RESUMEN
Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.
Asunto(s)
Polaridad Celular , Proteínas del Citoesqueleto/metabolismo , Gonorrea/microbiología , Neisseria gonorrhoeae/genética , Actinas/metabolismo , Cuello del Útero/microbiología , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Epitelio/microbiología , Femenino , Humanos , Microvellosidades/ultraestructura , Membrana Mucosa/microbiología , Neisseria gonorrhoeae/fisiología , FosforilaciónRESUMEN
Cytophaga hutchinsonii is a Gram-negative bacterium belonging to the phylum Bacteroidetes. It digests crystalline cellulose with an unknown mechanism and possesses a type IX secretion system (T9SS) that can recognize the C-terminal domain (CTD) of the cargo protein as a signal. In this study, the functions of the CTD in the secretion and localization of T9SS substrates in C. hutchinsonii were studied by fusing the green fluorescent protein (GFP) with the CTD from CHU_2708. The CTD is necessary for the secretion of GFP by C. hutchinsonii T9SS. The GFP-CTDCHU_2708 fusion protein was found to be glycosylated in the periplasm, with a molecular mass about 5 kDa higher than that predicted from its sequence. The glycosylated protein was sensitive to peptide-N-glycosidase F, which can hydrolyze N-linked oligosaccharides. Analyses of mutants obtained by site-directed mutagenesis of asparagine residues in the N-X-S/T motif of CTDCHU_2708 suggested that N-glycosylation occurred on the CTD. CTD N-glycosylation is important for the secretion and localization of GFP-CTD recombinant proteins in C. hutchinsonii. Glycosyltransferase-encoding gene chu_3842, a homologous gene of Campylobacter jejuni pglA, was found to participate in the N-glycosylation of C. hutchinsonii. Deletion of chu_3842 affected cell motility, cellulose degradation, and cell resistance to some chemicals. Our study provided evidence that the CTD as the signal of T9SS was N-glycosylated in the periplasm of C. hutchinsonii. IMPORTANCE The bacterial N-glycosylation system has previously been found only in several species of Proteobacteria and Campylobacterota, and the role of N-linked glycans in bacteria is still not fully understood. C. hutchinsonii has a unique cell contact cellulose degradation mechanism, and many cell surface proteins, including cellulases, are secreted by the T9SS. In this study, we found that C. hutchinsonii, a member of the phylum Bacteroidetes, has an N-glycosylation system. Glycosyltransferase CHU_3842 was found to participate in the N-glycosylation of C. hutchinsonii proteins and had effects on cell resistance to some chemicals, cell motility, and cellulose degradation. Moreover, N-glycosylation occurs on the CTD translocation signal of T9SS. The glycosylation of the CTD appears to play an important role in affecting T9SS substrate transportation and localization. This study enriched our understanding of the widespread existence and multiple biological roles of N-glycosylation in bacteria.
