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2.
Anal Methods ; 13(35): 3874-3884, 2021 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-34528947

RESUMO

The key factor in the development of antibody-based assays is to find an antibody that has an appropriate affinity, high specificity, and low cross-reactivity. However, this task is not easy to carry out since the research antibodies on the market may suffer from low specificity and reproducibility. Here, we report on a palm-sized dot blot-based device, called the affiblot, that has a specially designed lid that allows simultaneous semi-quantitative comparison of up to five antibodies from different suppliers regarding their affinity/avidity, cross-reactivity, and batch-to-batch reliability. The only required peripheral equipment is a vacuum pump, a camera, and densitometry software. The affiblot device was tested for its functionality and its measurements were compared against those obtained by standard dot blot and ELISA. The benefit over these methods, when various antibodies are evaluated, is in its simplicity. It allows easy antigen deposition, fast application and the discarding of the solutions, a compact undivided membrane, and therefore significant decrease of labor. The device was tested with specific anti-ApoE, anti-EpCAM, anti-Salmonella, anti-E. coli, and anti-Listeria antibodies from different suppliers. Their properties were compared for their ability to interact specifically with antigen and/or non-target structures and the best-suited antibody for the intended application was identified.


Assuntos
Anticorpos Monoclonais , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Reprodutibilidade dos Testes
3.
Invest Ophthalmol Vis Sci ; 62(12): 16, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34533562

RESUMO

Purpose: Over 90% of uveal melanomas harbor pathogenic variants of the GNAQ or GNA11 genes that activate survival pathways. As previous studies found that Ras-mutated cell lines were vulnerable to a combination of survival pathway inhibitors and the histone-deacetylase inhibitor romidepsin, we investigated whether this combination would be effective in models of uveal melanoma. Methods: A small-scale screen of inhibitors of bromodomain-containing protein 4 (BRD4; OTX-015), extracellular signal-related kinase (ERK; ulixertinib), mechanistic target of rapamycin (mTOR; AZD-8055), or phosphoinositide 3-kinase (PI3K; GDC-0941) combined with a clinically relevant administration of romidepsin was performed on a panel of uveal melanoma cell lines (92.1, Mel202, MP38, and MP41) and apoptosis was quantified by flow cytometry after 48 hours. RNA sequencing analysis was performed on Mel202 cells treated with romidepsin alone, AZD-8055 alone, or the combination, and protein changes were validated by immunoblot. Results: AZD-8055 with romidepsin was the most effective combination in inducing apoptosis in the cell lines. Increased caspase-3 and PARP cleavage were noted in the cell lines when they were treated with romidepsin and mTOR inhibitors. RNA sequencing analysis of Mel202 cells revealed that apoptosis was the most affected pathway in the romidepsin/AZD-8055-treated cells. Increases in pro-apoptotic BCL2L11 and decreases in anti-apoptotic BIRC5 and BCL2L1 transcripts noted in the sequencing analysis were confirmed at the protein level in Mel202 cells. Conclusions: Our data suggest that romidepsin in combination with mTOR inhibition could be an effective treatment strategy against uveal melanoma due in part to changes in apoptotic proteins.


Assuntos
Apoptose/efeitos dos fármacos , Depsipeptídeos/uso terapêutico , Inibidores de Histona Desacetilases/uso terapêutico , Histona Desacetilases/metabolismo , Melanoma/tratamento farmacológico , Morfolinas/uso terapêutico , Serina-Treonina Quinases TOR/antagonistas & inibidores , Neoplasias Uveais/tratamento farmacológico , Proteína 11 Semelhante a Bcl-2/genética , Caspase 3/metabolismo , Linhagem Celular Tumoral , Combinação de Medicamentos , Citometria de Fluxo , Subunidades alfa de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Immunoblotting , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Poli(ADP-Ribose) Polimerases/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Análise de Sequência de RNA , Survivina/genética , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismo , Neoplasias Uveais/patologia , Proteína bcl-X/genética
4.
mBio ; 12(5): e0233521, 2021 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-34544279

