isocitrato Desidrogenase/antagonistas & inibidores , Mutação/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Encefalopatias Metabólicas Congênitas/genética , Modelos Animais de Doenças , Isocitrato Desidrogenase/genética , Camundongos , Mutação/genéticaRESUMO
The c-Abl protein is a ubiquitously expressed nonreceptor tyrosine kinase involved in the development and function of many mammalian organ systems, including the immune system and bone. Here we show that homozygous Abl mutant embryos and newborns on the C57BL/6J background, but not on other backgrounds, display dramatically enlarged hearts and die perinatally. The heart defects can be largely rescued by cardiomyocyte-specific restoration of the full-length c-Abl protein. The cardiac hyperplasia phenotype is not caused by decreased apoptosis, but rather by abnormally increased cardiomyocyte proliferation during later stages of embryogenesis. Genes involved in cardiac stress and remodeling and cell cycle regulation are also up-regulated in the mutant hearts. These findings reveal an essential role for c-Abl in mammalian heart growth and development.
Assuntos
Coração/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-abl/metabolismo , Animais , Proliferação de Células , Genes Letais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Fenótipo , Proteínas Proto-Oncogênicas c-abl/genéticaRESUMO
Etiologic risk factors for hepatocellular carcinoma can be involved in the transformation process by directly targeting intracellular signaling pathways or by indirectly stimulating chronic cycles of hepatocyte destruction and regeneration. However, the contribution of these two routes to hepatocarcinogenesis has not been determined, partly because of the difficulty in distinguishing damaged and regenerated hepatocytes. Here we report that induced deletion of the damaged DNA binding protein 1 (DDB1) abrogates the self-renewing capacity of hepatocytes, resulting in compensatory proliferation of DDB1-expressing hepatocytes. Constitutive stimulation of this regeneration process leads to development of hepatocellular carcinoma, which surprisingly contains no disruption of the DDB1 gene, indicating a cell-nonautonomous role of DDB1 inactivation in tumor initiation. Our results suggest that viruses and hepatoxins may cause liver tumors by simply driving hepatocyte turnover without directly targeting cancer cells.
Assuntos
Carcinoma Hepatocelular/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Neoplasias Hepáticas/metabolismo , Fígado/metabolismo , Proteínas de Neoplasias/metabolismo , Regeneração , Animais , Carcinoma Hepatocelular/genética , Proliferação de Células , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Hepatócitos/metabolismo , Neoplasias Hepáticas/genética , Camundongos , Camundongos Mutantes , Proteínas de Neoplasias/genéticaRESUMO
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.
Assuntos
Neoplasias , Nicotinamida Fosforribosiltransferase , Humanos , Proliferação de Células , NAD , Neoplasias/tratamento farmacológico , ProteóliseRESUMO
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.
Assuntos
Antígenos CD28 , Receptor de Morte Celular Programada 1 , Animais , Linfócitos T CD8-Positivos , Fatores Imunológicos , Imunoterapia/métodos , Lenalidomida/farmacologia , CamundongosRESUMO
The Abl family nonreceptor tyrosine kinases, consisting of closely related Abl and Arg (Abl-related gene), play essential roles in mouse neurulation, but their functions in the subsequent development of CNS are poorly understood. Here, we show that conditional deletion of Abl in precursors of neurons and glia on an Arg knock-out background leads to striking cerebellar malformations, including defects in anterior cerebellar morphogenesis, granule cell ectopia, and hypoplasia. Time course analyses reveal that the abnormal anterior cerebellar foliation results from local disruptions of the basement membrane (BM) located between radial glial endfeet and the meninges during embryonic cerebellar development. Granule cell ectopia and hypoplasia are also associated with the breaches in the BM and abnormal Bergmann glial networks during postnatal cerebellar development. In vitro culture experiments indicate that Abl/Arg-deficient granule cells can interact with glial processes and proliferate normally in response to sonic hedgehog compared to cells isolated from control mice. Consistent with these findings, selective ablation of Abl family kinases in cerebellar granule cells alone does not cause any abnormality, suggesting that deletion of Abl/Arg from glia is likely required for the mutant phenotype. Together, these results provide compelling evidence that Abl and Arg play key redundant roles in BM maintenance and cortical lamination in the cerebellum.