Asunto(s)
Proteínas Bacterianas , Sistemas de Secreción Bacterianos , Cytophaga , Proteína C , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cytophaga/genética , Cytophaga/metabolismo , GlicosilaciónRESUMEN
Cytophaga hutchinsonii is an abundant soil cellulolytic bacterium that uses a unique cellulose degradation mechanism different from those that involve free cellulases or cellulosomes. Though several proteins have been identified as important for cellulose degradation, the mechanism used by C. hutchinsonii to digest crystalline cellulose remains a mystery. In this study, chu_0922 was identified by insertional mutation and gene deletion as an important gene locus indispensable for crystalline cellulose utilization. Deletion of chu_0922 resulted in defects in crystalline cellulose utilization. The Δ0922 mutant completely lost the ability to grow on crystalline cellulose, even with extended incubation, and selectively utilized the amorphous region of cellulose, leading to increased crystallinity. As a protein secreted by the type IX secretion system (T9SS), CHU_0922 was found to be located on the outer membrane, and the outer membrane localization of CHU_0922 relied on the T9SS. Comparative analysis of the outer membrane proteins revealed that the abundance of several cellulose-binding proteins, including CHU_1276, CHU_1277, and CHU_1279, was reduced in the Δ0922 mutant. Further study showed that CHU_0922 is crucial for the full expression of the gene cluster containing chu_1276, chu_1277, chu_1278, chu_1279, and chu_1280 (cel9C), which is essential for cellulose utilization. Moreover, CHU_0922 is required for the cell surface localization of CHU_3220, a cellulose-binding protein that is essential for crystalline cellulose utilization. Our study provides insights into the complex system that C. hutchinsonii uses to degrade crystalline cellulose. IMPORTANCE The widespread aerobic cellulolytic bacterium Cytophaga hutchinsonii, belonging to the phylum Bacteroidetes, utilizes a novel mechanism to degrade crystalline cellulose. No genes encoding proteins specialized in loosening or disruption the crystalline structure of cellulose were identified in the genome of C. hutchinsonii, except for chu_3220 and chu_1557. The crystalline cellulose degradation mechanism remains enigmatic. This study identified a new gene locus, chu_0922, encoding a typical T9SS substrate that is essential for crystalline cellulose degradation. Notably, CHU_0922 is crucial for the normal transcription of chu_1276, chu_1277, chu_1278, chu_1279, and chu_1280 (cel9C), which play important roles in the degradation of cellulose. Moreover, CHU_0922 participates in the cell surface localization of CHU_3220. These results demonstrated that CHU_0922 plays a key role in the crystalline cellulose degradation network. Our study will promote the uncovering of the novel cellulose utilization mechanism of C. hutchinsonii.
Asunto(s)
Proteínas Portadoras , Celulosa , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Celulosa/metabolismo , Cytophaga/genética , Cytophaga/metabolismoRESUMEN
OBJECTIVES: Adipokine resistin is highly expressed in the serum and synovial uid (SF) of patients with knee osteoarthritis (KOA) but its pathogenic role in KOA remains unclear. We aimed to explore the mechanism of resistin/CAP1 in human KOA chondrocytes. METHODS: We enrolled 103 patients with radiographic KOA and 86 healthy participants as controls. Resistin levels in serum and SF were determined by enzyme-linked immunosorbent assay (ELISA). CAP1 expression was measured in cartilage tissues using immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and western blot. Effects of resistin on chondrocytes and CAP1 were evaluated via qRT-PCR and co-immunoprecipitation. The roles of CAP1, p38-MAPK, and NF-κB signalling pathways in KOA development were evaluated using adenovirus-mediated CAP1 short hairpin RNA, qRT-PCR, western blot, and ELISA. RESULTS: Resistin expression in serum and SF was elevated in severe radiographic KOA. CAP1 levels were higher in KOA cartilage and were positively correlated with resistin expression. Resistin promoted CCL3, CCL4, MMP13, and ADAMTS-4 expression through the CAP1 receptor. Resistin also directly bound to CAP1, as confirmed by co-immunoprecipitation. CAP1 knockdown in chondrocytes attenuated resistin-induced expression of CCL3, CCL4, MMP13, and ADAMTS-4 and activated p38-MAPK and NF-κB signalling pathways. CONCLUSIONS: Resistin binds CAP1 and upregulates the expression of proinflammatory cytokines and matrix-degrading enzymes via p38-MAPK and NF-κB signalling in human chondrocytes.
