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1.
Background
Activation induced deaminase (AID) mediates class switch recombination and somatic hypermutation of immunoglobulin (Ig) genes in germinal centre B cells. In order to regulate its specific activity and as a means to keep off-target mutations low, several mechanisms have evolved, including binding to specific cofactors, phosphorylation and destabilization of nuclear AID protein. Although ubiquitination at lysine residues of AID is recognized as an essential step in initiating degradation of nuclear AID, any functional relevance of lysine modifications has remained elusive.Methodology/Principal Findings
Here, we report functional implications of lysine modifications of the human AID protein by generating a panel of lysine to arginine mutants of AID and assessment of their catalytic class switch activity. We found that only mutation of Lys22 to Arg resulted in a significant reduction of class switching to IgG1 in transfected primary mouse B cells. This decrease in activity was neither reflected in reduced hypermutation of Ig genes in AID-mutant transfected DT40 B cell lines nor recapitulated in bacterial deamination assays, pointing to involvement of post-translational modification of Lys22 for AID activity in B cells.Conclusions/Significance
Our results imply that lysine modification may represent a novel level of AID regulation and that Lys22 is important for effective AID activity. 相似文献2.
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Giulia Saraconi Francesco Severi Cesare Sala Giorgio Mattiuz Silvestro G Conticello 《Genome biology》2014,15(7)
Background
The AID/APOBECs are deaminases that act on cytosines in a diverse set of pathways and some of them have been linked to the onset of genetic alterations in cancer. Among them, APOBEC1 is the only family member to physiologically target RNA, as the catalytic subunit in the Apolipoprotein B mRNA editing complex. APOBEC1 has been linked to cancer development in mice but its oncogenic mechanisms are not yet well understood.Results
We analyze whether expression of APOBEC1 induces a mutator phenotype in vertebrate cells, likely through direct targeting of genomic DNA. We show its ability to increase the inactivation of a stably inserted reporter gene in a chicken cell line that lacks any other AID/APOBEC proteins, and to increase the number of imatinib-resistant clones in a human cellular model for chronic myeloid leukemia through induction of mutations in the BCR-ABL1 fusion gene. Moreover, we find the presence of an AID/APOBEC mutational signature in esophageal adenocarcinomas, a type of tumor where APOBEC1 is expressed, that mimics the one preferred by APOBEC1 in vitro.Conclusions
Our findings suggest that the ability of APOBEC1 to trigger genetic alterations represents a major layer in its oncogenic potential. Such APOBEC1-induced mutator phenotypes could play a role in the onset of esophageal adenocarcinomas. APOBEC1 could be involved in cancer promotion at the very early stages of carcinogenesis, as it is highly expressed in Barrett''s esophagus, a condition often associated with esophageal adenocarcinoma.Electronic supplementary material
The online version of this article (doi:10.1186/s13059-014-0417-z) contains supplementary material, which is available to authorized users. 相似文献5.
Glen M. Borchert Nathaniel W. Holton Kevin A. Edwards Laura A. Vogel Erik D. Larson 《PloS one》2010,5(7)
Background
Somatic hypermutation introduces base substitutions into the rearranged and expressed immunoglobulin (Ig) variable regions to promote immunity. This pathway requires and is initiated by the Activation Induced Deaminase (AID) protein, which deaminates cytidine to produce uracils and UG mismatches at the Ig genes. Subsequent processing of uracil by mismatch repair and base excision repair factors contributes to mutagenesis. While selective for certain genomic targets, the chromatin modifications which distinguish hypermutating from non-hypermutating loci are not defined.Methodology/Principal Findings
Here, we show that AID-targeted loci in mammalian B cells contain ubiquitinated chromatin. Chromatin immunoprecipitation (ChIP) analysis of a constitutively hypermutating Burkitt''s B cell line, Ramos, revealed the presence of monoubiquitinated forms of both histone H2A and H2B at two AID-associated loci, but not at control loci which are expressed but not hypermutated. Similar analysis using LPS activated primary murine splenocytes showed enrichment of the expressed VH and Sγ3 switch regions upon ChIP with antibody specific to AID and to monoubiquitinated H2A and H2B. In the mechanism of mammalian hypermutation, AID may interact with ubiquitinated chromatin because confocal immunofluorescence microscopy visualized AID colocalized with monoubiquitinated H2B within discrete nuclear foci.Conclusions/Significance
Our results indicate that monoubiquitinated histones accompany active somatic hypermutation, revealing part of the histone code marking AID-targeted loci. This expands the current view of the chromatin state during hypermutation by identifying a specific nucleosome architecture associated with somatic hypermutation. 相似文献6.
