RESUMO
The blood-brain barrier (BBB) is a unique system of the brain microvasculature that limits the exchange between the blood and the brain. Brain microvascular endothelial cells form the BBB as part of the neurovascular unit and express insulin receptors. The insulin receptor at the BBB has been studied in two different functional aspects. These functions include (1) the supplying of blood insulin to the brain and (2) the modulation of BBB function via insulin signaling. The first function involves drug delivery to the brain, while the second function is related to the association between central nervous system diseases and type 2 diabetes through insulin resistance. This chapter summarizes recent progress in research on the function of insulin receptors at the BBB.
Assuntos
Barreira Hematoencefálica , Receptor de Insulina , Transdução de Sinais , Barreira Hematoencefálica/metabolismo , Receptor de Insulina/metabolismo , Humanos , Transdução de Sinais/fisiologia , Animais , Transporte Biológico/fisiologia , Insulina/metabolismo , Células Endoteliais/metabolismoRESUMO
Data-independent acquisition (DIA) techniques such as sequential window acquisition of all theoretical mass spectra (SWATH) acquisition have emerged as the preferred strategies for proteomic analyses. Our study optimized the SWATH-DIA method using a narrow isolation window placement approach, improving its proteomic performance. We optimized the acquisition parameter combinations of narrow isolation windows with different widths (1.9 and 2.9 Da) on a ZenoTOF 7600 (Sciex); the acquired data were analyzed using DIA-NN (version 1.8.1). Narrow SWATH (nSWATH) identified 5916 and 7719 protein groups on the digested peptides, corresponding to 400 ng of protein from mouse liver and HEK293T cells, respectively, improving identification by 7.52 and 4.99%, respectively, compared to conventional SWATH. The median coefficient of variation of the quantified values was less than 6%. We further analyzed 200 ng of benchmark samples comprising peptides from known ratios ofEscherichia coli, yeast, and human peptides using nSWATH. Consequently, it achieved accuracy and precision comparable to those of conventional SWATH, identifying an average of 95,456 precursors and 9342 protein groups across three benchmark samples, representing 12.6 and 9.63% improved identification compared to conventional SWATH. The nSWATH method improved identification at various loading amounts of benchmark samples, identifying 40.7% more protein groups at 25 ng. These results demonstrate the improved performance of nSWATH, contributing to the acquisition of deeper proteomic data from complex biological samples.
Assuntos
Proteômica , Proteômica/métodos , Humanos , Animais , Camundongos , Células HEK293 , Fígado/metabolismo , Fígado/química , Peptídeos/química , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteoma/análise , Escherichia coli/metabolismo , Escherichia coli/genética , Espectrometria de Massas em Tandem/métodos , Espectrometria de Massas/métodosRESUMO
We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.
Assuntos
Antivirais , Influenza Humana , Fenóis , Animais , Cães , Humanos , Antivirais/farmacologia , Antivirais/química , Proteínas Mitocondriais/metabolismo , Proibitinas , Espectrometria de Massas em Tandem , Canal de Ânion 1 Dependente de Voltagem , Canal de Ânion 2 Dependente de Voltagem/metabolismo , Canais de Ânion Dependentes de Voltagem , Linhagem CelularRESUMO
Prototypic antidepressants, such as tricyclic/tetracyclic antidepressants (TCAs), have multiple pharmacological properties and have been considered to be more effective than newer antidepressants, such as selective serotonin reuptake inhibitors, in treating severe depression. However, the clinical contribution of non-monoaminergic effects of TCAs remains elusive. In this study, we discovered that amitriptyline, a typical TCA, directly binds to the lysophosphatidic acid receptor 1 (LPAR1), a G protein-coupled receptor, and activates downstream G protein signaling, while exerting a little effect on ß-arrestin recruitment. This suggests that amitriptyline acts as a G protein-biased agonist of LPAR1. This biased agonism was specific to TCAs and was not observed with other antidepressants. LPAR1 was found to be involved in the behavioral effects of amitriptyline. Notably, long-term infusion of mouse hippocampus with the potent G protein-biased LPAR agonist OMPT, but not the non-biased agonist LPA, induced antidepressant-like behavior, indicating that G protein-biased agonism might be necessary for the antidepressant-like effects. Furthermore, RNA-seq analysis revealed that LPA and OMPT have opposite patterns of gene expression changes in the hippocampus. Pathway analysis indicated that long-term treatment with OMPT activated LPAR1 downstream signaling (Rho and MAPK), whereas LPA suppressed LPAR1 signaling. Our findings provide insights into the mechanisms underlying the non-monoaminergic antidepressant effects of TCAs and identify the G protein-biased agonism of LPAR1 as a promising target for the development of novel antidepressants.
