RESUMO
The stepwise addition of monosaccharides to N-glycans attached to client proteins to generate a repertoire of mature proteins involves a concerted action of many glycosidases and glycosyltransferases. Here, we report that Golgi α-mannosidase II (GMII), a pivotal enzyme catalyzing the first step in the conversion of hybrid- to complex-type N-glycans, is activated by Zn2+ supplied by the early secretory compartment-resident ZNT5-ZNT6 heterodimers (ZNT5-6) and ZNT7 homodimers (ZNT7). Loss of ZNT5-6 and ZNT7 function results in marked accumulation of hybrid-type and complex/hybrid glycans with concomitant reduction of complex- and high-mannose-type glycans. In cells lacking the ZNT5-6 and ZNT7 functions, the GMII activity is substantially decreased. In contrast, the activity of its homolog, lysosomal mannosidase (LAMAN), is not decreased. Moreover, we show that the growth of pancreatic cancer MIA PaCa-2 cells lacking ZNT5-6 and ZNT7 is significantly decreased in a nude mouse xenograft model. Our results indicate the integral roles of ZNT5-6 and ZNT7 in N-glycosylation and highlight their potential as novel target proteins for cancer therapy.
Assuntos
Proteínas de Transporte de Cátions , Complexo de Golgi , Zinco , Humanos , Glicosilação , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/genética , Animais , Zinco/metabolismo , Camundongos , Complexo de Golgi/metabolismo , Manosidases/metabolismo , Manosidases/genética , Polissacarídeos/metabolismo , Linhagem Celular Tumoral , Camundongos Nus , Transportador 8 de ZincoRESUMO
Abemaciclib (ABM), a cyclin-dependent kinase 4/6 inhibitor, shows pharmacological effects in cell cycle arrest. Epithelial-mesenchymal transition is an important cellular event associated with pathophysiological states such as organ fibrosis and cancer progression. In the present study, we evaluated the contribution of factors associated with cell cycle arrest to ABM-induced epithelial-mesenchymal transition. Treatment with 0.6⯵M ABM induced both cell cycle arrest and epithelial-mesenchymal transition-related phenotypic changes. Interestingly, the knockdown of cyclin-dependent kinase 4/6, pharmacological targets of ABM or cyclin D1, which forms complexes with cyclin-dependent kinase 4/6, resulted in cell cycle arrest at the G1-phase and induction of epithelial-mesenchymal transition, indicating that downregulation of cyclin-dependent kinase 4/6-cyclin D1 complexes would mimic ABM. In contrast, knockdown of the Rb protein, which is phosphorylated by cyclin-dependent kinase 4/6, had no effect on the expression level of α-smooth muscle actin, an epithelial-mesenchymal transition marker. Furthermore, ABM-induced epithelial-mesenchymal transition was not affected by Rb knockdown, suggesting that Rb is not involved in the transition process. Our study is the first to suggest that cyclin-dependent kinase 4/6-cyclin D1 complexes, as pharmacological targets of ABM, may contribute to ABM-induced epithelial-mesenchymal transition, followed by clinical disorders such as organ fibrosis and cancer progression. This study suggests that blocking epithelial-mesenchymal transition might be a promising way to prevent negative side effects caused by a medication (ABM) without affecting its ability to treat the disease.
