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1.
Free Radic Biol Med ; 175: 206-215, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34506903

RESUMO

Flavonoids are natural polyphenolic compounds with a diverse array of biological activities and health-promoting effects. Recent studies have found that 4,4'-dimethoxychalcone (DMC) promoted longevity via autophagy; however, its targets are currently unknown. Herein, we employed an unbiased thermal proteome profiling (TPP) method and identified multiple targets of DMC, including ALDH1A3, ALDH2, and PTGES2. We further determined the dissociation constant (Kd) of DMC and ALDH1A3 to be 2.8 µM using microscale thermophoresis (MST) analysis, which indicated that DMC inhibited ALDH1A3 activity and aggravated cellular oxidative stress. DMC treatment significantly increased cellular reactive oxygen species (ROS) production and inhibited cancer cell growth. Quantitative proteomic analysis showed that DMC upregulated proteins associated with stress-responses and downregulated proteins associated with cell cycle progression, and this was confirmed using cell cycle analysis. Taken together, we showed that TPP is an effective tool with which to identify flavonoid targets and set a precedent for deciphering flavonoid function in the future. We have demonstrated that DMC inhibited cell proliferation via ROS-induced cell cycle arrest and is an anti-proliferative agent in cancer treatment.


Assuntos
Flavonoides , Proteômica , Apoptose , Proliferação de Células , Flavonoides/farmacologia , Estresse Oxidativo , Oxirredutases , Espécies Reativas de Oxigênio
2.
J Cell Mol Med ; 25(19): 9306-9318, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34435746

RESUMO

To determine the differentially expressed proteins (DEPs) between paired samples of cervical cancer (CC) and paracancerous tissue by quantitative proteomics and to examine the effects of DUSP7 expression on the tumorigenesis and progression of CC. Proteomic profiles of three paired samples of CC and paracancerous tissue were quantitatively analysed to identify DEPs. The relationship between DEP expression and patient clinicopathological characteristics and prognosis was evaluated. The effects of the selected DEPs on CC progression were examined in SIHA cells. A total of 129 DEPs were found. Western blot and immunohistochemistry (IHC) staining analyses confirmed the results from quantitative proteomic analysis showing that the selected DEP, HRAS, P-ERK1/2, and PLD1 levels were increased, whereas the DUSP7 level was decreased in CC tissue compared with the paired normal paracancerous tissues. The IHC results from the CC TMA analysis showed that the decreased expression of DUSP7 (p = 0.045 and 0.044) was significantly associated with a tumour size >2 cm and parametrial infiltration. In addition, the decreased expression of DUSP7 and increased expression of p-ERK1/2 were adversely related to patient relapse (p = 0.003 and 0.001) and survival (p = 0.034 and 0.006). The expression of HRAS and p-ERK1/2 was decreased in DUSP7-SIHA cells compared with NC-SIHA cells (p = 0.0003 and 0.0026). Biological functions in vitro, including invasion, migration and proliferation and tumour formation in vivo were decreased in DUSP7-SIHA cells (all p < 0.05) but increased in shDUSP7-SIHA cells (all p < 0.05). DUSP7 inhibits cervical cancer progression by inactivating the RAS pathway.

3.
PLoS One ; 16(8): e0250544, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34464391

RESUMO

Collagen is the major protein in the extracellular matrix and plays vital roles in tissue development and function. Collagen is also one of the most processed proteins in its biosynthesis. The most prominent post-translational modification (PTM) of collagen is the hydroxylation of Pro residues in the Y-position of the characteristic (Gly-Xaa-Yaa) repeating amino acid sequence of a collagen triple helix. Recent studies using mass spectrometry (MS) and tandem MS sequencing (MS/MS) have revealed unexpected hydroxylation of Pro residues in the X-positions (X-Hyp). The newly identified X-Hyp residues appear to be highly heterogeneous in location and percent occupancy. In order to understand the dynamic nature of the new X-Hyps and their potential impact on applications of MS and MS/MS for collagen research, we sampled four different collagen samples using standard MS and MS/MS techniques. We found considerable variations in the degree of PTMs of the same collagen from different organisms and/or tissues. The rat tail tendon type I collagen is particularly variable in terms of both over-hydroxylation of Pro in the X-position and under-hydroxylation of Pro in the Y-position. In contrast, only a few unexpected PTMs in collagens type I and type III from human placenta were observed. Some observations are not reproducible between different sequencing efforts of the same sample, presumably due to a low population and/or the unpredictable nature of the ionization process. Additionally, despite the heterogeneous preparation and sourcing, collagen samples from commercial sources do not show elevated variations in PTMs compared to samples prepared from a single tissue and/or organism. These findings will contribute to the growing body of information regarding the PTMs of collagen by MS technology, and culminate to a more comprehensive understanding of the extent and the functional roles of the PTMs of collagen.