Assuntos
Amitriptilina , Depressão , Camundongos , Animais , Amitriptilina/farmacologia , Depressão/tratamento farmacológico , Antidepressivos/farmacologia , Antidepressivos/uso terapêutico , Antidepressivos Tricíclicos , Proteínas de Ligação ao GTPRESUMO
In order to establish an in vitro model of the human blood-brain barrier (BBB), MDR1-overexpressing human induced pluripotent stem cells (hiPSCs) were generated, and they were differentiated to MDR1-expressing brain microvascular endothelial-like cells (MDR1-expressing hiPS-BMECs). MDR1-expressing hiPS-BMECs monolayers showed good barrier function in terms of tight junction protein expression and trans-epithelial electrical resistance (TEER). In sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS), MDR1 protein expression was markedly increased in MDR1-expressing hiPS-BMECs, whereas other ABC and SLC transporters showed almost identical expression between MDR1-expressing hiPS-BMECs and mock hiPS-BMECs, suggesting that MDR1 overexpression had little or no knock-on effect on other proteins. The basolateral-to-apical transport of MDR1 substrates, such as quinidine, [3H]digoxin and [3H]vinblastine, was higher than the apical-to-basolateral transport, and the efflux-dominant transport was attenuated by PSC833, an MDR1-specific inhibitor, indicating that MDR1-mediated efflux transport is functional. The robust MDR1 function was also supported by the efflux-dominant transports of [3H]cyclosporin A, loperamide, cetirizine, and verapamil by MDR1-expressing hiPS-BMECs. These results suggest that MDR1-expressing hiPS-BMECs can be used as an in vitro model of the human BBB.
Assuntos
Barreira Hematoencefálica , Células-Tronco Pluripotentes Induzidas , Humanos , Encéfalo , Linhagem Celular , Células CultivadasRESUMO
BACKGROUND: Blood-brain barrier (BBB) dysfunction is supposed to be an early event in the development of Alzheimer's disease (AD). This study aimed to investigate the relationship between BBB alterations and AD progression in terms of amyloid-ß peptide (Aß) accumulation in the brains of humanized amyloid precursor protein knock-in (APP-KI) mice. METHODS: Brain Aß accumulation was examined using immunohistochemical analysis. Alterations in differentially expressed proteins were determined using sequential window acquisition of all theoretical fragment ion mass spectroscopy (SWATH-MS)-based quantitative proteomics, and Metascape, STRING, Gene Ontology, and KEGG were used for network analyses of altered biological pathways and processes. Statistical significance was determined using the unpaired two-tailed Student's t-test and Welch's t-test for two groups and one-way analysis of variance followed by Tukey's test for more than two groups. Correlations between two groups were determined using Pearson's correlation analysis. RESULTS: Brain Aß accumulation in APP-KI mice was detectable at 2 months, increased significantly at 5 months, and remained elevated at 12 months of age. The levels of differentially expressed proteins in isolated brain capillaries were higher in younger mice, whereas those in the brain were higher in older mice. Network analyses indicated changes in basement membrane-associated and ribosomal proteins in the brain capillaries. There were no significant changes in key proteins involved in drug or Aß transport at the BBB. In contrast, solute carrier transporter levels in astrocytes, microglia, and neurons were altered in the brain of older mice. Moreover, the levels of the lipid transporters Apoe and Apoj were upregulated in both the brain and isolated brain capillaries after Aß accumulation. CONCLUSIONS: Our results suggest that changes in the brain occurred after advanced Aß accumulation, whereas initial Aß accumulation was sufficient to cause alterations in the BBB. These findings may help elucidate the role of BBB alterations in AD progression and predict the distribution of drugs across the BBB in the brain of patients with AD.
