1.
Ubiquitin-proteasome-rich cytoplasmic structures in neutrophils of patients with Shwachman-Diamond syndrome.
Source
Policlinico S. Matteo, Pavia;
Abstract
Background. Shwachman-Diamond syndrome is an autosomal recessive disorder with severe bone marrow dysfunction causing neutropenia and increased leukemia risk. Recently, novel particulate cytoplasmic structures, named PaCSs, rich in ubiquitinated and proteasomal proteins, have been detected in epithelial cells and neutrophils from Helicobacter pylori gastritis and several epithelial neoplasms.Design and Methods Blood neutrophils from 13 Shwachman-Diamond syndrome cases, 10 with and three without SBDS gene mutation, and 10 controls were investigated by confocal microscopy and ultrastructural immunocytochemistry using antibodies against ubiquitinated proteins, proteasomes, p62 protein, and Helicobacter pylori VacA, urease and outer membrane proteins.Results. Many extensively disseminated PaCSs, accounting for 22.78+/-5.57% (mean+/-SD) of the total cytoplasm, were found in blood neutrophils from mutated Shwachman-Diamond syndrome patients. PaCSs showed immunoreactivity for polyubiquitinated proteins and proteasomes, but no reactivity for Helicobacter pylori products, which are present in PaCSs of Helicobacter pylori-positive gastritis. Neutrophils from Shwachman-Diamond syndrome patients frequently showed p62-positive autophagic vacuoles and apoptotic changes in 5% of cells. No PaCSs were observed in most control neutrophils; however, in a few cells from two cases, we noted focal development of minute PaCSs, accounting for 0.74+/-0.56% of total cytoplasm (P<0.001 vs PaCSs from mutated Shwachman-Diamond syndrome patients). Neutrophils from non-mutated Shwachman-Diamond-syndrome-like patients resembled controls in two cases, and a third case showed PaCS patterns intermediate between those in control and mutated Shwachman-Diamond syndrome patients. Conclusions. PaCSs are a prominent feature of neutrophils from Shwachman-Diamond syndrome patients. They may help us to understand the mechanism of granulocyte dysfunction and the neoplastic risk of the disease.
Identification of a new causative gene of amyotrophic lateral sclerosis; optineurin.
Source
Department of Epidemiology, Research Institute for Radiation Biology and Medicine, Hiroshima University.
Abstract
Amyotrophic lateral sclerosis (ALS) is a devastating disorder characterized by degeneration of motor neurons of the primary motor cortex, brainstem and spinal cord. ALS patients die within 3 to 5 years without respiratory support. Detecting the causing gene is necessary to elucidate ALS. We identified mutations of optineurin (OPTN) in ALS. We found three types of mutation of OPTN: a homozygous deletion of exon 5, a homozygous Q398X nonsense mutation and a heterozygous E478G missense mutation within its ubiquitin-binding domain. Cell transfection experiments showed that the nonsense and missense mutations of OPTN abolished the inhibition of activation of nuclear factor kappa B. The missense mutation revealed a cytoplasmic distribution different from that of the wild type. A case with the E478G mutation showed OPTN-immunoreactive cytoplasmic retention, and Golgi fragmentation was identified in 70% of the anterior horn cells. TDP-43- or SOD1-positive inclusions of sporadic and SOD1 cases of ALS were also immunolabelled with anti-OPTN antibodies. Furthermore, optineurin is co-localized with fused in sarcoma (FUS) in basophilic inclusions of ALS with FUS mutation and in basophilic inclusion body disease. Our findings suggest that OPTN is involved in the great part of pathogenesis of ALS.
Comparison of phosphorylated TDP-43-positive inclusions in oculomotor neurons in patients with non-ALS and ALS disorders.
Source
Department of Neurology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan.
Abstract
TDP-43 has been identified as a major component of the pathological inclusions in most forms of frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis (ALS). In the present study, paraffin sections of the midbrain in 112 patients with various non-ALS disorders and 27 patients with sporadic ALS were immunostained with antibody against phosphorylated TDP-43 (pTDP-43). pTDP-43-positive inclusions in oculomotor neurons were detected in 18 of 112 patients with non-ALS disorders (16.1%). The appearance of the inclusions showed fine filamentous structures rather than the skein-like inclusions seen in the anterior horn cells of ALS spinal cords. The incidence was increased in the age range of 80-89years old (10/37 cases; 27.0%), in which 6 of 10 cases demonstrated AD pathology in the temporal lobes. Twenty-seven ALS patients were examined and the findings were compared with those of non-ALS patients. There were 13 cases demonstrating pTDP-43-positive inclusions (48.1%) which showed stronger immunoreactivities in ALS cases. This is the first report demonstrating fine filamentous pTDP-43-positive inclusions in oculomotor neurons in non-ALS disorders. Although the mechanisms underlying pTDP-43 in oculomotor neurons are currently unknown, its detection is of interest, and the expression may occur not only in ALS but also during the aging process.