Assuntos
Colágeno/química , Espectrometria de Massas , Prolina/química , Processamento de Proteína Pós-Traducional , Animais , Colágeno/metabolismo , Humanos , Hidroxilação , Prolina/metabolismo , Ratos
4.
Front Mol Biosci ; 8: 702107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34295923

RESUMO

Macrophage activation is an important process in controlling infection, but persistent macrophage activation leads to chronic inflammation and diseases, such as tumor progression, insulin resistance and atherosclerosis. Characterizing metabolic signatures of macrophage activation is important for developing new approaches for macrophage inactivation. Herein, we performed metabolomic analysis on lipopolysaccharide (LPS)-activated macrophages and identified the associated changes in metabolites. Notably, the cellular Nicotinamide adenine dinucleotide+ levels were decreased while NADPH was increased, proposing that NAD+ restoration can inhibit macrophage activation. Indeed, supplementation of nicotinamide mononucleotide (NMN) increased cellular NAD+ levels and decreased cytokine productions in LPS-activated cells. Quantitative proteomics identified that nicotinamide mononucleotide downregulated the expressions of LPS-responsive proteins, in which cyclooxygenase-2 (COX-2) expression was significantly decreased in NMN-treated cells. Consequently, the cellular levels of prostaglandin E2 (PGE2) was also decreased, indicating that NMN inactivated macrophages via COX-2-PGE2 pathway, which was validated in activated THP-1 cells and mouse peritoneal macrophages. In conclusion, the present study identified the metabolic characteristics of activated macrophages and revealed that NMN replenishment is an efficient approach for controlling macrophage activation.

5.
Oncol Lett ; 22(2): 630, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34267822

RESUMO

Colorectal cancer (CRC) is the fourth most lethal cancer in the world. Heat shock protein 60 (HSP60), a mitochondrial chaperone that maintains mitochondrial proteostasis, is highly expressed in tumors compared with in paracancerous tissues, suggesting that high HSP60 expression benefits tumor growth. To determine the effects of HSP60 expression on tumor progression, stable HSP60-knockdown HCT116 cells were constructed in the present study, revealing that knockdown of HSP60 inhibited cell proliferation. Proteomic analysis demonstrated that mitochondrial proteins were downregulated, indicating that knockdown of HSP60 disrupted mitochondrial homeostasis. Metabolomic analysis demonstrated that cellular adenine levels were >30-fold higher in HSP60-knockdown cells than in control cells. It was further confirmed that elevated adenine activated the AMPK signaling pathway, which inhibited mTOR-regulated protein synthesis to slow down cell proliferation. Overall, the current results provide a valuable resource for understanding mitochondrial function in CRC, suggesting that HSP60 may be a potential target for CRC intervention.

6.
Nat Commun ; 12(1): 4645, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34330918

RESUMO

A major part of the transcriptome complexity is attributed to multiple types of DNA or RNA fusion events, which take place within a gene such as alternative splicing or between different genes such as DNA rearrangement and trans-splicing. In the present study, using the RNA deep sequencing data, we systematically survey a type of non-canonical fusions between the RNA transcripts from the two opposite DNA strands. We name the products of such fusion events cross-strand chimeric RNA (cscRNA). Hundreds to thousands of cscRNAs can be found in human normal tissues, primary cells, and cancerous cells, and in other species as well. Although cscRNAs exhibit strong tissue-specificity, our analysis identifies thousands of recurrent cscRNAs found in multiple different samples. cscRNAs are mostly originated from convergent transcriptions of the annotated genes and their anti-sense DNA. The machinery of cscRNA biogenesis is unclear, but the cross-strand junction events show some features related to RNA splicing. The present study is a comprehensive survey of the non-canonical cross-strand RNA junction events, a resource for further characterization of the originations and functions of the cscRNAs.