Assuntos
Aminopiridinas , Benzimidazóis , Quinase 4 Dependente de Ciclina , Quinase 6 Dependente de Ciclina , Transição Epitelial-Mesenquimal , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Benzimidazóis/farmacologia , Humanos , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 4 Dependente de Ciclina/genética , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Aminopiridinas/farmacologia , Quinase 6 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/genética , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina D1/genéticaRESUMO
The transcorneal route is the main entry route for drugs to the intraocular parts, after topical administration. The outer surface, the corneal epithelium (CE), forms the rate-limiting barrier for drug permeability. Information about the role and protein expression of drug and amino acid transporter proteins in the CE is sparse and lacking. The aim of our study was to characterize transporter protein expression in rabbit and porcine CE to better understand potential drug and nutrient absorption after topical administration. Proteins, mainly Abc and Slc transporters, were characterized with quantitative targeted absolute proteomics and global untargeted proteomics methods. In the rabbit CE, 24 of 48 proteins were detected in the targeted approach, and 21 of these were quantified. In the porcine CE, 26 of 58 proteins were detected in the targeted approach, and 20 of these were quantified. Among these, 15 proteins were quantified in both animals: 4f2hc (Slc3a2), Aqp0, Asct1 (Slc1a4), Asct2 (Slc1a5), Glut1 (Slc2a1), Hmit (Slc2a13), Insr, Lat1 (Slc7a5), Mct1 (Slc16a1), Mct2 (Slc16a7), Mct4 (Slc16a3), Mrp 4 (Abcc4), Na+/K+-ATPase, Oatp3a1 (Slco3a1), and Snat2 (Slc38a2). Overall, the global proteomics results supported the targeted proteomics results. Organic anion transporting polypeptide Oatp3a1 was detected and quantified for the first time in both rabbit (1.4 ± 0.4 fmol/cm2) and porcine (11.1 ± 5.3 fmol/cm2) CE. High expression levels were observed for L-type amino acid transporter, Lat1, which was quantified with newly selected extracellular domain peptides in rabbit (48.9 ± 11.8 fmol/cm2) and porcine (37.6 ± 11.5 fmol/cm2) CE. The knowledge of transporter protein expression in ocular barriers is a key factor in the successful design of new ocular drugs, pharmacokinetic modeling, understanding ocular diseases, and the translation to human.
Assuntos
Epitélio Corneano , Proteômica , Animais , Coelhos , Suínos , Epitélio Corneano/metabolismo , Proteômica/métodos , Transporte Biológico , Proteínas de Membrana Transportadoras/metabolismo , Administração OftálmicaRESUMO
Organic anion transporting polypeptide (OATP)1A2 and OATP2B1 have potential N-glycosylation sites, but their influence remains unclear. This study aimed to identify the N-glycosylation sites of OATP1A2/2B1 and investigate their impact on the expression and function of OATP1A2/2B1. Human embryonic kidney cells expressing OATP1A2 or OATP2B1 (HEK293-OATP1A2/2B1) were exposed to tunicamycin, an N-glycosylation inhibitor, and a plasma membrane fraction (PMF) Western blot assay and an estrone 3-sulfate (E3S) uptake study were conducted. HEK293-OATP1A2/OATP2B1 cell lines with mutation(s) at potential N-glycosylation sites were established, and the Western blotting and uptake study were repeated. Tunicamycin reduced the PMF levels and E3S uptake of OATP1A2/OATP2B1. The Asn124Gln, Asn135Gln, and Asn492Gln mutations in OATP1A2 and Asn176Gln and Asn538Gln mutations in OATP2B1 reduced the molecular weights of the OATP molecules and their PMF levels. The PMF levels of OATP1A2 Asn124/135Gln, OATP1A2 Asn124/135/492Gln, and OATP2B1 Asn176/538Gln were further reduced. The maximum transport velocities of OATP1A2 Asn124Gln, OATP1A2 Asn135Gln, and OATP2B1 Asn176/538Gln were markedly reduced to 10 %, 4 %, and 10 % of the wild-type level, respectively. In conclusion, the N-glycans at Asn124 and Asn135 of OATP1A2 and those at Asn176 and Asn538 of OATP2B1 are essential for the plasma membrane expression of these molecules and also affect their transport function.
