Suppression of TCAB1 expression induced cellular senescence by lessening proteasomal degradation of p21 in cancer cells.

BACKGROUND: TCAB1, a.k.a. WRAP53ß or WDR79, is an important molecule for the maintenance of Cajal bodies and critically involved in telomere elongation and DNA repair. Upregulation of TCAB1 were discovered in a variety types of cancers. However, the function of TCAB1 in tumor cell senescence remains absent. METHODS: The TCAB1 knockdown cell lines were constructed. The expression levels of TCAB1, p21, p16 and p53 were detected by qRT-PCR and western blotting. Staining of senescence-associated ß-galactosidase was used to detect senescent cells. The ubiquitination of the p21 was analysed by immunoprecipitation and in vivo ubiquitination assay. TCGA databases were employed to perform in silico analyses for the mRNA expression of TCAB1, p21, p16 and p53. RESULTS: Here, we discovered that knockdown of TCAB1 induced rapid progression of cellular senescence in A549, H1299 and HeLa cells. In exploiting the mechanism underlining the role of TCAB1 on senescence, we found a significant increase of p21 at the protein levels upon TCAB1 depletion, whereas the p21 mRNA expression was not altered. We verified that TCAB1 knockdown was able to shunt p21 from proteasomal degradation by regulating the ubiquitination of p21. In rescue assays, it was demonstrated that decreasing the expression of p21 or increasing the expression of TCAB1 were able to attenuate the cellular senescence process induced by TCAB1 silencing. CONCLUSIONS: This study revealed the importance of TCAB1 for its biological functions in the regulation of cell senescence. Our results will be helpful to understand the mechanisms of senescence in cancer cells, which could provide clues for designing novel strategies for developing effective treatment regimens.

Engineering and characterization of a humanized antibody targeting TNF-α and RANKL.

To neutralize the pathological activities of tumor necrosis factor-α (TNF-α) and receptor activator of NF-κB ligand (RANKL), we engineered and characterized a humanized 8G12 (h8G12) antibody that targeted TNF-α and RANKL. Standard molecular biological and complementarity determining region (CDR)-grafting techniques were used to engineer the h8G12 antibody, and enzyme-linked immunosorbent assays (ELISAs) and Western blotting were employed to determine its binding activation and specificity. TNF-α-mediated cytotoxicity and RANKL-induced osteoclastogenesis assays were used to evaluate the neutralizing effects of the antibody. The cDNA sequences were established by grafting the murine monoclonal antibody (mAb) 8G12 CDRs into the heavy and light chain (HC and LC) variable regions (VH and VL) of the human mAbs 3DGG_B and 1I9R_L, respectively. The recombinant plasmids were transfected into Chinese hamster ovary (CHO) cells to produce the h8G12 antibody, which could simultaneously recognize TNF-α and RANKL. In addition, the h8G12 antibody reduced the TNF-α-mediated apoptosis of L929 cells by 25.84%. Furthermore, the h8G12 antibody significantly inhibited leukocyte infiltration in a murine allergic contact inflammation model. Concurrent with the inhibition of apoptosis, the h8G12 antibody significantly reduced the number of osteoclast-like cells in a dose-dependent manner. These results demonstrated that the h8G12 antibody neutralized the activities of TNF-α and RANKL and that it might be a potential candidate for the treatment of inflammatory bone diseases, such as rheumatoid arthritis (RA).

The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway.

Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity of Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice.

Baicalin protects mice from Staphylococcus aureus pneumonia via inhibition of the cytolytic activity of α-hemolysin.

α-Hemolysin (Hla) is a self-assembling, channel-forming toxin that is secreted by Staphylococcus aureus and is central to the pathogenesis of pulmonary, intraperitoneal, intramammary, and corneal infections in animal models. In this study, we report that baicalin (BAI), a natural compound that lacks anti-S. aureus activity, could inhibit the hemolytic activity of Hla. Using molecular dynamics simulations and mutagenesis assays, we further demonstrate that BAI binds to the binding sites of Y148, P151, and F153 in the Hla. This binding interaction inhibits heptamer formation. Furthermore, when added to S. aureus cultures, BAI prevents Hla-mediated human alveolar epithelial (A549) cell injury. In vivo studies further demonstrated that BAI protects mice from S. aureus pneumonia. These findings indicate that BAI hinders the cell lysis activity of Hla through a novel mechanism of interrupting the formation of heptamer, which may lead to the development of novel therapeutics that aim against S. aureus Hla.