RESUMO

Newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic with astonishing mortality and morbidity. The high replication and transmission of SARS-CoV-2 are remarkably distinct from those of previous closely related coronaviruses, and the underlying molecular mechanisms remain unclear. The innate immune defense is a physical barrier that restricts viral replication. We report here that the SARS-CoV-2 Nsp5 main protease targets RIG-I and mitochondrial antiviral signaling (MAVS) protein via two distinct mechanisms for inhibition. Specifically, Nsp5 cleaves off the 10 most-N-terminal amino acids from RIG-I and deprives it of the ability to activate MAVS, whereas Nsp5 promotes the ubiquitination and proteosome-mediated degradation of MAVS. As such, Nsp5 potently inhibits interferon (IFN) induction by double-stranded RNA (dsRNA) in an enzyme-dependent manner. A synthetic small-molecule inhibitor blunts the Nsp5-mediated destruction of cellular RIG-I and MAVS and processing of SARS-CoV-2 nonstructural proteins, thus restoring the innate immune response and impeding SARS-CoV-2 replication. This work offers new insight into the immune evasion strategy of SARS-CoV-2 and provides a potential antiviral agent to treat CoV disease 2019 (COVID-19) patients. IMPORTANCE The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteases 3C de Coronavírus/metabolismo , Proteína DEAD-box 58/metabolismo , Receptores Imunológicos/metabolismo , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células CACO-2 , Proteases 3C de Coronavírus/genética , Proteína DEAD-box 58/genética , Ensaio de Imunoadsorção Enzimática , Células HCT116 , Células HEK293 , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Immunoblotting , Interferon Tipo I/metabolismo , Camundongos , Receptores Imunológicos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitinação , Replicação Viral/genética , Replicação Viral/fisiologia
5.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576176

RESUMO

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.


Assuntos
Conexina 43/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cromatografia Líquida de Alta Pressão , Citalopram/farmacologia , Conexina 43/genética , Depressão/genética , Depressão/metabolismo , Feminino , Immunoblotting , Ratos , Receptores de Serotonina/metabolismo , Vortioxetina/farmacologia
6.
Biomolecules ; 11(8)2021 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-34439834

RESUMO

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.


Assuntos
Glutaratos/metabolismo , Lisina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Succinatos/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/isolamento & purificação , Especificidade de Anticorpos , Encéfalo/metabolismo , Cromatografia de Afinidade , Soros Imunes/química , Immunoblotting , Fígado/metabolismo , Masculino , Coelhos , Ratos
7.
Sci Rep ; 11(1): 16505, 2021 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-34389744

RESUMO

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. The two predominant histologic variants of RMS, embryonal and alveolar rhabdomyosarcoma (eRMS and aRMS, respectively), carry very different prognoses. While eRMS is associated with an intermediate prognosis, the 5-year survival rate of aRMS is less than 30%. The RMS subtypes are also different at the molecular level-eRMS frequently has multiple genetic alterations, including mutations in RAS and TP53, whereas aRMS often has chromosomal translocations resulting in PAX3-FOXO1 or PAX7-FOXO1 fusions, but otherwise has a "quiet" genome. Interestingly, mutations in RAS are rarely found in aRMS. In this study, we explored the role of oncogenic RAS in aRMS. We found that while ectopic oncogenic HRAS expression was tolerated in the human RAS-driven eRMS cell line RD, it was detrimental to cell growth and proliferation in the human aRMS cell line Rh28. Growth inhibition was mediated by oncogene-induced senescence and associated with increased RB pathway activity and expression of the cyclin-dependent kinase inhibitors p16 and p21. Unexpectedly, the human eRMS cell line RMS-YM, a RAS wild-type eRMS cell line, also exhibited growth inhibition in response to oncogenic HRAS in a manner similar to aRMS Rh28 cells. This work suggests that oncogenic RAS is expressed in a context-dependent manner in RMS and may provide insight into the differential origins and therapeutic opportunities for RMS subtypes.