Assuntos
Transportadores de Ânions Orgânicos , Humanos , Transporte Biológico , Estrona/metabolismo , Glicosilação , Células HEK293 , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Tunicamicina/metabolismoRESUMO
Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) triggers a noncanonical form of programmed cell death (PCD) called parthanatos, yet the mechanisms of its induction are not fully understood. We have recently demonstrated that the aggresome-like induced structures (ALIS) composed of the autophagy receptor SQSTM1/p62 and K48-linked polyubiquitinated proteins (p62-based ALIS) mediate parthanatos. In this study, we identified the D1 dopamine receptor agonist YM435 as a unique parthanatos inhibitor that acts as the disaggregating agent for the p62-based ALIS. We found that YM435 structurally reduces aggregability of the ALIS, and then increases its hydrophilicity and liquidity, which prevents parthanatos. Moreover, dopamine and L-DOPA, a dopamine precursor, also prevented parthanatos by reducing the aggregability of the ALIS. Together, these observations suggest that aggregability of the p62-based ALIS determines the sensitivity to parthanatos, and the pharmacological properties of YM435 that reduces the aggregability may be suitable for therapeutic drugs for parthanatos-related diseases such as neurodegenerative diseases.
RESUMO
One of the critical unmet medical needs in schizophrenia is the treatment for cognitive deficits. However, the neural circuit mechanisms of them remain unresolved. Previous studies utilizing animal models of schizophrenia did not consider the fact that patients with schizophrenia generally cannot discontinue antipsychotic medication due to the high risk of relapse. Here, we used multi-dimensional approaches, including histological analysis of the prelimbic cortex (PL), LC-MS/MS-based in vivo dopamine D2 receptor occupancy analysis for antipsychotics, in vivo calcium imaging, and behavioral analyses of mice using chemogenetics to investigate neural mechanisms and potential therapeutic strategies for working memory deficit in a chronic phencyclidine (PCP) mouse model of schizophrenia. Chronic PCP administration led to alterations in excitatory and inhibitory synapses, specifically in dendritic spines of pyramidal neurons, vesicular glutamate transporter 1 (VGLUT1) positive terminals, and parvalbumin (PV) positive GABAergic interneurons located in layer 2-3 of the PL. Continuous administration of olanzapine, which achieved a sustained therapeutic window of dopamine D2 receptor occupancy (60-80%) in the striatum, did not ameliorate these synaptic abnormalities and working memory deficit in the chronic PCP-treated mice. We demonstrated that chemogenetic activation of PV neurons in the PL, as confirmed by in vivo calcium imaging, ameliorated working memory deficit in this model even under clinically comparable olanzapine treatment which by itself inhibited only PCP-induced psychomotor hyperactivity. Our study suggests that targeting prefrontal PV neurons could be a promising therapeutic intervention for cognitive deficits in schizophrenia in combination with antipsychotic medication.
Assuntos
Antipsicóticos , Esquizofrenia , Animais , Humanos , Camundongos , Antipsicóticos/uso terapêutico , Cálcio , Cromatografia Líquida , Modelos Animais de Doenças , Interneurônios/metabolismo , Transtornos da Memória/tratamento farmacológico , Olanzapina/efeitos adversos , Parvalbuminas/metabolismo , Fenciclidina/farmacologia , Córtex Pré-Frontal/metabolismo , Receptores de Dopamina D2 , Esquizofrenia/induzido quimicamente , Esquizofrenia/tratamento farmacológico , Esquizofrenia/patologia , Espectrometria de Massas em TandemRESUMO
Orexins are a family of neuropeptides that regulate various physiological events, such as sleep/wakefulness as well as emotional and feeding behavior, and that act on two G-protein-coupled receptors, i.e., orexin 1 (OX1R) and orexin 2 receptors (OX2R). Since the discovery that dysfunction of the orexin/OX2R system causes the sleep disorder narcolepsy, several OX2R-selective and OX1/2R dual agonists have been disclosed. However, an OX1R-selective agonist has not yet been reported, despite the importance of the biological function of OX1R. Herein, we report the discovery of a potent OX1R-selective agonist, (R,E)-3-(4-methoxy-3-(N-(8-(2-(3-methoxyphenyl)-N-methylacetamido)-5,6,7,8-tetrahydronaphthalen-2-yl)sulfamoyl)phenyl)-N-(pyridin-4-yl)acrylamide [(R)-YNT-3708; EC50 = 7.48 nM for OX1R; OX2R/OX1R EC50 ratio = 22.5]. The OX1R-selective agonist (R)-YNT-3708 exhibited antinociceptive and reinforcing effects through the activation of OX1R in mice.