Background
Human immunodeficiency virus type 1 (HIV-1) induces a general dysregulation of immune system. Dysregulation of B cell compartment is generally thought to be induced by HIV-related immune activation and lymphopenia. However, a direct influence of HIV-1 particles on B cells was recently proposed as the third pathway of B cells dysregulation.Methods/Principal Findings
We evaluated the direct and specific consequences of HIV-1 contact on activation, survival, proliferation and phenotype of primary B cells in vitro. Moreover, we examined expression of activation-induced cytidine deaminase (AID) mRNA that is responsible for class switch recombination (CSR) and somatic hypermutation (SHM). Here, we report that changes observed in cellular proliferation, phenotypes and activation of B cells could be caused by direct contact between HIV-1 particles and primary B cells in vitro. Finally, direct HIV-1-derived B cells activation led to the increase of AID mRNA expression and its subsequent CSR function was detected in vitro.Conclusion/Significance
We showed that HIV-1 could directly induce primary B cells dysregulation triggering phenotypical and functional abilities of B cells in vitro that could explain in some extent early B-cell abnormalities in HIV disease. 相似文献7.
Ainsley A. MacFarlane George Orriss Natalie Okun Markus Meier Thomas Klonisch Mazdak Khajehpour J?rg Stetefeld 《PloS one》2012,7(11)
Background
COMPcc forms a pentameric left-handed coiled coil that is known to bind hydrophilic signaling molecules such as vitamin D3, and vitamin A.Principal Findings
In an integrated approach we reveal the unique binding properties of COMPcc for saturated and unsaturated fatty acids. Our observations suggest that residues Met33 (gating pore), Thr40/Asn41 (water chamber) and Gln54 (electrostatic trap) are key elements for the binding of fatty acids by COMPcc. In addition, this work characterizes the binding of various fatty acids to COMPcc using fluorescence spectroscopy. Our findings reveal a binding trend within the hydrophobic channel of COMPcc, namely, that is driven by length of the methylene tail and incorporation of unsaturation.Conclusion/Significance
The unique binding properties imply that COMPcc may be involved in signalling functions in which hydrophilic ligands are involved. The pentameric channel is a unique carrier for lipophilic compounds. This opens the exciting possibility that COMPcc could be developed as a targeted drug delivery system. 相似文献8.
Flück CE Pandey AV Dick B Camats N Fernández-Cancio M Clemente M Gussinyé M Carrascosa A Mullis PE Audi L 《PloS one》2011,6(5):e20178
Context
Steroidogenic acute regulatory protein (StAR) is crucial for transport of cholesterol to mitochondria where biosynthesis of steroids is initiated. Loss of StAR function causes lipoid congenital adrenal hyperplasia (LCAH).Objective
StAR gene mutations causing partial loss of function manifest atypical and may be mistaken as familial glucocorticoid deficiency. Only a few mutations have been reported.Design
To report clinical, biochemical, genetic, protein structure and functional data on two novel StAR mutations, and to compare them with published literature.Setting
Collaboration between the University Children''s Hospital Bern, Switzerland, and the CIBERER, Hospital Vall d''Hebron, Autonomous University, Barcelona, Spain.Patients
Two subjects of a non-consanguineous Caucasian family were studied. The 46,XX phenotypic normal female was diagnosed with adrenal insufficiency at the age of 10 months, had normal pubertal development and still has no signs of hypergonodatropic hypogonadism at 32 years of age. Her 46,XY brother was born with normal male external genitalia and was diagnosed with adrenal insufficiency at 14 months. Puberty was normal and no signs of hypergonadotropic hypogonadism are present at 29 years of age.Results
StAR gene analysis revealed two novel compound heterozygote mutations T44HfsX3 and G221S. T44HfsX3 is a loss-of-function StAR mutation. G221S retains partial activity (∼30%) and is therefore responsible for a milder, non-classic phenotype. G221S is located in the cholesterol binding pocket and seems to alter binding/release of cholesterol.Conclusions
StAR mutations located in the cholesterol binding pocket (V187M, R188C, R192C, G221D/S) seem to cause non-classic lipoid CAH. Accuracy of genotype-phenotype prediction by in vitro testing may vary with the assays employed. 相似文献9.
Background
Hepatitis B virus (HBV) is a major cause of chronic liver diseases, and frequently results in hepatitis, cirrhosis, and ultimately hepatocellular carcinoma. The role of HCV in associations with insulin signaling has been elucidated. However, the pathogenesis of HBV-associated insulin signaling remains to be clearly characterized. Therefore, we have attempted to determine the mechanisms underlying the HBV-associated impairment of insulin signaling.Methodology
The expressions of insulin signaling components were investigated in HBx-transgenic mice, HBx-constitutive expressing cells, and transiently HBx-transfected cells. Protein and gene expression was examined by Western blot, immunohistochemistry, RT-PCR, and promoter assay. Protein-protein interaction was detected by coimmunoprecipitation.Principal Findings
HBx induced a reduction in the expression of IRS1, and a potent proteasomal inhibitor blocked the downregulation of IRS1. Additionally, HBx enhanced the expression of SOCS3 and induced IRS1 ubiquitination. Also, C/EBPα and STAT3 were involved in the HBx-induced expression of SOCS3. HBx interfered with insulin signaling activation and recovered the insulin-mediated downregulation of gluconeogenic genes.Conclusions/Significance
These results provide direct experimental evidences for the contribution of HBx in the impairment of insulin signaling. 相似文献10.