Assuntos
Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Rabdomiossarcoma Alveolar/metabolismo , Rabdomiossarcoma Embrionário/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Senescência Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Immunoblotting
8.
Cells ; 10(8)2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34440937

RESUMO

Angiogenesis is a critical process in the formation of new capillaries and a key participant in rheumatoid arthritis (RA) pathogenesis. Vascular endothelial growth factor (VEGF) stimulation of endothelial progenitor cells (EPCs) facilitates angiogenesis and the progression of RA. Phosphorylation of sphingosine kinase 1 (SphK1) produces sphingosine-1-phosphate (S1P), which increases inflammatory cytokine production, although the role of S1P in RA angiogenesis is unclear. In this study, we evaluated the impact of S1P treatment on VEGF-dependent angiogenesis in osteoblast-like cells (MG-63 cells) and the significance of SphK1 short hairpin RNA (shRNA) on S1P production in an in vivo model. We found significantly higher levels of S1P and VEGF expression in synovial fluid from RA patients compared with those with osteoarthritis by ELISA analysis. Treating MG-63 cells with S1P increased VEGF production, while focal adhesion kinase (FAK) and Src siRNAs and inhibitors decreased VEGF production in S1P-treated MG-63 cells. Conditioned medium from S1P-treated osteoblasts significantly increased EPC tube formation and migration by inhibiting miR-16-5p synthesis via proto-oncogene tyrosine-protein kinase src (c-Src) and FAK signaling in chick chorioallantoic membrane (CAM) and Matrigel plug assays. Infection with SphK1 shRNA reduced angiogenesis, articular swelling and cartilage erosion in the ankle joints of mice with collagen-induced arthritis (CIA). S1P appears to have therapeutic potential in RA treatment.


Assuntos
Artrite Reumatoide/metabolismo , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Linhagem Celular , Células Progenitoras Endoteliais/citologia , Células Progenitoras Endoteliais/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Immunoblotting , Camundongos , MicroRNAs/metabolismo , Osteoblastos/citologia , Osteoblastos/metabolismo , Transdução de Sinais/fisiologia , Esfingosina/metabolismo
9.
Int J Cancer ; 149(10): 1787-1800, 2021 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-34346508

RESUMO

The splicing of microexons (very small exons) is frequently dysregulated in the brain of individuals with autism spectrum disorder. However, little is known of the patterns, regulatory mechanisms and roles of microexon splicing in cancer. We here examined the transcriptome-wide profile of microexon splicing in matched colorectal cancer (CRC) and normal tissue specimens. Out of 1492 microexons comprising 3 to 15 nucleotides, 21 (1%) manifested differential splicing between CRC and normal tissue. The 21 genes harboring the differentially spliced microexons were enriched in gene ontology terms related to cell adhesion and migration. RNA interference-mediated knockdown experiments identified two splicing factors, RBFOX2 and PTBP1, as regulators of microexon splicing in CRC cells. RBFOX2 and PTBP1 were found to directly bind to microexon-containing pre-mRNAs and to control their splicing in such cells. Differential microexon splicing was shown to be due, at least in part, to altered expression of RBFOX2 and PTBP1 in CRC tissue compared to matched normal tissue. Finally, we found that changes in the pattern of microexon splicing were associated with CRC metastasis. Our data thus suggest that altered expression of RBFOX2 and PTBP1 might influence CRC metastasis through the regulation of microexon splicing.


Assuntos
Processamento Alternativo , Neoplasias Colorretais/genética , Éxons/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Fatores de Processamento de RNA/genética , Proteínas Repressoras/genética , Linhagem Celular Tumoral , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Ontologia Genética , Células HCT116 , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Immunoblotting , Metástase Neoplásica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Ligação Proteica , Precursores de RNA/genética , Precursores de RNA/metabolismo , Fatores de Processamento de RNA/metabolismo , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
10.
Int J Nanomedicine ; 16: 4739-4753, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34267520

RESUMO

Background: Serological tests detecting severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use. Methods: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both <14 days and >14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD). Results: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with <14 days using FT-DBA was 94.7%, increasing to 100% for samples >14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen's Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits. Conclusion: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.