Assuntos
Doença de Alzheimer , Barreira Hematoencefálica , Animais , Camundongos , Doença de Alzheimer/genética , Proteômica , Encéfalo , Proteínas de Membrana Transportadoras , Modelos Animais de DoençasRESUMO
The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. In this study, we identify a functional nuclear localization signal in and interaction partners of the FLOS domain. Our screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells.
Assuntos
Leucemia , Mitose , Humanos , Mitose/genética , Ciclinas/genética , Ciclinas/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Leucemia/genética , Proteínas de Ligação a Poli-ADP-Ribose/genéticaRESUMO
Small extracellular vesicles (sEVs) are small, cell-derived particles with sizes of approximately 100 nm. Since these particles include cargos such as host cell-derived proteins, messenger RNAs, and micro RNAs, they serve as mediators of cell-cell communication. While the analysis of the pharmacokinetic of sEVs after the intravenous injection have been reported, the lymphatic transport of sEVs remains unclear. The objective of this study was to provide insights into the intra-lymphatic trafficking and distribution of sEVs when they are injected into an interstitial space both in normal skin tissue and in cancerous tissue. When sEVs were Subcutaneously administered into the tail base and the tumor tissue, they preferably accumulated in the lymph nodes (LNs), rather than in the liver and the spleen. The findings reported herein show that the lymphatic transport of sEVs was drastically changed in model mice, in which a surgical treatment was used to modify to allow the dominant lymphatic flow from the footpad directly to the axillary LN via the inguinal LN. Based on the results, we conclude that when sEVs are injected into the subcutis space, they are preferably delivered to the LN via the lymphatic system. Further, the extent of accumulation of sEVs in the LN after subcutaneous injection was reduced when they were preliminarily incubated with Proteinase K. These results suggest that the lymphatic drainage of sEVs in normal skin tissue is regulated by membrane proteins on their surface. This reduction, however, was not observed in the case of cancer tissue. This discrepancy can be attributed to the presence of highly permeable lymphatic vessels in the tumor tissue. Further, the major cell subtypes that captured sEVs in the LN were LN-resident medullary sinus macrophages. These collective findings indicate that the lymphatic drainage of sEVs are mediated by proteins and, that they may appear to contribute to the control of the function of immune-responsive cells in the LNs.
Assuntos
Vesículas Extracelulares , Vasos Linfáticos , Camundongos , Animais , Linfonodos/metabolismo , Vasos Linfáticos/metabolismo , Pele , Injeções SubcutâneasRESUMO
BACKGROUND: The functions and protein expressions of the blood-brain barrier are changed throughout brain development following birth. This study aimed to develop a method to isolate brain capillaries from a single frozen neonatal mouse brain and elucidate the enrichment of brain capillaries by quantitative proteomic analysis. We further compared the expression profile of proteins between neonatal and adult brain capillary fractions. METHODS: The brain capillary fraction was prepared by the optimized method from a single frozen mouse neonatal brain on postnatal day 7. The brain capillary fractions and brain lysates were digested by trypsin and analyzed by liquid chromatography-mass spectrometry for quantitative proteomics. RESULTS: By optimizing the isolation method, we observed brain capillaries in the fraction prepared from a single neonatal mouse brain (nBC fraction). A protein amount of 31.5 µg, which is enough for proteomic analysis, was recovered from the nBC fraction. By proteomics analysis, the brain capillary selective proteins, including Abcb1a/Mdr1, Slc2a1/Glut1, Claudin-5, and Pecam-1, were found to be concentrated > 13.4-fold more in nBC fractions than in whole brain lysates. The marker proteins for neurons and astrocytes were not concentrated in nBC fractions, while those of pericytes and microglia were concentrated. Compared to adult mouse brain capillary fractions (aBC fractions), the expressions of Abcb1a/Mdr1a, Abcc4/Mrp4, and Slc2a1/Glut1 were significantly lower in nBC fractions than in aBC fractions, whereas those of Slc1a4/Asct1, Slc1a5/Asct2, Slc7a1/Cat1, and Slc16a1/Mct1 were significantly higher. Amino acid transporters, Slc38a5/Snat5, showed the greatest nBC-to-aBC ratio among transporters (9.83-fold). Network analysis of proteins expressed differentially between nBC and aBC fractions revealed that the proteins with terms related to the extracellular matrix were enriched. CONCLUSIONS: We succeeded in isolating brain capillaries from a single frozen brain of a neonatal mouse at postnatal day 7. Proteomic analysis revealed the differential expression in brain capillaries between neonatal and adult mice. Specifically, amino acid transporters, including Slc1a5/Asct2 and Slc38a5/Snat5, were found to be induced in neonatal brain capillaries. The present isolation method will promote the study of the function and expression of the neonatal blood-brain barrier.