Assuntos
Perfilação da Expressão Gênica/métodos , Fusão Gênica , Splicing de RNA , RNA/genética , Trans-Splicing , Transcriptoma/genética , Células A549 , Linhagem Celular Tumoral , Biologia Computacional/métodos , Humanos , Hibridização in Situ Fluorescente/métodos , Modelos Genéticos , Células PC-3 , Precursores de RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos
7.
Cell Death Dis ; 12(7): 697, 2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34257278

RESUMO

The tripartite motif-containing protein 21 (TRIM21) plays important roles in autophagy and innate immunity. Here, we found that HECT and RLD domain containing E3 ubiquitin protein ligase 5 (HERC5), as an interferon-stimulated gene 15 (ISG15) E3 ligase, catalyzes the ISGylation of TRIM21 at the Lys260 and Lys279 residues. Moreover, IFN-ß also induces TRIM21 ISGylation at multiple lysine residues, thereby enhancing its E3 ligase activity for K63-linkage-specific ubiquitination and resulting in increased levels of TRIM21 and p62 K63-linked ubiquitination. The K63-linked ubiquitination of p62 at Lys7 prevents its self-oligomerization and targeting to the autophagosome. Taken together, our study suggests that the ISGylation of TRIM21 plays a vital role in regulating self-oligomerization and localization of p62 in the autophagy induced by IFN-ß.


Assuntos
Autofagossomos/enzimologia , Citocinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Processamento de Proteína Pós-Traducional , Ribonucleoproteínas/metabolismo , Proteína Sequestossoma-1/metabolismo , Ubiquitinas/metabolismo , Células A549 , Autofagossomos/efeitos dos fármacos , Autofagossomos/genética , Autofagia , Citocinas/genética , Células HEK293 , Humanos , Interferon beta/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lisina , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ribonucleoproteínas/genética , Proteína Sequestossoma-1/genética , Ubiquitinação , Ubiquitinas/genética
8.
Sci Rep ; 11(1): 14253, 2021 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-34244565

RESUMO

SIRT6 is an NAD+-dependent deacetylase that plays an important role in mitosis fidelity and genome stability. In the present study, we found that SIRT6 overexpression leads to mitosis defects and aneuploidy. We identified SIRT6 as a novel substrate of anaphase-promoting complex/cyclosome (APC/C), which is a master regulator of mitosis. Both CDH1 and CDC20, co-activators of APC/C, mediated SIRT6 degradation via the ubiquitination-proteasome pathway. Reciprocally, SIRT6 also deacetylated CDH1 at lysine K135 and promoted its degradation, resulting in an increase in APC/C-CDH1-targeted substrates, dysfunction in centrosome amplification, and chromosome instability. Our findings demonstrate the importance of SIRT6 for genome integrity during mitotic progression and reveal how SIRT6 and APC/C cooperate to drive mitosis.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Sirtuínas/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Proteínas Cdc20/genética , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Instabilidade Cromossômica/genética , Instabilidade Cromossômica/fisiologia , Células HeLa , Humanos , Ligação Proteica , Sirtuínas/genética
9.
Cell Rep ; 36(4): 109421, 2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34320342

RESUMO

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.

10.
Nat Microbiol ; 6(7): 921-931, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34127846

RESUMO

Zoonotic arenaviruses can lead to life-threating diseases in humans. These viruses encode a large (L) polymerase that transcribes and replicates the viral genome. At the late stage of replication, the multifunctional Z protein interacts with the L polymerase to shut down RNA synthesis and initiate virion assembly. However, the mechanism by which the Z protein regulates the activity of L polymerase is unclear. Here, we used cryo-electron microscopy to resolve the structures of both Lassa and Machupo virus L polymerases in complex with their cognate Z proteins, and viral RNA, to 3.1-3.9 Å resolutions. These structures reveal that Z protein binding induces conformational changes in two catalytic motifs of the L polymerase, and restrains their conformational dynamics to inhibit RNA synthesis, which is supported by hydrogen-deuterium exchange mass spectrometry analysis. Importantly, we show, by in vitro polymerase reactions, that Z proteins of Lassa and Machupo viruses can cross-inhibit their L polymerases, albeit with decreased inhibition efficiencies. This cross-reactivity results from a highly conserved determinant motif at the contacting interface, but is affected by other variable auxiliary motifs due to the divergent evolution of Old World and New World arenaviruses. These findings could provide promising targets for developing broad-spectrum antiviral drugs.