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Background
Specific delivery to synapses of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors with long-tailed subunits is believed to be a key event in many forms of activity-dependent changes in synaptic strength. GluA1, the best characterized long-tailed AMPA receptor subunit, contains a C-terminal class I PDZ binding motif, which mediates its interaction with scaffold and trafficking proteins, including synapse-associated protein 97 (SAP97). In GluA4, another long-tailed subunit implicated in synaptic plasticity, the PDZ motif is blocked by a single proline residue. This feature is highly conserved in vertebrates, whereas the closest invertebrate homologs of GluA4 have a canonical class I PDZ binding motif. In this work, we have examined the role of GluA4 in PDZ interactions.Methodology/Principal Findings
Deletion of the carboxy-terminal proline residue of recombinant GluA4 conferred avid binding to SAP97 in cultured cells as shown by coimmunoprecipitation, whereas wild-type GluA4 did not associate with SAP97. Native GluA4 and SAP97 coimmunoprecipitated from mouse brain independently of the GluA1 subunit, supporting the possibility of in vivo PDZ interaction. To obtain evidence for or against the exposure of the PDZ motif by carboxyterminal processing of native GluA4 receptors, we generated an antibody reagent specific for proline-deleted GluA4 C-terminus. Immunoprecipitation and mass spectrometric analyses indicated that the carboxyl-terminus of native GluA4 AMPA receptors is intact and that the postulated single-residue cleavage does not occur to any significant extent.Conclusion/Significance
We conclude that native GluA4 receptors are not capable of canonical PDZ interactions and that their association with SAP97 is likely to be indirect. 相似文献12.
Background
Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding.Methodology/Principal Findings
We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked.Conclusions/Significance
The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner. 相似文献13.
Stax MJ Naarding MA Tanck MW Lindquist S Hernell O Lyle R Brandtzaeg P Eggesbø M Pollakis G Paxton WA 《PloS one》2011,6(2):e17316
Objective
Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4+ T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms.Study Design
ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions.Results
DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein.Conclusion
The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding. 相似文献14.
Background
Sequence variation in the human 12/15 lipoxygenase (ALOX15) has been associated with atherosclerotic disease. We functionally characterized an ALOX15 promoter polymorphism, rs2255888, previously associated with carotid plaque burden.Methodology/Principal Findings
We demonstrate specific in vitro and in vivo binding of the cytoskeletal protein, vimentin, to the ALOX15 promoter. We show that the two promoter haplotypes carrying alternate alleles at rs2255888 exhibit significant differences in promoter activity by luciferase reporter assay in two cell lines. Differences in in-vitro vimentin-binding to and formation of DNA secondary structures in the polymorphic promoter sequence are also detected by electrophoretic mobility shift assay and biophysical analysis, respectively. We show regulation of ALOX15 protein by vimentin.Conclusions/Significance
This study suggests that vimentin binds the ALOX15 promoter and regulates its promoter activity and protein expression. Sequence variation that results in changes in DNA conformation and vimentin binding to the promoter may be relevant to ALOX15 gene regulation. 相似文献15.
Context
Levothyroxine monotherapy is the treatment of choice for hypothyroid patients because peripheral T4 to T3 conversion is believed to account for the overall tissue requirement for thyroid hormones. However, there are indirect evidences that this may not be the case in all patients.Objective
To evaluate in a large series of athyreotic patients whether levothyroxine monotherapy can normalize serum thyroid hormones and thyroid-pituitary feedback.Design
Retrospective study.Setting
Academic hospital.Patients
1,811 athyreotic patients with normal TSH levels under levothyroxine monotherapy and 3,875 euthyroid controls.Measurements
TSH, FT4 and FT3 concentrations by immunoassays.Results
FT4 levels were significantly higher and FT3 levels were significantly lower (p<0.001 in both cases) in levothyroxine-treated athyreotic patients than in matched euthyroid controls. Among the levothyroxine-treated patients 15.2% had lower serum FT3 and 7.2% had higher serum FT4 compared to euthyroid controls. A wide range of FT3/FT4 ratios indicated a major heterogeneity in the peripheral T3 production capacity in different individuals. The correlation between thyroid hormones and serum TSH levels indicated an abnormal feedback mechanism in levothyroxine-treated patients.Conclusions
Athyreotic patients have a highly heterogeneous T3 production capacity from orally administered levothyroxine. More than 20% of these patients, despite normal TSH levels, do not maintain FT3 or FT4 values in the reference range, reflecting the inadequacy of peripheral deiodination to compensate for the absent T3 secretion. The long-term effects of chronic tissue exposure to abnormal T3/T4 ratio are unknown but a sensitive marker of target organ response to thyroid hormones (serum TSH) suggests that this condition causes an abnormal pituitary response. A more physiological treatment than levothyroxine monotherapy may be required in some hypothyroid patients. 相似文献16.