Assuntos
Capilares , Proteômica , Camundongos , Animais , Animais Recém-Nascidos , Transportador de Glucose Tipo 1/metabolismo , Capilares/metabolismo , Proteômica/métodos , Encéfalo/metabolismo , Barreira Hematoencefálica/metabolismoRESUMO
Antibiotic administration affects pharmacokinetics through changes in the intestinal microbiota, and bile acids are involved in this regulation. The purpose of the present study was to clarify the effect of different periods of antibiotic administration on the hepatic bile acid profile and expression of pharmacokinetic-related proteins in mouse liver, kidney, and brain capillaries. Vancomycin and polymyxin B were orally administered to mice for either 5- or 25-days. The hepatic bile acid profile of the 25-day treatment group was distinct. In the liver, the protein expression of cytochrome P450 (Cyp)3a11 showed the greatest reduction to 11.4% after the 5-day treatment and further reduced to 7.01% after the 25-day treatment. Similar reductions were observed for sulfotransferase 1d1, Cyp2b10, carboxylesterase 2e, UDP-glucuronosyltransferase (Ugt)1a5, and Ugt1a9. In the kidney and brain capillaries, no drug-metabolizing enzymes or drug transporters were changed with >1.5-fold or <0.66-fold statistical significance in either period. These results suggest that bile acids and metabolizing enzymes in the liver are affected in a period-dependent manner by antibiotic treatment, while the blood-brain barrier and kidneys are less affected. Drug-drug interactions of antibiotics via the intestinal microbiota should be considered by changing drug metabolism in the liver.
Assuntos
Ácidos e Sais Biliares , Capilares , Camundongos , Animais , Ácidos e Sais Biliares/farmacologia , Capilares/metabolismo , Fígado/metabolismo , Glucuronosiltransferase/metabolismo , Encéfalo/metabolismo , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Rim/metabolismoRESUMO
OBJECTIVE: An extracellular matrix such as collagen is an essential component of the tumor microenvironment. Collagen alpha-2(I) chain (COL1A2) is a chain of type I collagen whose triple helix comprises two alpha-1 chains and one alpha-2 chain. The authors' proteomics data showed that COL1A2 is significantly higher in the blood of patients with glioblastoma (GBM) compared with healthy controls. COL1A2 has many different functions in various types of cancers. However, the functions of COL1A2 in GBM are poorly understood. In this study, the authors analyzed the functions of COL1A2 and its signaling pathways in GBM. METHODS: Surgical specimens and GBM cell lines (T98, U87, and U251) were used. The expression level of COL1A2 was examined using GBM tissues and normal brain tissues by quantitative real-time polymerase chain reaction. The clinical significance of these levels was evaluated using Kaplan-Meier analysis. Small interfering RNA (siRNA) and small hairpin RNA of COL1A2 were transfected into GBM cell lines to investigate the function of COL1A2 in vitro and in vivo. Flow cytometry was introduced to analyze the alteration of cell cycles. Western blot and immunohistochemistry were performed to analyze the underlying mechanisms. RESULTS: The expression level of COL1A2 was upregulated in GBM compared with normal brain tissues. A higher expression of COL1A2 was correlated with poor progression-free survival and overall survival. COL1A2 inhibition significantly suppressed cell proliferation in vitro and in vivo, likely due to G1 arrest. The invasion ability was notably deteriorated by inhibiting COL1A2. Cyclin D1, cyclin-dependent kinase 1, and cyclin-dependent kinase 4, which are involved in the cell cycle, were all downregulated after blockade of COL1A2 in vitro and in vivo. Phosphoinositide 3-kinase inhibitor reduced the expression of COL1A2. Although downregulation of COL1A2 decreased the protein kinase B (Akt) phosphorylation, Akt activator can phosphorylate Akt in siRNA-treated cells. This finding suggests that Akt phosphorylation is partially dependent on COL1A2. CONCLUSIONS: COL1A2 plays an important role in driving GBM progression. COL1A2 inhibition attenuated GBM proliferation by promoting cell cycle arrest, indicating that COL1A2 could be a promising therapeutic target for GBM treatment.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Colágeno Tipo I , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , RNA Interferente Pequeno/uso terapêutico , Microambiente TumoralRESUMO