Assuntos
Membrana Basal/fisiologia , Córtex Cerebelar/fisiologia , Genes abl/genética , Proteínas Oncogênicas v-abl/fisiologia , Animais , Antimetabólitos , Western Blotting , Bromodesoxiuridina , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Córtex Cerebelar/metabolismo , Feminino , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroglia/fisiologia , Proteínas Oncogênicas v-abl/genética , Gravidez , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Desempenho Psicomotor/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
OBJECTIVE: Study on the application of WAIS-RC short forms and adult intelligence disability scale in mental impairment assessment. METHODS: Mental impairment assessment cases between July 2009 and March 2011 in judicial appraisal institute of Taizhou University were collected. Assessment results obtained with the WAIS-RC short forms and adult intelligence disability scale were compared with the experts assessing conclusions and analyzed using SPSS 11.5 software. RESULTS: Assessment results with the two scales did not fully comply with the expert's conclusions, with reliability coefficient were 0.785 and 0.940 respectively, correlation coefficient were 0.850 and 0.922 respectively. CONCLUSION: The intelligence assessment was influenced by many factors. When the appraised individuals had nerve dysfunction and mild intelligence disability or mental disorders, the two scales should be used together. When the appraised individuals had moderate intelligence disability or mental disorders, adult intelligence disability scale had advantage.
Assuntos
Deficiência Intelectual/diagnóstico , Inteligência , Escalas de Wechsler , Acidentes de Trânsito , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Lesões Encefálicas/complicações , Avaliação da Deficiência , Prova Pericial , Feminino , Psiquiatria Legal , Humanos , Deficiência Intelectual/psicologia , Masculino , Transtornos Mentais/diagnóstico , Transtornos Mentais/psicologia , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Índice de Gravidade de Doença , Escalas de Wechsler/normas , Escalas de Wechsler/estatística & dados numéricos , Adulto JovemRESUMO
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.
Assuntos
Evasão da Resposta Imune , MicroRNAs/metabolismo , Neoplasias , Animais , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Heterogeneidade Genética , Humanos , Imunoterapia , Interferon gama , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Neoplasias/genética , Fosfoproteínas Fosfatases , Receptor de Morte Celular Programada 1 , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Linfócitos TRESUMO
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.
Assuntos
Agentes de Imunomodulação , Mieloma Múltiplo , Medula Óssea/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Mieloma Múltiplo/metabolismo , NF-kappa B/metabolismo , Recidiva Local de Neoplasia , Fator 2 Associado a Receptor de TNF/metabolismo , Microambiente TumoralRESUMO
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.
Assuntos
Adipócitos/efeitos dos fármacos , Proteínas de Bactérias/farmacologia , Colágeno/metabolismo , Colagenases/farmacologia , Lipólise/efeitos dos fármacos , Gordura Subcutânea/efeitos dos fármacos , Adipócitos/patologia , Animais , Proteínas de Bactérias/isolamento & purificação , Colagenases/isolamento & purificação , Escherichia coli/genética , Camundongos , Camundongos Obesos , Obesidade/tratamento farmacológico , Gordura Subcutânea/patologia , Suínos , Porco MiniaturaRESUMO
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.
Assuntos
Complexo do Signalossomo COP9/metabolismo , Sistemas CRISPR-Cas , Fator de Transcrição Ikaros/metabolismo , Fatores Imunológicos/farmacologia , Mieloma Múltiplo/tratamento farmacológico , Peptídeo Hidrolases/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Bortezomib/farmacologia , Complexo do Signalossomo COP9/antagonistas & inibidores , Complexo do Signalossomo COP9/genética , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Fator de Transcrição Ikaros/genética , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Peptídeo Hidrolases/genética , Prognóstico , Proteólise , Pirimidinas/farmacologia , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
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.