Assuntos
Neuropeptídeos , Receptores Acoplados a Proteínas G , Camundongos , Animais , Orexinas , Receptores de Orexina/agonistas , SonoRESUMO
Transcriptomic data of cultured cells treated with a chemical are widely recognized as useful numeric information that describes the effects of the chemical. This property is due to the high coverage and low arbitrariness of the transcriptomic data as profiles of chemicals. Considering the importance of posttranslational regulation, proteomic profiles could provide insights into the unrecognized aspects of the effects of chemicals. Therefore, this study aimed to address the question of how well the proteomic profiles obtained using data-independent acquisition (DIA) with the sequential window acquisition of all theoretical mass spectra, which can achieve comprehensive and arbitrariness-free protein quantification, can describe chemical effects. We demonstrated that the proteomic data obtained using DIA-MS exhibited favorable properties as profile data, such as being able to discriminate chemicals like the transcriptomic profiles. Furthermore, we revealed a new mode of action of a natural compound, harmine, through profile data analysis using the proteomic profile data. To our knowledge, this is the first study to investigate the properties of proteomic data obtained using DIA-MS as the profiles of chemicals. Our 54 (samples) × 2831 (proteins) data matrix would be an important source for further analyses to understand the effects of chemicals in a data-driven manner.
RESUMO
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
We previously developed an in vitro model of the human blood-brain barrier (BBB) based on the use of endothelial cells derived from CD34+-hematopoietic stem cells and cultured with brain pericytes. The purpose of the present study was to provide information on the protein expression levels of the transporters, receptors, tight junction/adherence junction molecules, and transporter-associated molecules of human brain-like endothelial cells (hBLECs). The absolute protein expression levels were determined by liquid chromatography-mass spectrometry-based quantitative targeted absolute proteomics and compared with those from human brain microvessels (hBMVs). The protein levels of CD144, CD147, MRP4, Annexin A6 and caveolin-1 showed more than 3-fold abundance in hBLECs, those of MCT1, Connexin 43, TfR1, and claudin-5 showed less than 3-fold differences, and the protein levels of other drug efflux transporters and nutrient transporters were less represented in hBLECs than in hBMVs. It is noteworthy that BCRP was more expressed than MDR1 in hBLECs, as this was the case for hBMVs. These results suggest that transports mediated by MCT1, TfR1, and claudin-5-related tight junction function reflect the in vivo BBB situation. The present study provided a better characterization of hBLECs and clarified the equivalence of the transport characteristics between in vitro BBB models and in vivo BBB models using LC-MS/MS-based protein quantification.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Humanos , Barreira Hematoencefálica/metabolismo , Células Endoteliais/metabolismo , Cromatografia Líquida , Proteômica/métodos , Claudina-5/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Espectrometria de Massas em Tandem/métodos , Proteínas de Neoplasias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Células-Tronco Hematopoéticas/metabolismoRESUMO
The blood-brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material (ssPalm). We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations.
RESUMO
A proton-coupled organic cation (H+/OC) antiporter working at the blood-brain barrier (BBB) in humans and rodents is thought to be a promising candidate for the efficient delivery of cationic drugs to the brain. Therefore, it is important to identify the molecular entity that exhibits this activity. Here, for this purpose, we established the Proteomics-based Identification of transporter by Crosslinking substrate in Keyhole (PICK) method, which combines photo-affinity labeling with comprehensive proteomics analysis using SWATH-MS. Using preselected criteria, the PICK method generated sixteen candidate proteins. From these, knockdown screening in hCMEC/D3 cells, an in vitro BBB model, identified two proteins, TM7SF3 and LHFPL6, as candidates for the H+/OC antiporter. We synthesized a novel H+/OC antiporter substrate for functional analysis of TM7SF3 and LHFPL6 in hCMEC/D3 cells and HEK293 cells. The results suggested that both TM7SF3 and LHFPL6 are components of the H+/OC antiporter.