Histone methyltransferase SETD1A is critical for acute myeloid leukemia (AML) cell survival, but the molecular mechanism driving SETD1A gene regulation remains elusive. To delineate the role of SETD1A, we utilize a protein degrader technology to induce rapid SETD1A degradation in AML cell lines. SETD1A degradation results in immediate downregulation of transcripts associated with DNA repair and heme biosynthesis pathways. CRISPR-based functional analyses and metabolomics reveal an essential role of SETD1A to maintain mitochondrial respiration in AML cells. These SETD1A targets are enriched in head-to-head (H2H) genes. SETD1A degradation disrupts a non-enzymatic SETD1A domain-dependent cyclin K function, increases the Ser5P RNA polymerase II (RNAPII) at the transcriptional start site (TSS), and induces the promoter-proximal pausing of RNAPII in a strand-specific manner. This study reveals a non-enzymatic role for SETD1A in transcriptional pause release and provides insight into the mechanism of RNAPII pausing and its function in cancer.
Assuntos
Leucemia , Humanos , Metabolômica , Regulação para Baixo , Reparo do DNA , RNA Polimerase II , Heme , Histona-Lisina N-Metiltransferase/genéticaRESUMO
PURPOSE: Quantitative targeted absolute proteomics (QTAP) quantifies proteins by measuring the signature peptides produced from target proteins by trypsin digestion. The selection of signature peptides is critical for reliable peptide quantification. The purpose of this study was to comprehensively assess the digestion efficiency and stability of tryptic peptides and to identify optimal signature peptides for human hepatic transporters and membrane marker proteins. METHODS: The plasma membrane fraction of the human liver was digested at different time points and the peptides were comprehensively quantified using quantitative proteomics. Transporters and membrane markers were quantified using the signature peptides by QTAP. RESULTS: Tryptic peptides were classified into clusters with low digestion efficiency, low stability, and high digestion efficiency and stability. Using the cluster information, we found that a proline residue next to the digestion site or the peptide position in or close to the transmembrane domains lowers digestion efficiency. A peptide containing cysteine at the N-terminus or arginine-glycine lowers peptide stability. Based on this information and the time course of peptide quantification, optimal signature peptides were identified for human hepatic transporters and membrane markers. The quantification of transporters with multiple signature peptides yielded consistent absolute values with less than 30% of coefficient variants in human liver microsomes and homogenates. CONCLUSIONS: The signature peptides selected in the present study enabled the reliable quantification of human hepatic transporters. The QTAP protocol using these optimal signature peptides provides quantitative data on hepatic transporters usable for integrated pharmacokinetic studies.
Assuntos
Peptídeos , Proteômica , Humanos , Proteômica/métodos , Proteínas de Membrana Transportadoras/metabolismo , Fígado/metabolismo , Proteínas de Membrana/metabolismo , Digestão , Tripsina/químicaRESUMO
P-glycoprotein, the encoded product of the MDR1 / ABCB1 gene in humans, is expressed in numerous tissues including brain capillary endothelial cells and restricts the distribution of xenobiotics into the brain as an efflux pump. Although a large number of single nucleotide polymorphisms in the MDR1 gene have been identified, the influence of the nonsynonymous 2677G>T/A single nucleotide polymorphism on P-glycoprotein at the blood-brain barrier has remained unclear. In the present study, we developed a novel P-glycoprotein humanized mouse line carrying the 2677G>T mutation by utilizing a mouse artificial chromosome vector constructed by genetic engineering technology and we evaluated the influence of 2677G>T on the expression and function of P-glycoprotein at the blood-brain barrier in vivo . The results of this study showed that the introduction of the 2677G>T mutation does not alter the expression levels of P-glycoprotein protein in the brain capillary fraction. On the other hand, the brain penetration of verapamil, a representative substrate of P-glycoprotein, was increased by the introduction of the 2677G>T mutation. These results suggested that the 2677G>T single nucleotide polymorphism may attenuate the function of P-glycoprotein, resulting in increased brain penetration of P-glycoprotein substrates, without altering the expression levels of P-glycoprotein protein in the blood-brain barrier. This mutant mouse line is a useful model for elucidating the influence of an MDR1 gene single nucleotide polymorphism on the expression and function of P-glycoprotein at the blood-brain barrier.