Structure of the branched-chain aminotransferase from Streptococcus mutans.

The branched-chain amino-acid aminotransferase from Streptococcus mutans (SmIlvE) was recombinantly expressed in Escherichia coli with high yield. An effective purification protocol was established. A bioactivity assay indicated that SmIlvE had aminotransferase activity. The specific activity of SmIlvE towards amino-acid substrates was found to be as follows (in descending order): Ile > Leu > Val > Trp > Gly. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. The structure of SmIlvE was solved at 1.97 Å resolution by the molecular-replacement method. Comparison with structures of homologous proteins enabled the identification of conserved structural elements that might play a role in substrate binding. Further work is needed to confirm the interaction between SmIlvE and its substrates by determining the structures of their complexes.

Crystallization and preliminary X-ray analysis of S-ribosylhomocysteinase from Streptococcus mutans.

S-Ribosylhomocysteinase (LuxS) encoded by the luxS gene from Streptococcus mutans plays a crucial role in the quorum-sensing system. LuxS was solubly expressed in Escherichia coli with high yield. The purity of the purified target protein, which was identified by SDS-PAGE and MALDI-TOF MS analysis, was >95%. The protein was crystallized using the hanging-drop vapour-diffusion method with PEG 3350 as the primary precipitant. X-ray diffraction data were collected at Beijing Synchrotron Radiation Facility (BSRF). Diffraction by the crystal extended to 2.4 Å resolution and the crystal belonged to space group C222(1), with unit-cell parameters a = 55.3, b = 148.7, c = 82.8 Å.

[Status of endothelial progenitor cell in murine model of Kawasaki disease].

OBJECTIVE: To assess whether the occurrence of coronary artery lesion was correlated with the changes of endothelial progenitor cell (EPC) number and function in murine model of Kawasaki disease (KD). METHODS: Lactobacillus casei cell wall extract (LCWE) was prepared and then C57BL/6 mice received a single intraperitoneal injection of LCWE for inducing KD. Twenty-four mice were categorized randomly into 3 groups: KD model group at Day 14 post-injection, KD model group at Day 56 post-injection and control group with an intraperitoneal injection of phosphate buffered solution (n = 8 each). The number of circulating EPC was defined as CD34(+)Flk-1(+)CD45(-) from mice. Meanwhile, bone marrow mononuclear cells were cultured in vitro to expand EPC for functional analysis. After 7 days of culturing, EPC were inoculated onto culture plate and thiazolyl blue assay was used to measure the absorbance value by enzyme labeling instrument to evaluate the proliferation. The adhesion of EPC was performed by replating cells on fibronectin coated dishes and then counting the number of adherent cells. The migration of EPC was assayed by Transwell. RESULTS: Focal inflammatory infiltrate was evident in coronary artery trunk and a series of branches at Day 14 post-injection. The inflammatory cell infiltrate consisted of mononuclear lymphocytes. The number of circulating EPC were significantly lower in the Day 14 LCWE-treating murine model versus the controls (0.017% ± 0.008% vs 0.028% ± 0.007%, P < 0.01). Disruption of elastin was consistently observed at Day 56 post-injection. And there was no apparent recovery in number of EPC (0.016% ± 0.007%, P < 0.01). When bone marrow mononuclear cells were cultured in vitro, the colony-forming ability of EPC decreased in the KD model group at Day 14 post-injection versus the controls. Test of proliferating ability showed that the absorbance was 0.39 ± 0.11 in MTT experiment and decreased than the controls (0.61 ± 0.14, P < 0.01). Adhesion and migration were also down-regulated versus the controls ((3.1 ± 0.6) and (3.2 ± 0.6) vs (6.4 ± 1.2) and (6.2 ± 0.5) cells/HPF, both P < 0.01). In the KD model group at Day 56 post-injection, the colony-forming ability of EPC was not recovered significantly. Proliferation ability, adhesion and migration were still decreased compared to the controls (0.38 ± 0.09, (3.12 ± 0.56) cells/HPF and (3.29 ± 0.63) cells/HPF, all P < 0.01). CONCLUSION: The occurrence of coronary artery lesion may be correlated with the down-regulation of EPC number and function in murine model of KD.