RESUMO
Structurally diverse small compounds are utilized to obtain hit compounds that have suitable pharmacophores in appropriate three-dimensional conformations for the target drug receptors. We have focused on the 1,3,5-trioxazatriquinane skeleton, which has a rigid bowl-like structure enabling the diverse orientation of side chain units, leading to a novel small-scale focused library based on the skeleton. In the library screening for the orexin receptor, some of the compounds showed orexin receptor antagonistic activity with a high hit rate of 7%. By optimizing the hit compounds, we discovered a potent dual orexin receptor antagonist, 38b, and a selective orexin 1 receptor antagonist, 41b carrying the same plane structure. Both compounds showed reasonable brain permeability and beneficial effects when administered intraperitoneally to wild-type mice. Docking simulations of their eutomers, (-)-38b and (+)-41b, with orexin receptors suggested that the interaction between the 1,3,5-trioxazatriquinane core structure and the hydrophobic subpocket in orexin receptors enables a U-shape structure, which causes tight van der Waals interactions with the receptors similar to SB-334867, a selective orexin 1 receptor antagonist. These results indicate that the library approach utilizing the 1,3,5-trioxazatriquinanes bearing multiple effective residues (TriMERs) might be useful for the hit discovery process targeting not only opioid and orexin receptors but other G-protein coupled receptors.
Assuntos
Antagonistas dos Receptores de Orexina , Animais , Compostos Heterocíclicos de 4 ou mais Anéis , Camundongos , Antagonistas dos Receptores de Orexina/química , Antagonistas dos Receptores de Orexina/farmacologia , Receptores de Orexina , Orexinas , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Cerebral amyloid angiopathy (CAA) occurs in 80% of patients with Alzheimer's disease (AD) and is mainly caused by the abnormal deposition of Aß in the walls of cerebral blood vessels. Cerebrovascular molecular mechanisms in CAA were investigated by using comprehensive and accurate quantitative proteomics. METHODS: Concerning the molecular mechanisms specific to CAA, formalin-fixed paraffin-embedded (FFPE) sections were prepared from patients having AD neuropathologic change (ADNC) with severe cortical Aß vascular deposition (ADNC +/CAA +), and from patients having ADNC without vascular deposition of Aß (ADNC +/CAA -; so called, AD). Cerebral cortical vessels were isolated from FFPE sections using laser microdissection (LMD), processed by pressure cycling technology (PCT), and applied to SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. RESULTS: The protein expression levels of 17 proteins in ADNC +/CAA +/H donors (ADNC +/CAA + donors with highly abundant Aß in capillaries) were significantly different from those in ADNC +/CAA - and ADNC -/CAA - donors. Furthermore, we identified 56 proteins showing more than a 1.5-fold difference in average expression levels between ADNC +/CAA + and ADNC -/CAA - donors, and were significantly correlated with the levels of Aß or Collagen alpha-2(VI) chain (COL6A2) (CAA markers) in 11 donors (6 ADNC +/CAA + and 5 ADNC -/CAA -). Over 70% of the 56 proteins showed ADNC +/CAA + specific changes in protein expression. The comparative analysis with brain parenchyma showed that more than 90% of the 56 proteins were vascular-specific pathological changes. A literature-based pathway analysis showed that 42 proteins are associated with fibrosis, oxidative stress and apoptosis. This included the increased expression of Heat shock protein HSP 90-alpha, CD44 antigen and Carbonic anhydrase 1 which are inhibited by potential drugs against CAA. CONCLUSIONS: The combination of LMD-based isolation of vessels from FFPE sections, PCT-assisted sample processing and SWATH analysis (FFPE-LMD-PCT-SWATH method) revealed for the first time the changes in the expression of many proteins that are involved in fibrosis, ROS production and cell death in ADNC +/CAA + (CAA patients) vessels. The findings reported herein would be useful for developing a better understanding of the pathology of CAA and for promoting the discovery and development of drugs and biomarkers for CAA.