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Condrocitos , FN-kappa B , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/farmacología , Células Cultivadas , Quimiocinas , Condrocitos/metabolismo , Proteínas del Citoesqueleto/metabolismo , Humanos , Metaloproteinasas de la Matriz/metabolismo , Metaloproteinasas de la Matriz/farmacología , FN-kappa B/metabolismo , Oligopéptidos , Resistina , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Cytophaga hutchinsonii is an important Gram-negative bacterium belonging to the Bacteroides phylum that can efficiently degrade cellulose. But the promoter that mediates the initiation of gene transcription has been unknown for a long time. In this study, we determined the transcription start site (TSS) of C. hutchinsonii by 5' rapid amplification of cDNA ends (5'RACE). The promoter structure was first identified as TAAT and TATTG which are located -5 and -31 bp upstream of TSS, respectively. The function of -5 and -31 regions and the spacer length of the promoter Pchu_1284 were explored by site directed ligase-independent mutagenesis (SLIM). The results showed that the promoter activities were sharply decreased when the TTG motif was mutated into guanine (G) or cytosine (C). Interestingly, we found that the strong promoter was accompanied with many TTTG motifs which could enhance the promoter activities within certain copies. These characteristics were different from other promoters of Bacteriodes species. Furthermore, we carried out genome scanning analysis for C. hutchinsonii and another Bacteroides species by Perl6.0. The results indicated that the promoter structure of C. hutchinsonii possessed more unique features than other species. Also, the screened inducible promoter Pchu_2268 was used to overexpress protein CHU_2196 with a molecular weight of 120 kDa in C. hutchinsonii. The present study enriched the promoter structure of Bacteroidetes species and also provided a novel method for the highly expressed large protein (cellulase) in vivo, which was helpful to elucidate the unique cellulose degradation mechanism of C. hutchinsonii.Key points⢠The conserved structure of strong promoter of C. hutchinsonii was elucidated.⢠Two novel regulation motifs of TTTG and AATTATG in the promoter were discovered.⢠A new method for induced expression of cellulase in vivo was established.⢠Helpful for explained the unique cellulose degradation mechanism of C. hutchinsonii.
Asunto(s)
Celulasa , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Celulasa/metabolismo , Celulosa/metabolismo , Cytophaga/genética , Cytophaga/metabolismoRESUMEN
The alpha-ketoglutarate-dependent (ALKB) homolog 5 (ALKBH5), an m6 A demethylase, has been reported to be involved in the pathogenesis of preeclampsia (PE), but the exact mechanism requires further investigation. RT-qPCR or Western blotting were used to determine ALKBH5 and peroxisome proliferator-activated receptor gamma (PPARG) expression in placentas from PE patients and normal volunteers, as well as in HTR-8/SVneo cells treated with hypoxia/reoxygenation (H/R). Our results showed that the expression of ALKBH5 was significantly upregulated and PPARG was downregulated in preeclamptic placentas and H/R-treated cells. ALKBH5 interference reduced m6 A levels of PPARG mRNA, and increased PPARG mRNA stability and promoted PPARG translation level. In addition, ALKBH5 silencing increased the cell proliferation, migration, and vimentin protein level, and inhibited cell apoptosis, oxidative stress, and protein levels of endoglin (ENG) and E-cadherin in H/R-treated cells, whereas PPARG interference reversed these effects. Furthermore, PPARG repressed the H3K9me2 levels at activated leukocyte cell adhesion molecule (ALCAM) promoter region by increasing the expression and activity of lysine demethylase 3B (KDM3B). ALCAM inhibition reversed the effects of PPARG overexpression on H/R-treated cell functions. PKF115-584 suppressed the effects of ALKBH5 interference on the behaviors of H/R-treated cells. Finally, inhibition of ALKBH5 alleviates PE-like features in pregnant mice. Inhibition of ALKBH5 promotes KDM3B-mediated ALCAM demethylation by facilitating PPARG mRNA m6 A modification, and further activates the Wnt/ß-catenin pathway, and in turn alleviates PE progression.