Assuntos
Ouro/química , Immunoblotting/métodos , Imunoglobulina G/análise , Nanopartículas Metálicas/química , Nucleocapsídeo/análise , SARS-CoV-2/isolamento & purificação , Adulto , Anticorpos Antivirais/sangue , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Valor Preditivo dos Testes , SARS-CoV-2/imunologia , Sensibilidade e Especificidade , Estudos Soroepidemiológicos
11.
Int J Med Microbiol ; 311(6): 151518, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34237624

RESUMO

Many models assessing the risk of sepsis utilize the knowledge of the constituents of the plasminogen system, as it is proven that some species of bacteria can activate plasminogen, as a result of interactions with bacterial outer membrane proteins. However, much is yet to be discovered about this interaction since there is little information regarding some bacterial species. This study is aimed to check if Klebsiella pneumoniae, one of the major factors of nosocomial pneumonia and a factor for severe sepsis, has the ability to bind to human plasminogen. The strain used in this study, PCM 2713, acted as a typical representative of the species. With use of various methods, including: electron microscopy, 2-dimensional electrophoresis, immunoblotting and peptide fragmentation fingerprinting, it is shown that Klebsiella pneumoniae binds to human plasminogen, among others, due to plasminogen-bacterial enolase-like protein interaction, occurring on the outer membrane of the bacterium. Moreover, the study reveals, that other proteins, such as: phosphoglucomutase, and phosphoenolpyruvate carboxykinase act as putative plasminogen-binding factors. These information may virtually act as a foundation for future studies investigating: the: pathogenicity of Klebsiella pneumoniae and means for prevention from the outcomes of Klebsiella-derived sepsis.


Assuntos
Klebsiella pneumoniae , Plasminogênio , Proteínas da Membrana Bacteriana Externa , Humanos , Immunoblotting , Fosfopiruvato Hidratase
12.
Commun Biol ; 4(1): 828, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34211117

RESUMO

The heterotrimeric Sec61 complex is a major site for the biogenesis of transmembrane proteins (TMPs), accepting nascent TMP precursors that are targeted to the endoplasmic reticulum (ER) by the signal recognition particle (SRP). Unlike most single-spanning membrane proteins, the integration of type III TMPs is completely resistant to small molecule inhibitors of the Sec61 translocon. Using siRNA-mediated depletion of specific ER components, in combination with the potent Sec61 inhibitor ipomoeassin F (Ipom-F), we show that type III TMPs utilise a distinct pathway for membrane integration at the ER. Hence, following SRP-mediated delivery to the ER, type III TMPs can uniquely access the membrane insertase activity of the ER membrane complex (EMC) via a mechanism that is facilitated by the Sec61 translocon. This alternative EMC-mediated insertion pathway allows type III TMPs to bypass the Ipom-F-mediated blockade of membrane integration that is seen with obligate Sec61 clients.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Biossíntese de Proteínas , Canais de Translocação SEC/metabolismo , Animais , Retículo Endoplasmático/efeitos dos fármacos , Glicoconjugados/farmacologia , Células HeLa , Humanos , Immunoblotting , Membranas Intracelulares/efeitos dos fármacos , Modelos Biológicos , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Canais de Translocação SEC/genética , Partícula de Reconhecimento de Sinal/metabolismo
13.
Commun Biol ; 4(1): 831, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215848

RESUMO

Gain of even a single chromosome leads to changes in human cell physiology and uniform perturbations of specific cellular processes, including downregulation of DNA replication pathway, upregulation of autophagy and lysosomal degradation, and constitutive activation of the type I interferon response. Little is known about the molecular mechanisms underlying these changes. We show that the constitutive nuclear localization of TFEB, a transcription factor that activates the expression of autophagy and lysosomal genes, is characteristic of human trisomic cells. Constitutive nuclear localization of TFEB in trisomic cells is independent of mTORC1 signaling, but depends on the cGAS-STING activation. Trisomic cells accumulate cytoplasmic dsDNA, which activates the cGAS-STING signaling cascade, thereby triggering nuclear accumulation of the transcription factor IRF3 and, consequently, upregulation of interferon-stimulated genes. cGAS depletion interferes with TFEB-dependent upregulation of autophagy in model trisomic cells. Importantly, activation of both the innate immune response and autophagy occurs also in primary trisomic embryonic fibroblasts, independent of the identity of the additional chromosome. Our research identifies the cGAS-STING pathway as an upstream regulator responsible for activation of autophagy and inflammatory response in human cells with extra chromosomes, such as in Down syndrome or other aneuploidy-associated pathologies.