Assuntos
Doença de Alzheimer , Angiopatia Amiloide Cerebral , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Angiopatia Amiloide Cerebral/metabolismo , Fibrose , Humanos , Proteômica , TecnologiaRESUMO
The cerebrovascular-specific molecular mechanism in Alzheimer's disease (AD) was investigated by employing comprehensive and accurate quantitative proteomics. Highly purified brain capillaries were isolated from cerebral gray and white matter of four AD and three control donors, and examined by SWATH (sequential window acquisition of all theoretical fragment ion spectra) proteomics. Of the 29 ribosomal proteins that were quantified, 28 (RPLP0, RPL4, RPL6, RPL7A, RPL8, RPL10A, RPL11, RPL12, RPL14, RPL15, RPL18, RPL23, RPL27, RPL27A, RPL31, RPL35A, RPS2, RPS3, RPS3A, RPS4X, RPS7, RPS8, RPS14, RPS16, RPS20, RPS24, RPS25, and RPSA) were significantly upregulated in AD patients. This upregulation of ribosomal protein expression occurred only in brain capillaries and not in brain parenchyma. The protein expression of protein processing and N-glycosylation-related proteins in the endoplasmic reticulum (DDOST, STT3A, MOGS, GANAB, RPN1, RPN2, SEC61B, UGGT1, LMAN2, and SSR4) were also upregulated in AD brain capillaries and was correlated with the expression of ribosomal proteins. The findings reported herein indicate that the ribosome complex, the subsequent protein processing and N-glycosylation-related processes are significantly and specifically upregulated in the brain capillaries of AD patients.
Assuntos
Doença de Alzheimer , Hexosiltransferases , Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Hexosiltransferases/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Regulação para CimaRESUMO
Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5-ZNT6 heterodimers [ZNT5-6] and ZNT7-ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.
Assuntos
Proteínas de Transporte de Cátions , Proteínas Ligadas por GPI , Zinco , Animais , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Galinhas/metabolismo , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis/genética , Proteínas de Membrana/metabolismo , Peixe-Zebra/metabolismo , Zinco/metabolismoRESUMO
PURPOSE: The purpose of the present study was to quantitatively determine the expression of transporters, receptors and tight junction molecules at the blood-arachnoid barrier (BAB) and blood-spinal cord barrier (BSCB) in cervical, thoracic and lumbar spines from dogs. METHODS: The expression levels of 31 transporters, 3 receptors, 1 tight junction protein, and 3 marker proteins in leptomeninges and capillaries isolated from spines (3 male and 2 female dogs) were determined by quantitative Targeted Absolute Proteomics (qTAP). The units were converted from fmol/µg protein to pmol/cm (absolute abundance at the BAB and the BSCB in a 1 cm section of spine). RESULTS: The expression of MDR1 and BCRP were greater at the BSCB compared to the BAB (especially in the cervical cord), and the expressions at the lumbar BSCB were lower than that for the cervical BSCB. Among the organic anionic and cationic drug transporters, OAT1, OAT3, MRP1, OCT2 and MATE1/2 were detected only in the BAB, and not at the BSCB). The expression of these transporters was higher in the order: lumbar > thoracic > cervical BAB. The expressions of GLUT1, 4F2hc, EAAT1, 2, PEPT2, CTL1, and MCT1 at the BSCB of the cervical cord were higher than the corresponding values for the cervical BAB, and these values decreased in going down the spinal cord. CONCLUSION: These results provide a better understanding of the molecular mechanisms underlying the concentration gradients of drugs and endogenous substances in the cerebrospinal fluid and parenchyma of the spinal cord.