Copyright © 2011 Elsevier B.V. All rights reserved.
Engineering and Structural Characterization of a Linear Polyubiquitin-SpecificAntibody.
Source
Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Abstract
Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibodyFab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.
Copyright © 2011. Published by Elsevier Ltd.
A DNA vaccine co-expressing Trichinella spiralis MIF and MCD-1 with murineubiquitin induces partial protective immunity in mice.
Source
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Abstract
Co-expression of Trichinella spiralis macrophage migration inhibitory factor (TsMIF) with T. spiralis cystatin-like domain protein (TsMCD-1) in a DNA vaccine induces a Th1 immune response and partial protection against T. spiralis infection. The present study evaluated whether co-expression of mouse ubiquitin (Ub) with TsMIF and TsMCD-1 might improve the immune response against T. spiralis infection. Groups of BALB/c mice were immunized twice at 2-week intervals with 100 μg of plasmid DNA encoding either a TsMIF-TsMCD-1 fusion protein (pVAX1-Tsmif-Tsmcd-1) or an Ub-co-expressing triple fusion protein Ub-TsMIF-TsMCD-1 (pVAX1-Ub-Tsmif-Tsmcd-1). Control animals were immunized with pVAX1-Ub or blank vector plasmid. Specific antibody levels (IgG, IgG1, IgG2a, IgG2b, IgM, IgA, IgE) against the recombinant protein TsMIF-TsMCD-1, serum cytokines (interferon (IFN)-γ, interleukin (IL)-4, IL-5, transforming growth factor (TGF)-β1 and IL-17), CD4+/CD8+ T cells and cytotoxic T lymphocyte (CTL) responses were monitored. Challenge infection was performed 2 weeks after the second immunization and worm burden was assayed at 35 days post-challenge. Antibodyresponses induced by pVAX1-Ub-Tsmif-Tsmcd-1 were significantly lower than for TsMIF-TsMCD-1, but the vaccine induced increased levels of Th1 cytokine (IFN-γ) and increased T-cell cytotoxicity. The reduction of worm burden (37.95%) following immunization with pVAX1-Ub-Tsmif-Tsmcd-1 was significantly greater than that induced by the pVAX1-Tsmif-Tsmcd-1 vaccine (23.17%; P < 0.05).
Widespread neuronal and glial hyperphosphorylated tau deposition in ALS with cognitive impairment.
Source
Robarts Research Institute, The University of Western Ontario.
Abstract
Although the biological basis of frontotemporal syndromes associated with amyotrophic lateral sclerosis (ALS) is considered to be altered metabolism of TDP-43, in ALS with cognitive impairment (ALSci) the metabolism of tau protein is also altered. This includes neuronal hyperphosphorylation (pThr(175)). Using novel polyclonal phospho-tau antibodies(pSer(208, 210), pThr(217) and pThr(175)) and antibodies directed against PHF tau (pSer(202)), TDP-43 or ubiquitin, we characterized tau deposition in ALS and ALSci. In ALS, we observed pThr(175) tau immunoreactive intraneuronal and neuritic aggregates throughout the amygdala and entorhinal cortex. In ALSci, this extended to the anterior cingulate gyrus, superior frontal cortex and substantia nigra. The pThr(217) antibody detected widespread astrocytic tau deposition, including punctuate or fibrillary aggregates, or intensely immunoreactive tufted astrocytes in the superior frontal cortex, anterior cingulate gyrus, entorhinal cortex, amygdala and basal ganglia of ALS. In ALSci, a similar but more widely distributed pThr(217) pathology was observed. There was no correlation between the extent of pathological tau deposition and TDP-43 pathology, although nuclear TDP-43 immunoreactivity was absent in neurons with tau pathology. In conclusion, ALSci is unique in possessing both tau and TDP-43 pathology. The presence of widespread astrocytic tau pathology suggests that ALSci may initially be characterized by astrocytic pathology.
- PMID:
- 22214313
- [PubMed - as supplied by publisher]
In this issue.