Assuntos
Arenavirus do Novo Mundo/química , Vírus Lassa/química , RNA Polimerase Dependente de RNA/química , Proteínas Virais/química , Motivos de Aminoácidos , Antivirais/farmacologia , Arenavirus do Novo Mundo/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Vírus Lassa/metabolismo , Mutação , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , RNA Viral/química , RNA Viral/metabolismo , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo
11.
Angew Chem Int Ed Engl ; 60(33): 17932-17936, 2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34060691

RESUMO

We present a method for atomically precise nanocluster synthesis. As an illustration, we introduced the reducing-ligand induction combined method and synthesized a novel nanocluster, which was determined to be Au28 (SCH2 Ph-t Bu)22 with the same number of gold atoms as existing Au28 (SR)20 nanoclusters but different ligands (hetero-composition-homo-size). Compared with the latter, the former has distinct properties and structures. In particular, a novel kernel evolution pattern is reported, i.e., the quasi-linear growth of Au4 -tetrahedron by sharing one vertex and structural features, including a tritetrahedron kernel with two bridging thiolates and two Au6 (SCH2 Ph-t Bu)6 hexamer chair-like rings on the kernel surface were also first reported, which endow Au28 (SCH2 Ph-t Bu)22 with the best photoluminescence quantum yield among hydrophobic thiolated gold nanoclusters so far, probably due to the enhanced charge transfer from the bi-ring to the kernel via Au-Au bonds.

12.
Nucleic Acids Res ; 49(11): 6511-6528, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34048589

RESUMO

The zinc uptake regulator (Zur) is a member of the Fur (ferric uptake regulator) family transcriptional regulators that plays important roles in zinc homeostasis and virulence of bacteria. Upon zinc perception, Zur binds to the promoters of zinc responsive genes and controls their transcription. However, the mechanism underlying zinc-mediated Zur activation remains unclear. Here we report a 2.2-Å crystal structure of apo Zur from the phytopathogen Xanthomonas campestris pv. campestris (XcZur), which reveals the molecular mechanism that XcZur exists in a closed inactive state before regulatory zinc binding. Subsequently, we present a 1.9-Å crystal structure of holo XcZur, which, by contrast, adopts an open state that has enough capacity to bind DNA. Structural comparison and hydrogen deuterium exchange mass spectrometry (HDX-MS) analyses uncover that binding of a zinc atom in the regulatory site, formed by the hinge region, the dimerization domain and the DNA binding domain, drives a closed-to-open conformational change that is essential for XcZur activation. Moreover, key residues responsible for DNA recognition are identified by site-directed mutagenesis. This work provides important insights into zinc-induced XcZur activation and valuable discussions on the mechanism of DNA recognition.


Assuntos
Proteínas de Bactérias/química , Zinco/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Alinhamento de Sequência , Transcrição Genética , Xanthomonas campestris
13.
Angew Chem Int Ed Engl ; 60(31): 17171-17177, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34021957

RESUMO

Activity-based E2 conjugating enzyme (E2)-ubiquitin (Ub) probes have recently emerged as effective tools for studying the molecular mechanism of E3 ligase (E3)-catalyzed ubiquitination. However, the preparation of existing activity-based E2-Ub probes depends on recombination technology and bioconjugation chemistry, limiting their structural diversity. Herein we describe an expedient total chemical synthesis of an E2 enzyme variant through a hydrazide-based native chemical ligation, which enabled the construction of a structurally new activity-based E2-Ub probe to covalently capture the catalytic site of Cys-dependent E3s. Chemical cross-linking coupled with mass spectrometry (CXMS) demonstrated the utility of this new probe in structural analysis of the intermediates formed during Nedd4 and Parkin-mediated transthiolation. This study exemplifies the utility of chemical protein synthesis for the development of protein probes for biological studies.