Assuntos
Barreira Hematoencefálica , Junções Íntimas , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Aracnoide-Máter/metabolismo , Barreira Hematoencefálica/metabolismo , Cães , Feminino , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Neoplasias/metabolismo , Medula Espinal/metabolismo , Junções Íntimas/metabolismoRESUMO
One of the major reasons why central nervous system (CNS)-drug development has been challenging in the past, is the barriers that prevent substances entering from the blood circulation into the brain. These barriers include the blood-brain barrier (BBB), blood-spinal cord barrier (BSCB), blood-cerebrospinal fluid barrier (BCSFB), and blood-arachnoid barrier (BAB), and they differ from each other in their transporter protein expression and function as well as among the species. The quantitative expression profiles of the transporters in the CNS-barriers have been recently revealed, and in this review, it is described how they affect the pharmacokinetics of compounds and how these expression differences can be taken into account in the prediction of brain drug disposition in humans, an approach called pharmacoproteomics. In recent years, also structural biology and computational resources have progressed remarkably, enabling a detailed understanding of the dynamic processes of transporters. Molecular dynamics simulations (MDS) are currently used commonly to reveal the conformational changes of the transporters and to find the interactions between the substrates and the protein during the binding, translocation in the transporter cavity, and release of the substrate on the other side of the membrane. The computational advancements have also aided in the rational design of transporter-utilizing compounds, including prodrugs that can be actively transported without losing potency towards the pharmacological target. In this review, the state-of-art of these approaches will be also discussed to give insights into the transporter-mediated drug delivery to the CNS.
Assuntos
Barreira Hematoencefálica , Encéfalo , Sistemas de Liberação de Medicamentos , Proteínas de Membrana Transportadoras , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Proteômica , Medula Espinal/metabolismoRESUMO
The purpose of this study was to develop a method to comprehensively determine the localization of apical and basolateral membrane proteins, using a combination of apical/basolateral membrane separation and accurate SWATH (Sequential Window Acquisition of all THeoretical fragment ion spectra) proteomics. The SWATH analysis of basolateral and apical plasma membrane fractions in mouse liver quantified the protein expression of 1373 proteins. The basolateral/apical ratios of the protein expression levels were compared with the reported immunohistochemical localization for 41 model proteins (23 basolateral, 11 apical and 7 both membrane-localized proteins). Three groups were perfectly distinguished. Border lines to distinguish the apical-, both- and basolateral localizations were determined to be 0.766 and 1.42 based on probability density. The method that was established was then applied to the comprehensive determination of the proteins in mouse liver. The findings indicated that 154 and 125 proteins were localized in the apical and basolateral membranes, respectively. The levels of receptors, CD antigens and integrins, enzymes and Ras-related molecules were much higher in apical membranes than in basolateral membranes. In contrast, the levels of adhesion molecules, scaffold proteins and transporters in basolateral membranes were much higher than in apical membranes.
RESUMO
The purpose of this study was to elucidate the absolute abundance of transporters, enzymes, receptors, and tight junction and marker proteins at human blood-arachnoid barrier (BAB) and compare with those of dogs and pigs. Protein expression levels in plasma membrane fractions of brain leptomeninges were determined by quantitative targeted absolute proteomics. To realistically compare the absolute abundance of target molecules at the BAB among humans, dogs, and pigs, the unit was converted from fmol/µg-protein to pmol/cm2 -leptomeninges. Of a total of 70 proteins, 52 were detected. OAT1, OAT3, GLUT1, 4F2hc, EAAT1, EAAT2, MCT8, SMVT, CTL2, GFAP, Claudin-5, Na+ /K+ -ATPase, COMT, GSTP1, and CES1 were abundantly expressed at the human BAB (>1 pmol/cm2 ). The protein expression levels were within a 3-fold difference for 16 out of 33 proteins between humans and dogs and for 13 out of 28 proteins between humans and pigs. Both human-dog and human-pig differences in protein expression levels were within 3-fold for OAT1, OAT3, 4F2hc, xCT, OCT2, MDR1, BCRP, PEPT2, SYP, and MCT1. In contrast, OCT3, MCT4, and OATP1A2 were detected in humans but not in dogs or pigs. MRP3 was detected in dogs and pigs but not in humans. The absolute level of GLUT1 in humans was nearly the same as that in dogs but was 6.14-fold greater in pigs. No significant differences in the levels were observed between male and female dogs for nearly all molecules. These results should be helpful in understanding the physiological roles of BAB and cerebrospinal fluid pharmacokinetics in humans and their differences from dogs and pigs.