Expression, crystallization and preliminary X-ray analysis of the phosphoribosylglycinamide formyltransferase from Streptococcus mutans.

Phosphoribosylglycinamide formyltransferase (PurN) from Streptococcus mutans was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The purified protein, which had a purity of >95%, was identified by SDS-PAGE and MALDI-TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 3350 as the primary precipitant. X-ray diffraction data were collected to 2.1 Šresolution. Preliminary X-ray analysis indicated that the crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 52.25, b = 63.29, c = 131.81 Å.

High-yield expression, purification, characterization, and structure determination of tag-free Candida utilis uricase.

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2(1)2(1)2(1) with unit cell parameters a = 69.16 Å, b = 139.31 Å, c = 256.33 Å, and α = ß = γ = 90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.

Purification, characterization, and crystallization of the adhesive domain of SdrD from Staphylococcus aureus.

The adhesive domain of SdrD from Staphylococcus aureus was solubly expressed in Escherichia coli in high yield. After a series of purification steps, the purified protein was >95% pure, which was SdrD from S. aureus identified by SDS-PAGE and MALDI-TOF MS. Crystals were grown at 18 degrees C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystal extends to 1.65A resolution, and the crystal belongs to the space group C2, with the unit cell parameters a=133.3, b=58.3, c=112.3A, alpha=90.00, beta=111.14, gamma=90.00.

Characterization, crystallization and preliminary X-ray analysis of the adhesive domain of SdrE from Staphylococcus aureus.

The adhesive domain of SdrE from Staphylococcus aureus was recombinantly expressed in Escherichia coli. The purified protein was identified by SDS-PAGE and MALDI-TOF MS. The protein was crystallized using the vapour-diffusion method in hanging-drop mode with PEG 8000 as the primary precipitating agent. X-ray diffraction data were collected to 1.8 A resolution from a single crystal of the protein. Preliminary X-ray analysis indicated that the crystal belonged to space group P1, with unit-cell parameters a = 40.714, b = 66.355, c = 80.827 A, alpha = 111.19, beta = 93.99, gamma = 104.39 degrees.

Effect of miR-184 on Proliferation and Apoptosis of Pancreatic Ductal Adenocarcinoma and Its Mechanism.

OBJECTIVE: Previous studies have shown that abnormal expression of microRNA-184 leads to a variety of cancers, including pancreatic ductal adenocarcinoma, suggesting microRNA-184 as a new treatment target for pancreatic ductal adenocarcinoma. However, the molecular mechanism of microRNA-184 in pancreatic ductal adenocarcinoma remains unclear. It is important to investigate the effect and role of microRNA-184 in pancreatic ductal adenocarcinoma. METHODS: The clinical and laboratory inspection data of 120 patients with pancreatic cancer admitted to the First Affiliated Hospital of Anhui Medical University were compared. MicroRNA-184 expression in tumor tissues and cells was evaluated using reverse transcription polymerase chain reaction. Flow cytometry and Annexin V/propidium iodide staining were performed to examine cell cycle and apoptosis. Western blotting analysis was conducted to measure the protein expression of p-PI3K, p-AKT, JNK1, C-Myc, C-Jun, caspase-9, and caspase-3. RESULTS: MicroRNA-184 expression was low in patients with pancreatic ductal adenocarcinoma. Survival curve showed that patients with lower expression of microRNA-184 in tumor tissues had a worse prognosis and shorter survival time (P < .05), and the multivariate analysis identified that microRNA-184 was an independent prognostic indicator (P < .05). In vitro studies showed that microRNA-184 overexpression induced apoptosis and suppressed cell cycle transition from G1 to S and G2 phases in pancreatic ductal adenocarcinoma cells. Furthermore, molecular studies revealed that inhibition of microRNA-184 promoted the gene expression of p-PI3K, p-AKT, JNK1, C-Myc, and C-Jun compared with the control group. Overexpression of microRNA-184 led to significantly increased expression of caspase-9 and caspase-3 and significantly decreased expression of Bcl-2. CONCLUSION: This study suggests that microRNA-184 inhibits the proliferation and promotes the apoptosis of pancreatic ductal adenocarcinoma cells by downregulating the expression of C-Myc, C-Jun, and Bcl-2. Our verification of the role of microRNA-184 may provide a novel biomarker for the diagnosis, therapy, and prognosis of pancreatic ductal adenocarcinoma.