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP , Barreira Hematoencefálica , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Humanos , Camundongos , Mutação , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Uncontrolled proliferation of intestinal epithelial cells caused by mutations in genes of the WNT/ß-catenin pathway is associated with development of intestinal cancers. We previously reported that intestinal stromal cell-derived angiopoietin-like protein 2 (ANGPTL2) controls epithelial regeneration and intestinal immune responses. However, the role of tumor cell-derived ANGPTL2 in intestinal tumorigenesis remained unclear. Here, we show that tumor cell-derived ANGPTL2 promotes ß-catenin-driven intestinal tumorigenesis. ANGPTL2 deficiency suppressed intestinal tumor development in an experimental mouse model of sporadic colon cancer. We also found that increased ANGPTL2 expression in colorectal cancer (CRC) cells augments ß-catenin pathway signaling and promotes tumor cell proliferation. Relevant to mechanism, our findings suggest that tumor cell-derived ANGPTL2 upregulates expression of OB-cadherin, which then interacts with ß-catenin, blocking destruction complex-independent proteasomal degradation of ß-catenin proteins. Moreover, our observations support a model whereby ANGPTL2-induced OB-cadherin expression in CRC cells is accompanied by decreased cell surface integrin α5ß1 expression. These findings overall provide novel insight into mechanisms of ß-catenin-driven intestinal tumorigenesis.
Assuntos
Neoplasias Colorretais , Neoplasias Intestinais , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Intestinais/genética , Camundongos , Via de Sinalização Wnt/genética , beta Catenina/metabolismoRESUMO
Recent advances in single-cell proteomics highlight the promise of sensitive analyses in limited cell populations. However, technical challenges remain for sample recovery, throughput, and versatility. Here, we first report a water droplet-in-oil digestion (WinO) method based on carboxyl-coated beads and phase transfer surfactants for proteomic analysis using limited sample amounts. This method was developed to minimize the contact area between the sample solution and the container to reduce the loss of proteins and peptides by adsorption. This method increased protein and peptide recovery 10-fold. The proteome profiles obtained from 100 cells using the WinO method highly correlated with those from 10,000 cells using the in-solution digestion method. We successfully applied the WinO method to single-cell proteomics and quantified 462 proteins. Using the WinO method, samples can be easily prepared in a multi-well plate, making it a widely applicable and suitable method for single-cell proteomics.
Assuntos
Proteoma , Proteômica , Digestão , Peptídeos/análise , Proteoma/análise , Proteômica/métodos , ÁguaRESUMO
Circadian rhythms influence the transport function of the blood-brain barrier (BBB) and peripheral organs. However, the influence of circadian rhythms on protein expression in the BBB remains to be completely elucidated. Therefore, we aimed to investigate diurnal changes in protein expression in the mouse BBB using quantitative proteomics. Quantitative proteomics showed that the expression of 67, 10, and 20 proteins in the isolated mouse brain capillary fraction changed significantly at zeitgeber time (ZT) 6, 12, and 18, respectively, compared to ZT0. Among them, the levels of 44 proteins were significantly increased at ZT6 and then returned to the same level as ZT0 at ZT12 and ZT18. Gene ontology analysis indicated that the proteins significantly increased at ZT6 were majorly related to translation. The brain capillary endothelial cell-selective proteins sepiapterin reductase and vascular endothelial growth factor receptor 2 showed diurnal variation. In contrast, the expression of ABC transporters, SLC transporters, and receptors associated with receptor-mediated transcytosis, and tight junction proteins did not change within a day. The present findings demonstrated that protein expression related to transport function and physical barrier at the BBB was maintained throughout the day, although the proteins involved in some biological processes exhibited diurnal variation at the BBB.