Abstract
COVER IMAGE: The cover shows a histological analysis of an ear section from a C57BL/6 mouse that was first sensitised with 2,4-dinitrofluorobenzene on the belly and then challenged 4 days later with 2,4-dinitrofluorobenzene on the ear. Haematoxylin and eosin staining shows massive skin infiltration by mononuclear cells and granulocytes 24 h after challenge. The image is from Rouzaire et al. (pp. 80-88) in which the authors show that NK cells and T cells induce different types of inflammatory reactions in the skin during recall responses to haptens; of note, NK cell-mediated pathogenesis does not rely on cellular infiltrate. A 'MÉNAGE À TROIS' IN THE GERMINAL CENTRE INVOLVING HUMAN ΓΔ T CELLS: Vγ9/Vδ2 T cells comprise a minor subset of unconventional T cells in the blood which uniquely respond to HMB-PP, a non-peptide metabolite produced by a large range of microbial pathogens. For reasons that are not understood Vγ9/Vδ2 T cells and immune responsiveness to HMB-PP are found only in higher primates and are absent in all other vertebrates including mice. In this issue, Bansal et al. demonstrate that HMB-PP induces upregulation of the IL-21 receptor on human Vγ9/Vδ2 T cells. The authors show that IL-21 plays a co-stimulatory role in the induction of CXCL13, CXCR5 and ICOS expression by HMB-PP-activated Vγ9/Vδ2 T cells and in the potential of these cells to provide B-cell help. These findings suggest that the 'ménage à trois' of Vγ9/Vδ2 T cells, T(FH) cells and B cells in the germinal centres of secondary lymphoid tissues is likely to impact on the generation of high-affinity, class-switched antibodies in microbial infections. pp. 110-119 THE HEART OF HIV-ASSOCIATED TUBERCULOSIS: Compared with those not infected with HIV, HIV-infected individuals are up to 30 times more likely to develop tuberculosis, with an increased risk of extrapulmonary disease. In this issue, Matthews et al. show that in the pericardial form of extrapulmonary tuberculosis, increased HIV replication at the tuberculous disease site leads to the elimination of the CCR5(+) effector memory CD4(+) T-cell population by the virus. This elimination of the memory population is accompanied by the recruitment of less differentiated cells to the disease site. The presence of polyfunctional CD4(+) T cells expressing TNF, IL-2 and IFN-γ is consistent with the less mature phenotype of CD4(+) memory T cells at the disease site of HIV-1-infected patients. Interestingly, and unlike other extrapulmonary tuberculous disease sites, there are instances where the compartmentalisation of antigen-specific T cells is not detectable. As this lack of compartmentalisation might limit diagnostic application in pericardial tuberculosis, the underlying reasons require further elucidation. pp. 147-157 HYBRIDS, NOT JUST FOR CARS: ΓΔ T-CELL TRANSCRIPTOME IS A HYBRID OF ΑΒ T-CELL AND NK-CELL SIGNATURES: Human γδ T cells reactive to non-peptide phosphoantigens represent promising new anti-cancer immunotherapeutics, but they are hitherto poorly characterized. In this issue, Pont et al. perform the first Affymetrix-based transcriptome analysis of human phosphoantigen-specific γδ T cells in resting and activated conditions. In comparison with a broad collection of transcriptomes from both healthy lymphocytes and lymphoma cells, phosphoantigen-specific γδ T cells are found to cluster with - and between - healthy αβ T and NK cells. γδ T cells show gene expression signatures corresponding to both αβ Th1 and NK cell functional activities, but not to phagocytic or antigen-presenting cell (APC) bioactivities. Functional studies show that Th1 cytokine, chemokine and cytotoxic activities reflect a high mitotic activity at later time points rather than antigen-presenting functions. The transcriptomes from these phosphoantigen-specific γδ T cells are therefore strikingly distinct from that of NK/T or peripheral T-cell lymphomas of the γδ subtype. pp. 228-240 NATURAL ANTIBODY-PRODUCING B-1 CELLS EXIST IN THE BONE MARROW: A small subset of B cells, B-1 cells, is thought to generate "natural antibodies" (nAbs) - antibodies that are generated without overt immunization and importantly contribute to antimicrobial defenses and tissue homeostasis. However, while nAb production occurs mainly in the spleen and bone marrow (BM), B-1 cells are most prominent in the body cavity of mice and are not thought to exist in BM. In this issue, Choi et al. resolve this apparent conundrum by identifying, for the first time, nAb-secreting B-1 cells in the BM. BM B-1 cells share many characteristics with splenic B-1 cells, but are distinct from their counterparts in body cavities, which despite their relative abundance contribute little to the nAb pools. nAb-producing B-1 cells are also distinct from conventional plasma cells. These data highlight the significant contribution of BM B-1 cells as nAb-producers and supports the view that body cavity B-1 cells function as rapidly inducible cellular reservoirs, but not pools, of nAb-producing cells. pp. 120-129 FROM OUR SISTER JOURNALS - COME ON IN! HOW ANTIBODIES CAN ALSO ACT INSIDE CELLS: It was recently discovered that - besides providing extracellular protection against pathogens - antibodies bound to adenovirus can also enter the cell and lead to the disposal of the virus intracellularly. McEwan et al. describe how this antibody-dependent intracellular neutralisation (ADIN) works: inside the cell the antibodies are recognized by the receptor TRIM21, which binds to IgG with high affinity. The binding of TRIM21 to IgG subsequently recruits the ubiquitin-proteasome system, leading to the degradation of the virus. Furthermore the authors discuss how this discovery alters our view of the way in which antibodies neutralize viral infection and what implications this may have for future research. pp. 803-809.
Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Altered intracellular localization and valosin-containing protein (p97 VCP) interaction underlie ATP7A-related distal motor neuropathy.
Source
Unit on Human Copper Metabolism, Molecular Medicine Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, MD, USA.