Assuntos
Autofagia/genética , Dano ao DNA , Imunidade Inata/genética , Proteínas de Membrana/genética , Nucleotidiltransferases/genética , Trissomia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular , Células Cultivadas , Regulação da Expressão Gênica , Células HCT116 , Humanos , Immunoblotting , Proteínas de Membrana/metabolismo , Microscopia Confocal , Nucleotidiltransferases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética
14.
Biomolecules ; 11(7)2021 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206715

RESUMO

The early diagnosis of Alzheimer's disease (AD) remains a challenge for medical scientists worldwide, leading to a number of research efforts that focus on biosensor development for AD biomarkers. However, the application of these complicated biosensors is limited in medical diagnosis, due to the difficulties in robust sensing platform development, high costs, and the necessity for technical professionals. We successfully developed a robust straightforward manufacturing process for the fabrication of multi-chamber paper devices using the wax printing method and exploited it to detect amyloid beta 42 oligomers (AßO42, a significant biomarker of AD) using copper-enhanced gold nanoprobe colorimetric immunoblotting. Small hydrophilic reaction chambers could concentrate the target sample to the desired size to improve the sensing performance. The copper-enhanced gold nanoprobe immunoblot using the designed multi-chamber platform exhibited a highly sensitive performance with a limit of detection of 320 pg/mL by the naked eye and 23.7 pg/mL by a smartphone camera. This process from sensing manufacture to sensing conduction is simple to perform whenever medical technicians require time- and cost-savings, without complicated instruments or the need for technical professionals, making it feasible to serve as a diagnostic tool worldwide for the early monitoring of AD and scalable devices for the sensing application of various biomarkers in clinical settings.


Assuntos
Peptídeos beta-Amiloides/análise , Peptídeos beta-Amiloides/imunologia , Immunoblotting/métodos , Doença de Alzheimer/diagnóstico , Biomarcadores , Técnicas Biossensoriais/instrumentação , Cobre/química , Ouro/química , Humanos , Limite de Detecção , Nanopartículas Metálicas , Fragmentos de Peptídeos
15.
Anal Chem ; 93(31): 10807-10815, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34328735

RESUMO

To assess low-abundance protein biomarkers associated with tumor progression, we have developed artificial catalytic antibodies based on well-defined metal clusters modified with rationally designed peptides, termed clusterbodies. Such clusterbodies possess favorable integrated features of matched ultrasmall sizes, intrinsic fluorescence, and enzyme-like catalytic and selective recognition properties that are inaccessible to traditional antibodies. Consequently, a quantitative assay with high accuracy and high sensitivity is established by measuring the fluorescence and catalytic chemiluminescence of metal clusters preferentially recognizing the protein biomarker, which is confirmed by the molecular-weight marker references of immunoblotting. The results of quantitative immunoblotting are highly close to that derived from the enzyme-linked immunosorbent assay, implying the reliability of this protocol. Remarkably, the detection limit of the aimed protein achieved is as low as 1.0 pg, one magnitude lower than that of the conventional immunoassay. The significant variation of expression levels of the biomarker in tumor cells evidently indicates their distinguished invasion ability. This platform has potential application in analyzing low-abundance protein biomarkers in complex biological matrixes, which is essential to corroborate tumor malignancy in early stage. It inspires the construction of clusterbody-based precise bioprobes with customized structures and integrative functions for advanced quantitative biosensing.