Mihai Bragaru C. P. van Wilgen Jan H. B. Geertzen Suzette G. J. B. Ruijs Pieter U. Dijkstra Rienk Dekker 《PloS one》2013,8(3)
Introduction
Although individuals with lower limb amputation may benefit from participation in sports, less than 40% do so.Aim
To identify the barriers and facilitators that influence participation in sports for individuals with lower limb amputation.Design
Qualitative study.Participants
Twenty six individuals with lower limb amputation, all originating from the Dutch provinces of Groningen and Drenthe, of which 13 athletes.Methods
Semi-structured interviews were used to gather information. Following thematic analysis, emerging themes were organized in three categories Technical, Social and Personal.Results
Sport was perceived as enjoyable activity that would help participants to become and stay healthy, improve the number of social contacts, reduce phantom pain and decrease daily tension. Inadequate facilities, problematic transportation, trivialization from others, poor health and lack of motivation or the lack of a sports partner were barriers commonly mentioned by non-athletes. Remarkably, while all athletes were successful prosthetic users, the majority chose to participate in sports for which prosthesis was neither required nor needed.Conclusions
Each individual with lower limb amputation needs to be counselled according to the barriers and facilitators he/she personally experiences. Athletes appeared to be more proactive in searching for a solution and also appeared less discouraged by failing. 相似文献17.
Fernagut PO Li Q Dovero S Chan P Wu T Ravenscroft P Hill M Chen Z Bezard E 《PloS one》2010,5(11):e14053
Background
Radiotracer imaging of the presynaptic nigrostriatal dopaminergic system is used to assess disease progression in Parkinson''s disease (PD) and may provide a useful adjunct to clinical assessment during therapeutic trials of potential neuroprotective agents. Several clinical trials comparing dopamine agonists to L-DOPA or early vs. late L-DOPA have revealed differences between clinical assessment and imaging of the presynaptic dopaminergic system, hence questioning the comparability of these measures as neuroprotection outcome variables. Thus, results of these studies may have been affected by factors other than the primary biological process investigated.Methodology/Principal Findings
We tested the possibility that L-DOPA might interfere with DAT binding. Post-mortem DAT binding was conducted in normal and MPTP-treated macaque monkeys that were administered L-DOPA, acutely or chronically. In parallel, DAT SPECT was conducted in MPTP-treated animals that were administered chronic L-DOPA. [99mTc]TRODAT-1 SPECT binding was similarly reduced in all MPTP monkeys regardless of L-DOPA treatment. L-DOPA had no significant effect on post-mortem DAT binding either in saline or in MPTP-lesioned animals.Conclusions/Significance
These data indicate that L-DOPA does not induce modifications of DAT expression detectable by SPECT of by DAT binding autoradiography, suggesting that differences between clinical assessment and radiotracer imaging in clinical trials may not be specifically related to L-DOPA treatment. 相似文献18.
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Irena Auersperger Branko ?kof Bojan Lesko?ek Bojan Knap Ale? Jerin Mitja Lainscak 《PloS one》2013,8(3)
Background and Aims
Exercise-induced iron deficiency is a common finding in endurance athletes. It has been suggested recently that hepcidin may be an important mediator in this process.Objective
To determine hepcidin levels and markers of iron status during long-term exercise training in female runners with depleted and normal iron stores.Methods
Fourteen runners were divided into two groups according to iron status. Blood samples were taken during a period of eight weeks at baseline, after training and after ten days’ recovery phase.Results
Of 14 runners, 7 were iron deficient at baseline and 10 after training. Hepcidin was lower at recovery compared with baseline (p<0.05). The mean cell haemoglobin content, haemoglobin content per reticulocyte and total iron binding capacity all decreased, whereas soluble transferrin receptor and hypochromic red cells increased after training and recovery (p<0.05 for all).Conclusion
The prevalence of depleted iron stores was 71% at the end of the training phase. Hepcidin and iron stores decreased during long-term running training and did not recover after ten days, regardless of baseline iron status. 相似文献20.