Abstract
ATP7A is a P-type ATPase that regulates cellular copper homeostasis by activity at the trans-Golgi network (TGN) and plasma membrane (PM), with the location normally governed by intracellular copper concentration. Defects in ATP7A lead to Menkes disease or its milder variant, occipital horn syndrome or to a newly discovered condition, ATP7A-related distal motor neuropathy (DMN), for which the precise pathophysiology has been obscure. We investigated two ATP7A motor neuropathy mutations (T994I, P1386S) previously associated with abnormal intracellular trafficking. In the patients' fibroblasts, total internal reflection fluorescence microscopy indicated a shift in steady-state equilibrium of ATP7A(T994I) and ATP7A(P1386S), with exaggerated PM localization. Transfection of Hek293T cells and NSC-34 motor neurons with the mutant alleles tagged with the Venus fluorescent protein also revealed excess PM localization. Endocytic retrieval of the mutant alleles from the PM to the TGN was impaired. Immunoprecipitation assays revealed an abnormal interaction between ATP7A(T994I) and p97/VCP, an ubiquitin-selective chaperone which is mutated in two autosomal dominant forms of motor neuron disease: amyotrophic lateral sclerosis and inclusion body myopathy with early-onset Paget disease and fronto-temporal dementia. Small-interfering RNA (SiRNA) knockdown of p97/VCP corrected ATP7A(T994I) mislocalization. Flow cytometry documented that non-permeabilized ATP7A(P1386S) fibroblasts bound a carboxyl-terminal ATP7A antibody, consistent with relocation of the ATP7A di-leucine endocytic retrieval signal to the extracellular surface and partially destabilized insertion of the eighth transmembrane helix. Our findings illuminate the mechanisms underlying ATP7A-related DMN and establish a link between p97/VCP and genetically distinct forms of motor neuron degeneration.
Production of bioactive, SUMO-modified, and native-like TNF-α of the rhesus monkey, Macaca mulatta, in Escherichia coli.
Source
Key Lab of Transplant Engineering and Immunology, Ministry of Health West China Hospital, Sichuan University, Chengdu, 610041, China.
Abstract
Biotechnologically produced tumor necrosis factor alpha (TNF-α) neutralizing agents have proven efficient in patients suffering from disparate autoimmune diseases. The rhesus monkey (Macaca mulatta) could be developed as a model for human autoimmune disease. Consequently, a large amount of M. mulatta TNF-α (mmTNFα) is required to further understand TNF-α-related pathogenesis and evaluate novel human TNF-α (hTNFα) neutralizing agents. We therefore attempted to express mmTNFα by using a small ubiquitin-like modifier (SUMO) fusion system. The synthetic gene, encoding the fusion protein SUMO-mmTNFα, was inserted into a pQE30 plasmid and was transformed into Escherichia coli M15. The fusion protein was expressed as both soluble and insoluble protein in E. coli. Approximately 10-12 mg of SUMO-mmTNFα was obtained from the soluble fraction of 1 L of bacterial culture. Cleavage of the fusion protein with SUMO protease produced native-like mmTNFα. Both native-like and SUMO-modified mmTNFα formed functional trimers and showed excellent cytotoxicity (ED(50), 0.05-0.1 ng/ml) in standard L929 cells. In addition, SUMO-mmTNFα and mmTNFα also exhibited cytotoxicity in human cancer cell types, such as, breast, lung, and liver cancer cells. The hTNFα neutralizing agents, including soluble receptors of hTNFα and antibodies against hTNFα, interacted with the mmTNFα. These results demonstrate that the bioactive mmTNFα produced with the SUMO fusion system is useful for further research, especially for the in vitro preclinical evaluation of biological hTNFα neutralizing agents.
Ubiquitin-intein and SUMO2-intein fusion systems for enhanced protein production and purification.
Source
School of Life Sciences, Tsinghua University, Beijing 100084, PR China; The Shenzhen Key Lab of Gene and Antibody Therapy, Center for Biotech and Division of Life Sciences, Graduate School at Shenzhen, Tsinghua University, Shenzhen, Guangdong 518055, China.
Abstract
Although most commonly used for protein production, expression of soluble and functional recombinant protein in Escherichia coli is still a major challenge. The development and application of fusion tags that can facilitate protein expression and solubility partly solve this problem, however, under most circumstance, the fusion tags have to be removed by proteases in order to use the proteins. Because the tag removal using proteases increases cost and introduces extra purification steps, it remains a significant problem that must be resolved before being widely used in industry production. Ubiquitin and SUMO have been successfully used to enhance protein expression and solubility. In the last decades, intein has also been widely used in protein production for its self-cleavage property, which could help to remove the fusion tag without any protease. Here, we take the advantages of ubiquitin, SUMO2 and intein in protein expression. We constructed tandem ubiquitin-intein and SUMO2-intein fusion tags, and chose human MMP13 (amino acid 104-274) and eGFP as the passenger proteins that fused to the C-terminus of the tags. These constructs were expressed in E. coli and both MMP13 and eGFP expression and solubility were evaluated. Both tags showed the ability to enhance the solubility of MMP13 and eGFP and improve the expression of eGFP, and the SUMO2-intein having a more significant effect. Both ubiquitin-intein-eGFP and SUMO2-intein-eGFP were purified using Ni-NTA column chromatography and self-cleavaged by changing pH. The recombinant un-tagged eGFP were released and eluted with high homogeneity. In summary, ubiquitin-intein and SUMO2-intein are convenient and useful fusion tags that can enhance the expression, solubility and improve the purification process of the model heterologous protein and these tags may have a good prospect in protein production.