Asunto(s)
Desmetilasa de ARN, Homólogo 5 de AlkB , Trofoblastos , Desmetilasa de ARN, Homólogo 5 de AlkB/genética , Desmetilasa de ARN, Homólogo 5 de AlkB/metabolismo , Animales , Apoptosis/genética , Femenino , Humanos , Hipoxia/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/farmacología , Ratones , Estrés Oxidativo/genética , PPAR gamma/genética , PPAR gamma/metabolismo , Placenta/metabolismo , Embarazo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Trofoblastos/metabolismoRESUMEN
Sexually transmitted infections are a critical public health issue. However, the mechanisms underlying sexually transmitted infections in women and the link between the infection mechanism and the wide range of clinical outcomes remain elusive due to a lack of research models mimicking human infection in vivo. We established a human cervical tissue explant model to mimic local Neisseria gonorrhoeae (GC) infections. We found that GC preferentially colonize the ectocervix by activating integrin-ß1, which inhibits epithelial shedding. GC selectively penetrate into the squamocolumnar junction (TZ) and endocervical epithelia by inducing ß-catenin phosphorylation, which leads to E-cadherin junction disassembly. Epithelial cells in various cervical regions differentially express carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), the host receptor for GC opacity-associated proteins (OpaCEA). Relatively high levels were detected on the luminal membrane of ecto/endocervical epithelial cells but very low levels intracellularly in TZ epithelial cells. CEACAM-OpaCEA interaction increased ecto/endocervical colonization and reduced endocervical penetration by increasing integrin-ß1 activation and inhibiting ß-catenin phosphorylation respectively, through CEACAM downstream signaling. Thus, the intrinsic properties of cervical epithelial cells and phase-variation of bacterial surface molecules both play a role in controlling GC infection mechanisms and infectivity, preferential colonization or penetration, potentially leading to asymptomatic or symptomatic infection.
Asunto(s)
Adhesión Bacteriana/fisiología , Cuello del Útero/microbiología , Gonorrea/microbiología , Membrana Mucosa/microbiología , Neisseria gonorrhoeae/patogenicidad , Cuello del Útero/metabolismo , Femenino , Humanos , Membrana Mucosa/metabolismo , Técnicas de Cultivo de ÓrganosRESUMEN
Transmembrane proteins (TMEMs), spanning the entire width of lipid bilayers and anchored to them permanently, exist in diverse cell types to implement a series of essential physiological functions. Recently, TMEM48, a member of the TMEM family, has been demonstrated to be closely associated with tumorigenesis. However, little is known about the specific role of TMEM48 in cervical cancer (CC). This study aimed to investigate the biological functions of TMEM48 in CC. The CCK-8 assay was performed to detect CC cell proliferation. The wound healing and transwell assays were conducted to measure cell migration and invasion, respectively. The levels of TMEM48, ß-catenin, T cell factor 1(TCF1) and axis formation inhibitor 2 (AXIN2) were examined by the western blot analysis. Xenograft models were established for the tumorigenesis assay in vivo. The results showed that TMEM48 was overexpressed in CC tissues and cell lines. Knockdown of TMEM48 significantly inhibited CC cell proliferation, migration and invasion in vitro and suppressed CC cell growth in vivo. In addition, the investigation on the molecular mechanisms indicated that TMEM48 down-regulation remarkably decreased the protein levels of ß-catenin, TCF1 and AXIN2 in CC cells and TMEM48 exerted its promoting effect on CC progression via activation of the Wnt/ß-catenin pathway. Taken together, our study suggested TMEM48 as a promising therapeutic target for CC treatment.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Proteínas de Complejo Poro Nuclear/biosíntesis , Neoplasias del Cuello Uterino/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animales , Proteína Axina/biosíntesis , Proteína Axina/metabolismo , Movimiento Celular , Proliferación Celular , Progresión de la Enfermedad , Femenino , Células HeLa , Factor Nuclear 1-alfa del Hepatocito/biosíntesis , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Humanos , Inmunohistoquímica , Membrana Dobles de Lípidos , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias , Cicatrización de HeridasRESUMEN
INTRODUCTION: Laparoscopic salpingo-oophorectomy and pelvic lymphadenectomy place the ureter at risk. Although traditional anatomic studies indicate that ureters generally cross over the common iliac artery in the pelvic inlet, the view is often different during surgery. Hence, digital three-dimensional models have been utilized to solve this conundrum. Our study aimed to evaluate the anatomic relation between the iliac artery and the ureter in the pelvic inlet. MATERIAL AND METHODS: A medical image processing system processed data collected from 129 female patients, and digital 3D models of the iliac artery and ureter were used to study their anatomic relation. Left- and right-side data were compared using Pearson's χ2 analysis. RESULTS: Two different anatomic relations were found immediately posterior to the ureter: Common iliac arteries were present in 33.2% and external iliac arteries in 76.8%. The mean distances from the point at which the ureter crossed over the iliac artery until the bifurcation of the artery was 8.8 ± 12.2 mm in the left pelvis and 12.4 ± 10.8 mm in the right pelvis - different from that of the traditional anatomic study. CONCLUSION: In the future, these models and digital anatomical data could help avoid surgical complications.