Assuntos
Técnicas Biossensoriais , Ensaio de Imunoadsorção Enzimática , Imunoensaio , Immunoblotting , Reprodutibilidade dos Testes
16.
PLoS One ; 16(7): e0254783, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34314438

RESUMO

An array of isoforms of the nuclear estrogen receptor alpha (ER-α) protein contribute to heterogeneous response in breast cancer (BCa); yet, a single-cell analysis tool that distinguishes the full-length ER-α66 protein from the activation function-1 deficient ER-α46 isoform has not been reported. Specific detection of protein isoforms is a gap in single-cell analysis tools, as the de facto standard immunoassay requires isoform-specific antibody probes. Consequently, to scrutinize hormone response heterogeneity among BCa tumor cells, we develop a precision tool to specifically measure ER-α66, ER- α46, and eight ER-signaling proteins with single-cell resolution in the highly hetero-clonal MCF-7 BCa cell line. With a literature-validated pan-ER immunoprobe, we distinguish ER-α66 from ER-α46 in each individual cell. We identify ER-α46 in 5.5% of hormone-sensitive (MCF-7) and 4.2% of hormone-insensitive (MDA-MB-231) BCa cell lines. To examine whether the single-cell immunoblotting can capture cellular responses to hormones, we treat cells with tamoxifen and identify different sub-populations of ER-α46: (i) ER-α46 induces phospho-AKT at Ser473, (ii) S6-ribosomal protein, an upstream ER target, activates both ER-α66 and ER-α46 in MCF-7 cells, and (iii) ER-α46 partitions MDA-MB-231 subpopulations, which are responsive to tamoxifen. Unlike other single-cell immunoassays, multiplexed single-cell immunoblotting reports-in the same cell-tamoxifen effects on ER signaling proteins and on distinct isoforms of the ER-α protein.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Análise de Célula Única/métodos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Receptor alfa de Estrogênio/genética , Feminino , Humanos , Immunoblotting , Fosforilação/efeitos dos fármacos , Análise de Componente Principal , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Análise de Célula Única/instrumentação , Tamoxifeno/farmacologia
17.
Nat Cell Biol ; 23(8): 846-858, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34257406

RESUMO

The integral membrane protein ATG9A plays a key role in autophagy. It displays a broad intracellular distribution and is present in numerous compartments, including the plasma membrane (PM). The reasons for the distribution of ATG9A to the PM and its role at the PM are not understood. Here, we show that ATG9A organizes, in concert with IQGAP1, components of the ESCRT system and uncover cooperation between ATG9A, IQGAP1 and ESCRTs in protection from PM damage. ESCRTs and ATG9A phenocopied each other in protection against PM injury. ATG9A knockouts sensitized the PM to permeabilization by a broad spectrum of microbial and endogenous agents, including gasdermin, MLKL and the MLKL-like action of coronavirus ORF3a. Thus, ATG9A engages IQGAP1 and the ESCRT system to maintain PM integrity.


Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/genética , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Imunoprecipitação , Proteínas de Membrana/genética , Microscopia Confocal , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/genética
18.
Biochem Biophys Res Commun ; 569: 72-78, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34237430

RESUMO

The membrane protein SIRPα is a cold stress-responsive signaling molecule in neurons. Cold stress directly induces tyrosine phosphorylation of SIRPα in its cytoplasmic region, and phosphorylated SIRPα is involved in regulating experience-dependent behavioral changes in mice. Here, we examined the mechanism of cold stress-induced SIRPα phosphorylation in vitro and in vivo. The levels of activated Src family protein tyrosine kinases (SFKs), which phosphorylate SIRPα, were not increased by lowering the temperature in cultured neurons. Although the SFK inhibitor dasatinib markedly reduced SIRPα phosphorylation, low temperature induced an increase in SIRPα phosphorylation even in the presence of dasatinib, suggesting that SFK activation is not required for low temperature-induced SIRPα phosphorylation. However, in the presence of pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases), SIRPα phosphorylation was significantly reduced by lowering the temperature, suggesting that either the inactivation of PTPase(s) that dephosphorylate SIRPα or increased protection of phosphorylated SIRPα from the PTPase activity is important for low temperature-induced SIRPα phosphorylation. Inactivation of PTPase Shp2 by the allosteric Shp2 inhibitor SHP099, but not by the competitive inhibitor NSC-87877, reduced SIRPα phosphorylation in cultured neurons. Shp2 knockout also reduced SIRPα phosphorylation in the mouse brain. Our data suggest that Shp2, but not SFKs, positively regulates cold stress-induced SIRPα phosphorylation in a PTPase activity-independent manner.