Copyright © 2011 Elsevier Inc. All rights reserved.
Expression of the novel human gene, UBE2Q1, in breast tumors.
Source
Department of Biochemistry, Shiraz University of Medical Sciences, P.O. Box 1167, Shiraz, Iran.
Abstract
The novel human gene, designated ubiquitin-conjugating enzyme E2Q family member 1 (UBE2Q1) maps to chromosome 1q21.3. The gene has an open reading frame corresponding to 422 amino acids and contains a RWD domain and an E2 ubiquitin conjugating enzyme domain. Here, we investigated the expression levels of both mRNA and protein of UBE2Q1 gene in cancerous versus normal parts of breast specimens from 26 patients. Real-time PCR data showed that the relative expression level of UBE2Q1 mRNA was significantly greater in cancers than in non-cancerous tissues of breast specimens (Mean ± SEM, 0.064 ± 0.015 for cancers and 0.026 ± 0.01 for noncancerous tissues, P < 0.05 Mann-Whitney test). A rabbit polyclonal antibody was generated against an amino acid sequence predicted from the DNA sequence of UBE2Q1 gene. This antibody was used to perform Western blotting on 21 cases in our cohort of breast specimens. Thus, 13 (61.904%) of the cases showed an increase in the UBE2Q1 immunoreactivity in their cancerous tissues as compared with the corresponding normal tissues. This result along with the real-time PCR data shows that the novel human gene, UBE2Q1, is expressed in human breast and may have implications for pathogenesis of breast cancer.
Profiling of autoantibodies in IgA nephropathy, an integrative antibiomics approach.
Source
Departments of Pediatrics-Nephrology, Stanford University School of Medicine, Stanford, CA 94304, USA.
Abstract
BACKGROUND AND OBJECTIVES:
IgG commonly co-exists with IgA in the glomerular mesangium of patients with IgA nephropathy (IgAN) with unclear clinical relevance. Autoantibody (autoAb) biomarkers to detect and track progression of IgAN are an unmet clinical need. The objective of the study was to identify IgA-specific autoAbs specific to IgAN.
DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS:
High-density protein microarrays were evaluated IgG autoAbs in the serum of IgAN patients (n = 22) and controls (n = 10). Clinical parameters, including annual GFR and urine protein measurements, were collected on all patients over 5 years. Bioinformatic data analysis was performed to select targets for further validation by immunohistochemistry (IHC).
RESULTS:
One hundred seventeen (1.4%) specific antibodies were increased in IgAN. Among the most significant were the autoAb to the Ig family of proteins. IgAN-specific autoAbs (approximately 50%) were mounted against proteins predominantly expressed in glomeruli and tubules, and selected candidates were verified by IHC. Receiver operating characteristic analysis of our study demonstrated that IgG autoAb levels (matriline 2, ubiquitin-conjugating enzyme E2W, DEAD box protein, and protein kinase D1) might be used in combination with 24-hour proteinuria to improve prediction of the progression of IgAN (area under the curve = 0.86, P = 0.02).
CONCLUSIONS:
IgAN is associated with elevated IgG autoAbs to multiple proteins in the kidney. This first analysis of the repertoire of autoAbs in IgAN identifies novel, immunogenic protein targets that are highly expressed in the kidney glomerulus and tubules that may bear relevance in the pathogenesis and progression of IgAN.
Congenital cataract causing mutants of αA-crystallin/sHSP form aggregates and aggresomes degraded through ubiquitin-proteasome pathway.
Source
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States of America.
Abstract
BACKGROUND:
Mutations of human αA-crystallin cause congenital cataract by protein aggregation. How mutations of αA-crystallin cause disease pathogenesis through protein aggregation is not well understood. To better understand the cellular events leading to protein aggregation, we transfected cataract causing mutants, R12C, R21L, R21W, R49C, R54C, R116C and R116H, of human αA-crystallin in HeLa cells and examined the formation of intracellular protein aggregates and aggresomes by confocal microscopy.