Asunto(s)
Arteria Ilíaca , Uréter , Aorta Abdominal , Femenino , Humanos , Arteria Ilíaca/cirugía , Escisión del Ganglio Linfático , Pelvis , Uréter/diagnóstico por imagen , Uréter/cirugíaRESUMEN
As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2) has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR) signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO) mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk) and phosphorylated SH2-containing inositol phosphatase (pSHIP), are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin) is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.
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Actinas/metabolismo , Linfocitos B/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Citoesqueleto/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Agammaglobulinemia Tirosina Quinasa , Animales , Compuestos Bicíclicos Heterocíclicos con Puentes , Membrana Celular/metabolismo , Inmunidad Humoral , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Polimerizacion , Proteína Asociada al mTOR Insensible a la Rapamicina , TiazolidinasRESUMEN
Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.
Asunto(s)
Cuello del Útero/patología , Gonorrea/microbiología , Uniones Intercelulares/microbiología , Miosina Tipo II/metabolismo , Neisseria gonorrhoeae/patogenicidad , Adhesión Bacteriana , Calcio/metabolismo , Cuello del Útero/microbiología , Células Epiteliales/microbiología , Células Epiteliales/patología , Epitelio/microbiología , Epitelio/patología , Femenino , Humanos , Uniones Intercelulares/patología , Membrana Mucosa/microbiología , Membrana Mucosa/patologíaRESUMEN
BACKGROUND: Resistin may be involved in the pathogenesis of osteoarthritis (OA), but a systematic understanding of the role of resistin in OA is lacking. METHODS: We reviewed studies that evaluated the role of resistin in OA. The expression levels of resistin in vitro experiments and OA/rheumatoid arthritis (RA) patients were analyzed. We also studied potential resistin receptors and the signaling pathways that these receptors activate, ultimately leading to cartilage degeneration. RESULTS: Resistin levels in both the serum and synovial fluid were higher in OA and RA patients than in healthy subjects. Overall, resistin levels are much higher in serum than in synovial fluid. In human cartilage, resistin induces the expression of proinflammatory factors such as degradative enzymes, leading to the inhibition of cartilage matrix synthesis, perhaps by binding to Toll-like receptor 4 and the adenylyl cyclase-associated protein 1 receptor, which then activates the p38-mitogen-activated phosphate kinase, protein kinase A-cyclic AMP, nuclear factor-κB, and C/enhancer-binding protein ß signaling pathways. CONCLUSION: Resistin levels are higher in OA patients than in healthy controls; however, the precise role of resistin in the pathogenesis of OA needs to be studied further. Resistin may be a novel therapeutic target in OA in the future.