Assuntos
Compostos de Sulfidrila/metabolismo , Ubiquitina-Proteína Ligases/análise , Ubiquitina/química , Biocatálise , Humanos , Estrutura Molecular , Compostos de Sulfidrila/química , Ubiquitina/síntese química , Ubiquitina-Proteína Ligases/metabolismo
14.
J Proteome Res ; 20(5): 2596-2606, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33793246

RESUMO

Decreased cellular NAD+ levels are causally linked to aging and aging-associated diseases. NAD+ precursors in oxidized form such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) have gained much attention and been well studied for their ability to restore NAD+ levels in model organisms. Less is known about whether NAD+ precursors in reduced form can also efficiently increase the tissue and cellular NAD+ levels and have different effects on cellular processes than NMN or NR. In the present study, we developed a chemical method to produce dihydronicotinamide mononucleotide (NMNH), which is the reduced form of NMN. We demonstrated that NMNH was a better NAD+ enhancer than NMN both in vitro and in vivo, mediated by nicotinamide mononucleotide adenylyltransferase (NMNAT). Additionally, NMNH increased the reduced NAD (NADH) levels in cells and in mouse livers. Metabolomic analysis revealed that NMNH inhibited glycolysis and the TCA cycle. In vitro experiments demonstrated that NMNH induced cell cycle arrest and suppressed cell growth. Nevertheless, NMNH treatment did not cause an observable difference in mouse weight. Taken together, our work demonstrates that NMNH is a potent NAD+ enhancer and suppresses glycolysis, the TCA cycle, and cell growth.


Assuntos
NAD , Mononucleotídeo de Nicotinamida , Animais , Ciclo Celular , Ciclo do Ácido Cítrico , Glicólise , Camundongos , NAD/metabolismo , Mononucleotídeo de Nicotinamida/metabolismo
15.
Mol Microbiol ; 116(2): 438-458, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33811693

RESUMO

Streptococcus pneumoniae resides in the human upper airway as a commensal but also causes pneumonia, bacteremia, meningitis, and otitis media. It remains unclear how pneumococci adapt to nutritional conditions of various host niches. We here show that MetR, a LysR family transcriptional regulator, serves as a molecular adaptor for pneumococcal fitness, particularly in the upper airway. The metR mutant of strain D39 rapidly disappeared from the nasopharynx but was marginally attenuated in the lungs and bloodstream of mice. RNA-seq and ChIP-seq analyses showed that MetR broadly regulates transcription of the genes involved in methionine synthesis and other functions under methionine starvation. Genetic and biochemical analyses confirmed that MetR is essential for the activation of methionine synthesis but not uptake. Co-infection of influenza virus partially restored the colonization defect of the metR mutant. These results strongly suggest that MetR is particularly evolved for pneumococcal carriage in the upper airway of healthy individuals where free methionine is severely limited, but it becomes dispensable where environmental methionine is relatively more abundant (e.g., inflamed upper airway and sterile sites). To the best of our knowledge, MetR represents the first known regulator particularly for pneumococcal carriage in healthy individuals.

16.
Cell Rep ; 35(3): 109025, 2021 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-33882315

RESUMO

Ablation of Slc22a14 causes male infertility in mice, but the underlying mechanisms remain unknown. Here, we show that SLC22A14 is a riboflavin transporter localized at the inner mitochondrial membrane of the spermatozoa mid-piece and show by genetic, biochemical, multi-omic, and nutritional evidence that riboflavin transport deficiency suppresses the oxidative phosphorylation and reprograms spermatozoa energy metabolism by disrupting flavoenzyme functions. Specifically, we find that fatty acid ß-oxidation (FAO) is defective with significantly reduced levels of acyl-carnitines and metabolites from the TCA cycle (the citric acid cycle) but accumulated triglycerides and free fatty acids in Slc22a14 knockout spermatozoa. We demonstrate that Slc22a14-mediated FAO is essential for spermatozoa energy generation and motility. Furthermore, sperm from wild-type mice treated with a riboflavin-deficient diet mimics those in Slc22a14 knockout mice, confirming that an altered riboflavin level causes spermatozoa morphological and bioenergetic defects. Beyond substantially advancing our understanding of spermatozoa energy metabolism, our study provides an attractive target for the development of male contraceptives.

17.
Nat Chem Biol ; 17(5): 567-575, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33664520

RESUMO

The discovery of effective therapeutic treatments for cancer via cell differentiation instead of antiproliferation remains a great challenge. Cyclin-dependent kinase 2 (CDK2) inactivation, which overcomes the differentiation arrest of acute myeloid leukemia (AML) cells, may be a promising method for AML treatment. However, there is no available selective CDK2 inhibitor. More importantly, the inhibition of only the enzymatic function of CDK2 would be insufficient to promote notable AML differentiation. To further validate the role and druggability of CDK2 involved in AML differentiation, a suitable chemical tool is needed. Therefore, we developed first-in-class CDK2-targeted proteolysis-targeting chimeras (PROTACs), which promoted rapid and potent CDK2 degradation in different cell lines without comparable degradation of other targets, and induced remarkable differentiation of AML cell lines and primary patient cells. These data clearly demonstrated the practicality and importance of PROTACs as alternative tools for verifying CDK2 protein functions.


Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Células Progenitoras Mieloides/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Triazóis/farmacologia , Antineoplásicos/síntese química , Aurora Quinase A/genética , Aurora Quinase A/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Desenho de Fármacos , Descoberta de Drogas , Humanos , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Concentração Inibidora 50 , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/enzimologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Células Progenitoras Mieloides/enzimologia , Células Progenitoras Mieloides/patologia , Piperazinas/farmacologia , Cultura Primária de Células , Piridinas/farmacologia , Pirimidinas/farmacologia , Quinazolinas/farmacologia , Transdução de Sinais , Relação Estrutura-Atividade , Transcriptoma , Triazóis/síntese química
18.
Artigo em Inglês | MEDLINE | ID: mdl-33647481

RESUMO

Clear cell renal cell carcinoma (ccRCC) is a frequently occurring renal cancer. Von Hippel-Lindau disease tumor suppressor (VHL), a known tumor suppressor, is frequently mutated in about 50% of patients with ccRCC. However, it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL. In the present study, we found that higher expression of VHL was correlated with the better disease-free survival (DFS) in ccRCC patients using The Cancer Genome Atlas (TCGA) datasets. We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition (EMT), sterol regulatory element-binding protein 1 (SREBP1) regulated triglyceride synthesis, and cell proliferation. Proteomic analysis provided us a global view that VHL regulated four biological processes including metabolism, immune regulation, apoptosis, and cell movement. Importantly, we found that VHL overexpression led to upregulation of proteins associated with antigen processing and interferon-responsive proteins, rendering ccRCC cells with high VHL expression more sensitive to interferon treatment. We defined an interferon-responsive signature (IRS) with ten proteins, whose expression levels were positively correlated with DFS in ccRCC patients. Taken together, our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.

19.
Mol Cell Proteomics ; 20: 100066, 2021 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-33631294

RESUMO

Plague is a zoonotic disease that primarily infects rodents via fleabite. Transmission from flea to host niches requires rapid adaption of Yersinia pestis to the outer environments to establish infection. Here, quantitative proteome and secretome analyses of Y. pestis grown under conditions mimicking the two typical niches, i.e., the mammalian host (Mh) and the flea vector (Fv), were performed to understand the adaption strategies of this deadly pathogen. A secretome of Y. pestis containing 308 proteins has been identified using TMT-labeling mass spectrometry analysis. Although some proteins are known to be secreted, such as the type III secretion substrates, PsaA and F1 antigen, most of them were found to be secretory proteins for the first time. Comparative proteomic analysis showed that membrane proteins, chaperonins and stress response proteins are significantly upregulated under the Mh condition, among which the previously uncharacterized proteins YP_3416∼YP_3418 are remarkable because they cannot only be secreted but also translocated into HeLa cells by Y. pestis. We further demonstrated that the purified YP_3416 and YP_3418 exhibited E3 ubiquitin ligase activity in in vitro ubiquitination assay and yp_3416∼3418 deletion mutant of Y. pestis showed significant virulence attenuation in mice. Taken together, our results represent the first Y. pestis secretome, which will promote the better understanding of Y. pestis pathogenesis, as well as the development of new strategies for treatment and prevention of plague.

20.
Commun Biol ; 4(1): 226, 2021 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-33597714

RESUMO

NKCC and KCC transporters mediate coupled transport of Na++K++Cl- and K++Cl- across the plasma membrane, thus regulating cell Cl- concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.


Assuntos
Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Simportadores/metabolismo , Animais , Ânions , Sítios de Ligação , Cátions , Microscopia Crioeletrônica , Células HEK293 , Humanos , Ativação do Canal Iônico , Transporte de Íons , Simulação de Dinâmica Molecular , Fosforilação , Ligação Proteica , Conformação Proteica , Células Sf9 , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/ultraestrutura , Relação Estrutura-Atividade , Simportadores/genética , Simportadores/ultraestrutura
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