METHODOLOGY/PRINCIPAL FINDINGS:
YFP-tagged human αA-wild-type (αA-wt) was sub-cloned and the mutants were generated by site-directed mutagenesis. The αA-wt and the mutants were individually transfected or co-transfected with CFP-tagged αA-wt or αB-wild-type (αB-wt) in HeLa cells. Overexpression of these mutants forms multiple small dispersed cytoplasmic aggregates as well as aggresomes. Co-expression of αB-wt with these mutants significantly inhibited protein aggregates where as co-expression with αA-wt enhanced protein aggregates which seems to be due to co-aggregation of the mutants with αA-wt. Aggresomes were validated by double immunofluorescence by co-localization of γ-tubulin, a centrosome marker protein with αA-crystallin. Furthermore, increased ubiquitination was detected in R21W, R116C and R116H as assessed by western blot analyses. Immunostaining with an ubiquitin antibody revealed that ubiquitin inclusions in the perinuclear regions were evident only in R116C transfected cells. Pulse chase assay, after cycloheximide treatment, suggested that R116C degraded faster than the wild-type control.
CONCLUSIONS/SIGNIFICANCE:
Mutants of αA-crystallin form aggregates and aggresomes. Co-expression of αA-wt with the mutants increased aggregates and co-expression of αB-wt with the mutants significantly decreased the aggregates. The mutant, R116C protein degraded faster than wild-type control and increased ubiquitination was evident in R116C expressing cells.
Ubiquitin conjugation of hepatitis B virus core antigen DNA vaccine leads to enhanced cell-mediated immune response in BALB/c mice.
Source
Department of Infectious Diseases, Sixth People's Hospital Affiliated to Shanghai Jiaotong University, Shanghai, China.
Abstract
BACKGROUND:
Nearly 350 million persons worldwide are chronically infected with hepatitis B virus (HBV). Ubiquitin(Ub) is a highly conserved small regulatory protein, ubiquitous in eukaryotes, that usually serves as a signal for the target protein that is recognised and degraded in proteasomes . The Ub-mediated processing of antigens is rapid and efficient and stimulates cell-mediated immune responses. Accordingly, Ub-mediated processing of antigens has been widely used in chronic-infection and cancer studies to improve immune response.
OBJECTIVES:
Many clinical trials have shown that DNA vaccine potency needs to be greatly enhanced. Here, we report a new strategy for designing an HBV DNA vaccine using the ubiquitin (Ub) sequence. The aim of this study was to investigate a novel DNA vaccination, based on the expression of HBV core antigen (HBcAg), fused to Ub to enhance DNA vaccine potency.
MATERIALS AND METHODS:
Mouse ubiquitin fused to the HBcAg gene and cloned into the eukaryotic vector pcDNA3.1 (-). BALB/c mice were immunized with recombinant pUb-HBcAg or pHBcAg DNA vaccine. Lymphocyte proliferation assay, intracellular IFN-γ assay, CTL cytotoxicity assay, and antibody assay were performed to analyze the cellular and humoral immune responses to our DNA constructs.
RESULTS:
HBcAg was expressed effectively in the COS-7 cells that were transiently transfected with pUb-HBcAg. Strong anti-HBc IgG responses were elicited in mice that were immunized with pUb-HBcAg. The endpoint titers of anti-HBc peaked at 1:656100 on the 42nd day after the third immunization. pUb-HBcAg stimulated greater lymphocyte proliferation and induced higher levels of IL-2 and IFN-γ and a greater percentage of HBcAg-specific CD8+ T cells in mice than pHBcAg. In the CTL assay, the specific lysis rate reached 56.5% at an effector:target ratio of 50:1 in mice that were immunized with pUb-HBcAg.
CONCLUSIONS:
pUb-HBcAg elicits specific anti-HBc responses and induces HBc-specific CTL responses in immunized BALB/c mice. Our results imply that Ub can be used as a molecular adjuvant that enhances the potency of DNA vaccines.
miR-135A regulates preimplantation embryo development through down-regulation of E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (SIAH1A) expression.
Source
Department of Obstetrics and Gynaecology, The University of Hong Kong, Pokfulam, Hong Kong, People's Republic of China.
Abstract
BACKGROUND:
MicroRNAs (miRNAs) are small non-coding RNA molecules capable of regulating transcription and translation. Previously, a cluster of miRNAs that are specifically expressed in mouse zygotes but not in oocytes or other preimplantation stages embryos are identified by multiplex real-time polymerase chain reaction-based miRNA profiling. The functional role of one of these zygote-specific miRNAs, miR-135a, in preimplantation embryo development was investigated.
METHODOLOGY/PRINCIPAL FINDINGS:
Microinjection of miR-135a inhibitor suppressed first cell cleavage in more than 30% of the zygotes. Bioinformatics analysis identified E3 Ubiquitin Ligase Seven In Absentia Homolog 1A (Siah1a) as a predicted target of miR-135a. Western blotting and 3'UTR luciferase functional assays demonstrated that miR-135a down-regulated the expression of Siah1 in HeLa cells and in mouse zygotes. Siah1a was expressed in preimplantation embryos and its expression pattern negatively correlated with that of miR-135a. Co-injection of Siah1a-specific antibodywith miR-135a inhibitor partially nullified the effect of miR-135a inhibition. Proteasome inhibition by MG-132 revealed that miR-135a regulated proteasomal degradation and potentially controlled the expression of chemokinesin DNA binding protein (Kid).