Asunto(s)
Osteoartritis/metabolismo , Osteoartritis/patología , Resistina/metabolismo , Artritis Reumatoide/sangre , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Decorina/sangre , Decorina/metabolismo , Femenino , Humanos , Masculino , Osteoartritis/sangre , Resistina/sangre , Transducción de Señal/efectos de los fármacos , Líquido Sinovial/metabolismo , Receptor Toll-Like 4/sangre , Receptor Toll-Like 4/metabolismoRESUMEN
Wiskott-Aldrich syndrome (WAS) pediatric patients exhibit a deficiency in humoral immune memory. However, the mechanism by which Wiskott-Aldrich syndrome protein (WASP) regulates the differentiation and activation of memory B cells remains elusive. Here we examine the early activation events of memory B cells from the peripheral blood mononuclear cells of WAS patients and age-matched healthy controls (HCs) using total internal reflection fluorescence microscopy. In response to stimulation through the B-cell receptor (BCR), memory B cells from HCs showed significantly higher magnitudes of BCR clustering and cell spreading than naive B cells from the same individuals. This was associated with increases in CD19 recruitment to the BCR and the activation of its downstream signaling molecule Btk and decreases in FcγRIIB recruitment and the activation of its downstream molecule Src homology 2-containing inositol 5' phosphatase (SHIP). However, these enhanced signaling activities mediated by CD19 and Btk are blocked in memory B cells from WAS patients, whereas the activation of FcγRIIB and SHIP was increased. Although the expression levels of CD19, Btk, and FcγRIIB did not change between CD27(-) and CD27(+) B cells of HCs, the protein and mRNA levels of CD19 but not Btk and FcγRIIB were significantly reduced in both CD27(-) and CD27(+) B cells of WAS patients, compared with those of HCs. Overall, our study suggests that WASP is required for memory B-cell activation, promoting the activation by positive regulating CD19 transcription and CD19 recruitment to the BCR.
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Antígenos CD19/metabolismo , Linfocitos B/inmunología , Memoria Inmunológica , Síndrome de Wiskott-Aldrich/inmunología , Citoesqueleto de Actina/metabolismo , Antígenos CD19/genética , Linfocitos B/metabolismo , Estudios de Casos y Controles , Preescolar , Regulación hacia Abajo , Humanos , Lactante , Activación de Linfocitos , Mutación , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de IgG/metabolismo , Transducción de Señal , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
Wiskott-Aldrich syndrome protein (WASp) is a hematopoietic-specific regulator of actin nucleation. Wiskott-Aldrich syndrome (WAS) patients show immunodeficiencies, most of which have been attributed to defective T-cell functions. T follicular helper (Tfh) cells are the major CD4(+) T-cell subset with specialized B-cell helper capabilities. Aberrant Tfh cells activities are involved in immunopathologies such as autoimmunity, immunodeficiencies, and lymphomas. We found that in WAS patients, the number of circulating Tfh cells was significantly reduced due to reduced proliferation and increased apoptosis, and Tfh cells were Th2 and Th17 polarized. The expression of inducible costimulator (ICOS) in circulating Tfh cells was higher in WAS patients than in controls. BCL6 expression was decreased in total CD4(+) T and Tfh cells of WAS patients. Mirroring the results in patients, the frequency of Tfh cells in WAS knockout (KO) mice was decreased, as was the frequency of BCL6(+) Tfh cells, but the frequency of ICOS(+) Tfh cells was increased. Using WAS chimera mice, we found that the number of ICOS(+) Tfh cells was decreased in WAS chimera mice, indicating that the increase in ICOS(+) Tfh cells in WAS KO mice was cell extrinsic. The data from in vivo CD4(+) naive T-cell adoptive transfer mice as well as in vitro coculture of naive B and Tfh cells showed that the defective function of WASp-deficient Tfh cells was T-cell intrinsic. Consistent findings in both WAS patients and WAS KO mice suggested an essential role for WASp in the development and memory response of Tfh cells and that WASp deficiency causes a deficient differentiation defect in Tfh cells by downregulating the transcription level of BCL6.