Assuntos
Temperatura Baixa , Neurônios/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Receptores Imunológicos/metabolismo , Tirosina/metabolismo , Animais , Células Cultivadas , Resposta ao Choque Frio , Dasatinibe/farmacologia , Immunoblotting , Camundongos Knockout , Camundongos Transgênicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Pirimidinas/farmacologia
19.
Biochem J ; 478(14): 2825-2842, 2021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34195792

RESUMO

Cullin ubiquitin ligases drive replisome disassembly during DNA replication termination. In worm, frog and mouse cells, CUL2LRR1 is required to ubiquitylate the MCM7 subunit of the CMG helicase. Here, we show that cullin ligases also drive CMG-MCM7 ubiquitylation in human cells, thereby making the helicase into a substrate for the p97 unfoldase. Using purified human proteins, including a panel of E2 ubiquitin-conjugating enzymes, we have reconstituted CMG helicase ubiquitylation, dependent upon neddylated CUL2LRR1. The reaction is highly specific to CMG-MCM7 and requires the LRR1 substrate targeting subunit, since replacement of LRR1 with the alternative CUL2 adaptor VHL switches ubiquitylation from CMG-MCM7 to HIF1. CUL2LRR1 firstly drives monoubiquitylation of CMG-MCM7 by the UBE2D class of E2 enzymes. Subsequently, CUL2LRR1 activates UBE2R1/R2 or UBE2G1/G2 to extend a single K48-linked ubiquitin chain on CMG-MCM7. Thereby, CUL2LRR1 converts CMG into a substrate for p97, which disassembles the ubiquitylated helicase during DNA replication termination.


Assuntos
Proteínas Culina/metabolismo , DNA Helicases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Animais , Linhagem Celular , Clonagem Molecular/métodos , Proteínas Culina/genética , DNA Helicases/genética , Humanos , Immunoblotting , Lisina/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Células Sf9 , Spodoptera , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Proteína com Valosina/genética , Proteína com Valosina/metabolismo
20.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069309

RESUMO

We identified a novel heterozygous hypofibrinogenemia, γY278H (Hiroshima). To demonstrate the cause of reduced plasma fibrinogen levels (functional level: 1.12 g/L and antigenic level: 1.16 g/L), we established γY278H fibrinogen-producing Chinese hamster ovary (CHO) cells. An enzyme-linked immunosorbent assay demonstrated that synthesis of γY278H fibrinogen inside CHO cells and secretion into the culture media were not reduced. Then, we established an additional five variant fibrinogen-producing CHO cell lines (γL276P, γT277P, γT277R, γA279D, and γY280C) and conducted further investigations. We have already established 33 γ-module variant fibrinogen-producing CHO cell lines, including 6 cell lines in this study, but only the γY278H and γT277R cell lines showed disagreement, namely, recombinant fibrinogen production was not reduced but the patients' plasma fibrinogen level was reduced. Finally, we performed fibrinogen degradation assays and demonstrated that the γY278H and γT277R fibrinogens were easily cleaved by plasmin whereas their polymerization in the presence of Ca2+ and "D:D" interaction was normal. In conclusion, our investigation suggested that patient γY278H showed hypofibrinogenemia because γY278H fibrinogen was secreted normally from the patient's hepatocytes but then underwent accelerated degradation by plasmin in the circulation.


Assuntos
Afibrinogenemia/genética , Fibrinogênios Anormais/genética , Fibrinogênios Anormais/metabolismo , Mutação , Adulto , Afibrinogenemia/sangue , Animais , Testes de Coagulação Sanguínea , Células CHO , Cricetulus , Fator XIIIa/química , Fator XIIIa/metabolismo , Feminino , Fibrina/metabolismo , Fibrinogênios Anormais/química , Fibrinolisina/metabolismo , Heterozigoto , Humanos , Immunoblotting , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/metabolismo
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