CONCLUSIONS/SIGNIFICANCE:
The present study demonstrated for the first time that zygotic specific miRNA modulates the first cell cleavage through regulating expression of Siah1a.
Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen and Angiogenin Interact with Common Host Proteins, Including Annexin A2, Which Is Essential for Survival of Latently Infected Cells.
Source
Address correspondence to Bala Chandran, bala.chandran@rosalindfranklin.edu.
Abstract
Kaposi's sarcoma-associated herpesvirus (KSHV) infection and latency-associated nuclear antigen (LANA-1) upregulate the multifunctional protein angiogenin (ANG). Our studies demonstrate that silencing ANG or inhibiting its nuclear translocation downregulates KSHV LANA-1 expression and ANG is necessary for KSHV latency, anti-apoptosis and angiogenesis (Sadagopan et al., J. Virol. 83:3342-3364, 2009; Sadagopan et al., J Virol. 85:2666-2685, 2011). Here we show that LANA-1 interacts with ANG and colocalizes in latently infected endothelial telomerase-immortalized human umbilical vein endothelial (TIVE-LTC) cells. Mass spectrometric analyses of TIVE-LTC proteins immunoprecipitated by anti-LANA-1 and ANG antibodies identified 28 common cellular proteins such as ribosomal proteins, structural proteins, tRNA synthetases, metabolic pathway enzymes, chaperons, transcription factors, antioxidants, and ubiquitinproteosome proteins. LANA-1 and ANG interaction with one of the proteins, annexin A2, was validated. Annexin A2 has been shown to play roles in cell proliferation, apoptosis, plasmin generation, exocytosis, endocytosis, and cytoskeleton reorganization. It is also known to associate with glycolytic enzyme 3-phosphoglyceratekinase in the primer recognition protein (PRP) complex that interacts with DNA polymerase α in the lagging strand of DNA during replication. A higher level of annexin A2 is expressed in KSHV(+) but not in Epstein-Barr virus (EBV)(+) B-lymphoma cell lines. Annexin A2 colocalized with several LANA-1 punctate spots in KSHV(+) body cavity B-cell lymphoma (BCBL-1) cells. In triple-staining analyses, we observed annexin A2-ANG-LANA-1, annexin A2-ANG, and ANG-LANA-1 colocalizations. Annexin A2 appeared as punctate nuclear dots in LANA-1-positive TIVE-LTC cells. In LANA-1-negative TIVE-LTC cells, annexin A2 was detected predominately in the cytoplasm, with some nuclear spots, and colocalization with ANG was observed mostly in the cytoplasm. Annexin A2 coimmunoprecipitated with LANA-1 and ANG in TIVE-LTC and BCBL-1 cells and with ANG in 293T cells independent of LANA-1. This suggested that annexin A2 forms a complex with LANA-1 and ANG as well as a separate complex with ANG. Silencing annexin A2 in BCBL-1 cells resulted in significant cell death, downregulation of cell cycle-associated Cdk6 and of cyclin D, E, and A proteins, and downregulation of LANA-1 and ANG expression. No effect was seen in KSHV(-) lymphoma (BJAB and Ramos) and 293T cells. These studies suggest that LANA-1 association with annexin A2/ANG could be more important than ANG association with annexin A2, and KSHV probably uses annexin A2 to maintain the viability and cell cycle regulation of latently infected cells. Since the identified LANA-1- and ANG-interacting common cellular proteins are hitherto unknown to KSHV and ANG biology, this offers a starting point for further analysis of their roles in KSHV biology, which may lead to identification of potential therapeutic targets to control KSHV latency and associated malignancies.
Immunohistochemical analysis of Marinesco bodies, using antibodies against proteins implicated in the ubiquitin-proteasome system, autophagy and aggresome formation.
Source
Departments of Neuropathology Neuroanatomy, Cell Biology and Histology, Hirosaki University Graduate School of Medicine, Hirosaki Department of Pathological Neuroscience, Center for Bioresource-based Researches Department of Pathology, Brain Research Institute, University of Niigata, Niigata, Japan Center for Molecular Chaperone, Medical College of Georgia, Augusta, GA, USA.
Abstract
Marinesco bodies (MBs) are spherical eosinophilic intranuclear inclusions in pigmented neurons in the substantia nigra and locus ceruleus. Previous immunohistochemical studies have shown that MBs are positive for ubiquitin, p62 and SUMO-1, suggesting the involvement of ubiquitination and related proteins in the formation or disaggregation of MBs. However, the involvement is not thoroughly understood. Therefore, we immunohistochemically examined the midbrain from five control subjects ranged from 53 to 84 years old. MBs were positive for various proteins implicated in theubiquitin-proteasome system (ubiquitin, p62, EDD1, NEDD8, NUB1, SUMO-1 and SUMO-2), aggresome formation (HDAC6) and autophagy (ubiquitin, p62, LC3, GABARAP and GATE-16). These findings suggest that proteins related to ubiquitination, proteasomal degradation and autophagy are involved in the formation or disaggregation of MBs.