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Centro Germinal/patología , Linfocitos T Colaboradores-Inductores/patología , Linfocitos T Colaboradores-Inductores/fisiología , Síndrome de Wiskott-Aldrich/inmunología , Animales , Linfocitos B , Linfocitos T CD4-Positivos/patología , Linfocitos T CD4-Positivos/fisiología , Estudios de Casos y Controles , Diferenciación Celular , Células Cultivadas , Centro Germinal/inmunología , Humanos , Proteína Coestimuladora de Linfocitos T Inducibles/metabolismo , Interleucinas/metabolismo , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/patología , Ratones , Ratones Noqueados , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Receptores CXCR5/metabolismo , Proteínas Represoras/metabolismo , Síndrome de Wiskott-Aldrich/sangre , Síndrome de Wiskott-Aldrich/patología , Proteína del Síndrome de Wiskott-Aldrich/genéticaRESUMEN
High dosage of enzyme is required to achieve effective lignocellulose hydrolysis, especially at high-solid loadings, which is a significant barrier to large-scale bioconversion of lignocellulose. Here, we screened four chemical additives and three accessory proteins for their effects on the enzymatic hydrolysis of various lignocellulosic materials. The effects were found to be highly dependent on the composition and solid loadings of substrates. For xylan-extracted lignin-rich corncob residue, the enhancing effect of PEG 6000 was most pronounced and negligibly affected by solid content, which reduced more than half of enzyme demand at 20% dry matter (DM). Lytic polysaccharide monooxygenase enhanced the hydrolysis of ammonium sulfite wheat straw pulp, and its addition reduced about half of protein demand at the solid loading of 20% DM. Supplementation of the additives in the hydrolysis of pure cellulose and complex lignocellulosic materials revealed that their effects are tightly linked to pretreatment strategies.
Asunto(s)
Celulasa/química , Proteínas Fúngicas/química , Lignina/química , Penicillium/enzimología , Zea mays/química , Hidrólisis , Polietilenglicoles/químicaRESUMEN
Prolonged or uncontrolled B-cell receptor (BCR) signaling is associated with autoimmunity. We previously demonstrated a role for actin in BCR signal attenuation. This study reveals that actin-binding protein 1 (Abp1/HIP-55/SH3P7) is a negative regulator of BCR signaling and links actin to negative regulatory pathways of the BCR. In both Abp1(-/-) and bone marrow chimeric mice, in which only B cells lack Abp1 expression, the number of spontaneous germinal center and marginal zone B cells and the level of autoantibody are significantly increased. Serum levels of T-independent antibody responses and total antibody are elevated, whereas T-dependent antibody responses are markedly reduced and fail to undergo affinity maturation. Upon activation, surface BCR clustering is enhanced and B-cell contraction delayed in Abp1(-/-) B cells, concurrent with slow but persistent increases in F-actin at BCR signalosomes. Furthermore, BCR signaling is enhanced in Abp1(-/-) B cells compared with wild-type B cells, including Ca(2+) flux and phosphorylation of B-cell linker protein, the mitogen-activated protein kinase kinase MEK1/2, and ERK, coinciding with reductions in recruitment of the inhibitory signaling molecules hematopoietic progenitor kinase 1 and SH2-containing inositol 5-phosphatase to BCR signalosomes. Our results indicate that Abp1 negatively regulates BCR signaling by coupling actin remodeling to B-cell contraction and activation of inhibitory signaling molecules, which contributes to the regulation of peripheral B-cell development and antibody responses.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Anticuerpos/sangre , Linfocitos B/citología , Centro Germinal/citología , Inositol Polifosfato 5-Fosfatasas , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Linfocitos T/inmunologíaRESUMEN
Upon recognizing cognate antigen, B cells mobilize multiple cellular apparatuses to propagate an optimal response. Antigen binding is transduced into cytoplasmic signaling events through B-cell antigen receptor (BCR)-based signalosomes at the B-cell surface. BCR signalosomes are dynamic and transient and are subsequently endocytosed for antigen processing. The function of BCR signalosomes is one of the determining factors for the fate of B cells: clonal expansion, anergy, or apoptosis. Accumulating evidence underscores the importance of the actin cytoskeleton in B-cell activation. We have begun to appreciate the role of actin dynamics in regulating BCR-mediated tonic signaling and the formation of BCR signalosomes. Our recent studies reveal an additional function of the actin cytoskeleton in the downregulation of BCR signaling, consequently contributing to the generation and maintenance of B-cell self-tolerance. In this review, we discuss how actin remodels its organization and dynamics in close coordination with BCR signaling and how actin remodeling in turn amplifies the activation and subsequent downregulation process of BCR signaling, providing vital feedback for optimal BCR activation.