Assuntos
Barreira Hematoencefálica , Ritmo Circadiano , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Camundongos , Proteômica , Receptor 2 de Fatores de Crescimento do Endotélio VascularRESUMO
Blood-brain barrier (BBB)-permeable middle- or macromolecules (middle/macromolecules) have recently attracted significant attention as new drug delivery carriers into the human brain via receptor-mediated transcytosis (RMT). During the development process of such carriers, it is necessary to thoroughly evaluate their human BBB permeability levels. In such evaluations, our recently established human immortalized cell-based multicellular spheroidal BBB models (hiMCS-BBB models) have shown high potential. However, the specifics of those capabilities have yet to be elucidated. Therefore, in this study, we characterize the ability of the hiMCS-BBB models to evaluate RMT-mediated BBB penetration properties of middle/macromolecules. More specifically, we began by validating transferrin receptor (TfR)-mediated RMT functionalities using transferrin in the hiMCS-BBB models and then examined the BBB permeability levels of MEM189 antibodies (known BBB-permeable anti-TfR antibodies). The obtained results showed that, as with the case of transferrin, temperature-dependent uptake of MEM189 antibodies was observed in the hiMCS-BBB models, and the extent of that uptake increased in a time-dependent manner until reaching a plateau after around 2 h. To further expand the evaluation applicability of the models, we also examined the BBB permeability levels of the recently developed SLS cyclic peptide and observed that peptide uptake was also temperature-dependent. To summarize, our results show that the hiMCS-BBB models possess the ability to evaluate the RMT-mediated BBB-permeable properties of antibodies and peptides and thus have the potential to provide valuable tools for use in the exploration and identification of middle/macromolecules showing excellent BBB permeability levels, thereby contributing powerfully to the development of new drug delivery carriers for transporting drugs into the human brain.
Assuntos
Barreira Hematoencefálica , Receptores da Transferrina , Anticorpos/química , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Humanos , Receptores da Transferrina/metabolismo , Transcitose , Transferrina/metabolismoRESUMO
Targeted proteomics is a method that measures the amount of target proteins via liquid chromatography-tandem mass spectrometry and is used to verify and validate the candidate cancer biomarker proteins. Compared with antibody-based quantification methods such as ELISA, targeted proteomics enables rapid method development, simultaneous measurement of multiple proteins, and high-specificity detection of modifications. Moreover, by spiking the internal standard peptide, targeted proteomics detects the absolute amounts of marker proteins, which is essential for determining the cut-off values for diagnosis and thus for multi-institutional validation. With these unique features, targeted proteomics can seamlessly transfer cancer biomarker candidate proteins from the discovery phase to the verification and validation phases, thereby resulting in an accelerated cancer biomarker pipeline. Furthermore, understanding the basic principles, advantages, and disadvantages is necessary to effectively utilize targeted proteomics in cancer biomarker pipelines. This review aimed to introduce the technical principles of targeted proteomics for cancer biomarker verification and validation.
Assuntos
Neoplasias , Proteômica , Biomarcadores Tumorais , Humanos , Espectrometria de Massas/métodos , Neoplasias/diagnóstico , Proteínas/análise , Proteômica/métodosRESUMO
Podocalyxin (PODXL) is a highly sialylated transmembrane protein that is expressed on the luminal membrane of brain microvascular endothelial cells. To clarify the role of PODXL in the blood-brain barrier (BBB), the present study aimed to investigate the effect of PODXL-knockdown on protein expression, especially the expression of ABCB1/MDR1, in human microvascular endothelial cells (hCMEC/D3). By quantitative proteomics, gene ontology enrichment with differentially expressed proteins showed that PODXL-knockdown influenced the immune response and intracellular trafficking. Among transporters, the protein expression of ABCB1/MDR1 and ABCG2/BCRP was significantly elevated by approximately 2-fold in the PODXL-knockdown cells. In the knockdown cells, the efflux activity of ABCB1/MDR1 was significantly increased, while its mRNA expression was not significantly different from that of the control cells. As receptors and tight junction proteins, levels of low-density lipoprotein receptor-related protein 1 and occludin were significantly increased, while those of transferrin receptor and claudin-11 were significantly decreased in the knockdown cells. The present results suggest that PODXL functions as a modulator of BBB function, including transport, tight junctions, and immune responses. Furthermore, PODXL post-transcriptionally regulates the protein expression and efflux activity of ABCB1/MDR1 at the BBB, which may affect drug distribution in the brain.