© 2011 Japanese Society of Neuropathology.
Immune Responses in Wild-type Mice Against Prion Proteins Induced Using a DNA Prime-Protein Boost Strategy.
Source
State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China; Medical School, Anhui University of Science and Technology, Huainan 231001, China.
Abstract
OBJECTIVE:
To break immune tolerance to prion (PrP) proteins using DNA vaccines.
METHODS:
Four different human prion DNA vaccine candidates were constructed based on the pcDNA3.1 vector: PrP-WT expressing wild-type PrP, Ubiq-PrP expressing PrP fused to ubiquitin, PrP-LII expressing PrP fused to the lysosomal integral membrane protein type II lysosome-targeting signal, and PrP-ER expressing PrP locating the ER. Using a prime-boost strategy, three-doses of DNA vaccine were injected intramuscularly into Balb/c mice, followed by two doses of PrP protein. Two weeks after the last immunization, sera and spleens were collected and PrP-specific humoral and cellular immune responses evaluated by ELISA and ELISPOT tests.
RESULTS:
Higher levels of serum PrP antibodies were detected in mice vaccinated using the strategy of DNA priming followed by protein boosting. Of these, WT-PrP, Ubiq-PrP, and PrP-LII induced significantly higher humoral responses. ELISPOT tests showed markedly increased numbers of IFN-γ-secreting T cells in mice vaccinated using the strategy of DNA priming followed by protein boosting after stimulation with recombinant PrP23-90 and PrP23-231. PrP-ER induced the strongest T-cell response.
CONCLUSION:
Prion vaccines can break tolerance to PrP proteins and induce PrP-specific humoral and cellular immune responses.
Copyright © 2011 The Editorial Board of Biomedical and Environmental Sciences. Published by Elsevier B.V. All rights reserved.
Diagnostic value of markers of muscle degeneration in sporadic inclusion body myositis.
Source
Laboratoire de Neuropathologie, Institut de Myologie, Assistance Publique, Hôpitaux de Paris, Hôpital Pitié-Salpêtrière, Université Pierre et Marie Curie, Paris, France. odile.dubourg@psl.aphp.fr
Abstract
Sporadic inclusion body myositis (s-IBM) is characterized histologically by the association of concomitant inflammatory and degenerative processes. We evaluated the sensitivity and specificity of different markers of the degenerative process in order to refine the histological diagnosis. We performed an immunohistochemical study with antibodies directed against ubiquitin, amyloid-beta precursor protein (AbetaPP), amyloid-beta (Abeta), SMI-31, SMI-310, Tar-DNA binding protein-43 (TDP-43) and p62 on s-IBM and control muscle biopsies. Based on conventional stains 36 patients with characteristic clinical features of s-IBM were subclassified as presumed definite s-IBM (d s-IBM, n = 17) or possible s-IBM (p s-IBM, n = 19) according to the presence or absence of vacuolated muscle fibers. Immunohistochemically, TDP-43 and p62 were the most sensitive markers, accumulating in all d s-IBM and in 31% and 37%, respectively, of the p s-IBM cases and thus enabling reclassification of these cases as d s-IBM. We recommend using TDP-43 and p62antibodies in the histological diagnosis workup of s-IBM. The specificity of these markers has to be further validated in prospective series.
UHRF1 phosphorylation by cyclin A2/cyclin-dependent kinase 2 is required for zebrafish embryogenesis.
Source
Division of Pediatric Hepatology, Department of Pediatrics, Mount Sinai School of Medicine, New York, NY 10029 Division of Liver Diseases, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029 Division of Gastroenterology, Hepatology and Endoscopy, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115.
Abstract
Ubiquitin-like, containing PHD and RING finger domains 1 (uhrf1) is regulated at the transcriptional level during the cell cycle and in developing zebrafish embryos. We identify phosphorylation as a novel means of regulating UHRF1 and demonstrate that Uhrf1 phosphorylation is required for gastrulation in zebrafish. Human UHRF1 contains a conserved cyclin-dependent kinase 2 (CDK2) phosphorylation site at Ser-661 that is phosphorylated in vitro by CDK2 partnered with cyclin A2 (CCNA2), but not cyclin E. An antibody specific for phospho-Ser-661 recognizes UHRF1 in both mammalian cancer cells and in nontransformed zebrafish cells, but not in zebrafish bearing a mutation in ccna2. Depleting Uhrf1 from zebrafish embryos by morpholino injection causes arrest before gastrulation and early embryonic death. This phenotype is rescued by wild-type UHRF1, but not by UHRF1 in which the phospho-acceptor site is mutated, demonstrating that UHRF1 phosphorylation is essential for embryogenesis. UHRF1 was detected in the nucleus and cytoplasm, whereas nonphosphorylatable UHRF1 is unable to localize to the cytoplasm, suggesting the importance of localization in UHRF1 function. Together, these data point to an essential role for UHRF1 phosphorylation by CDK/CCNA2 during early vertebrate development.
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