Asunto(s)
Actinas/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Retroalimentación Fisiológica , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Animales , Humanos , Unión ProteicaRESUMEN
Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR) signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.
Asunto(s)
Linfocitos B/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Transducción de Señal , Proteína Neuronal del Síndrome de Wiskott-Aldrich/fisiología , Actinas/metabolismo , Animales , Anticuerpos Antinucleares/sangre , Antígenos/inmunología , Autoanticuerpos/sangre , Linfocitos B/inmunología , Células Cultivadas , Humanos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Transporte de Proteínas , Síndrome de Wiskott-Aldrich/inmunología , Síndrome de Wiskott-Aldrich/metabolismo , Proteína del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
AIM: We have shown that rutaecarpine extracted from the dried fruit of Chinese herb Evodia rutaecarpa (Juss) Benth (Wu Zhu Yu) promotes glucose consumption and anti-inflammatory cytokine expression in insulin-resistant primary skeletal muscle cells. In this study we investigated whether rutaecarpine ameliorated the obesity profiles, lipid abnormality, glucose metabolism and insulin resistance in rat model of hyperlipidemia and hyperglycemia. METHODS: Rats fed on a high-fat diet for 8 weeks, followed by injection of streptozotocin (30 mg/kg, ip) to induce hyperlipidemia and hyperglycemia. One week after streptozotocin injection, the fat-fed, streptozotocin-treated rats were orally treated with rutaecarpine (25 mg·kg(-1)·d(-1)) or a positive control drug metformin (250 mg·kg(-1)·d(-1)) for 7 weeks. The body weight, visceral fat, blood lipid profiles and glucose levels, insulin sensitivity were measured. Serum levels of inflammatory cytokines were analyzed. IRS-1 and Akt/PKB phosphorylation, PI3K and NF-κB protein levels in liver tissues were assessed; pathological changes of livers and pancreases were examined. Glucose uptake and AMPK/ACC2 phosphorylation were studied in cultured rat skeletal muscle cells in vitro. RESULTS: Administration of rutaecarpine or metformin significantly decreased obesity, visceral fat accumulation, water consumption, and serum TC, TG and LDL-cholesterol levels in fat-fed, streptozotocin-treated rats. The two drugs also attenuated hyperglycemia and enhanced insulin sensitivity. Moreover, the two drugs significantly decreased NF-κB protein levels in liver tissues and plasma TNF-α, IL-6, CRP and MCP-1 levels, and ameliorated the pathological changes in livers and pancreases. In addition, the two drugs increased PI3K p85 subunit levels and Akt/PKB phosphorylation, but decreased IRS-1 phosphorylation in liver tissues. Treatment of cultured skeletal muscle cells with rutaecarpine (20-180 µmol/L) or metformin (20 µmol/L) promoted the phosphorylation of AMPK and ACC2, and increased glucose uptake. CONCLUSION: Rutaecarpine ameliorates hyperlipidemia and hyperglycemia in fat-fed, streptozotocin-treated rats via regulating IRS-1/PI3K/Akt signaling pathway in liver and AMPK/ACC2 signaling pathway in skeletal muscles.