Crystal structure of the aromatic-amino-acid aminotransferase from Streptococcus mutans.

Streptococcus mutans, a facultatively aerobic and Gram-positive bacterium, is the primary causative agent of dental caries and contributes to the multispecies biofilm known as dental plaque. In this study, the aromatic-amino-acid aminotransferase from Streptococcus mutans (SmAroAT) was recombinantly expressed in Escherichia coli. An effective purification protocol was established. The recombinant protein was crystallized using the hanging-drop vapor-diffusion method with PEG 3350 as the primary precipitant. The crystal structure of SmAroAT was solved at 2.2 Šresolution by the molecular-replacement method. Structural analysis indicated that the proteins of the aromatic-amino-acid aminotransferase family have conserved structural elements that might play a role in substrate binding. These results may help in obtaining a better understanding of the catabolism and biosynthesis of aromatic amino acids.

Adeno-associated virus 2 bound to its cellular receptor AAVR.

Adeno-associated virus (AAV) is a leading vector for virus-based gene therapy. The receptor for AAV (AAVR; also named KIAA0319L) was recently identified, and the precise characterization of AAV-AAVR recognition is in immediate demand. Taking advantage of a particle-filtering algorithm, we report here the cryo-electron microscopy structure of the AAV2-AAVR complex at 2.8 Å resolution. This structure reveals that of the five Ig-like polycystic kidney disease (PKD) domains in AAVR, PKD2 binds directly to the spike region of the AAV2 capsid adjacent to the icosahedral three-fold axis. Residues in strands B and E, and the BC loop of AAVR PKD2 interact directly with the AAV2 capsid. The interacting residues in the AAV2 capsid are mainly in AAV-featured variable regions. Mutagenesis of the amino acids at the AAV2-AAVR interface reduces binding activity and viral infectivity. Our findings provide insights into the biology of AAV entry with high-resolution details, providing opportunities for the development of new AAV vectors for gene therapy.

Divergent engagements between adeno-associated viruses with their cellular receptor AAVR.

Adeno-associated virus (AAV) receptor (AAVR) is an essential receptor for the entry of multiple AAV serotypes with divergent rules; however, the mechanism remains unclear. Here, we determine the structures of the AAV1-AAVR and AAV5-AAVR complexes, revealing the molecular details by which PKD1 recognizes AAV5 and PKD2 is solely engaged with AAV1. PKD2 lies on the plateau region of the AAV1 capsid. However, the AAV5-AAVR interface is strikingly different, in which PKD1 is bound at the opposite side of the spike of the AAV5 capsid than the PKD2-interacting region of AAV1. Residues in strands F/G and the CD loop of PKD1 interact directly with AAV5, whereas residues in strands B/C/E and the BC loop of PKD2 make contact with AAV1. These findings further the understanding of the distinct mechanisms by which AAVR recognizes various AAV serotypes and provide an example of a single receptor engaging multiple viral serotypes with divergent rules.

Crystal structure of human transgelin.

Transgelin (TAGLN), also known as smooth muscle protein 22 (SM22), is a highly conserved protein found in smooth muscle tissues of adult vertebrates. Abolition of transgelin gene expression by the oncogenic Ras may be an important early event in tumor progression and a diagnostic marker for breast and colon cancer development. Transgelin contains a single calponin homology (CH) domain. However, the question of whether this single CH domain can bind actin remains open. Here we report the 2.3 A resolution crystal structure of full length human transgelin, whose main structural feature is confirmed to be a CH domain. Secondary structures of CH domains from different proteins were analyzed and conserved residues were identified that maintain similar tertiary structures.

The crystal structure of MCAT from Mycobacterium tuberculosis reveals three new catalytic models.

The malonyl coenzyme A (CoA)-acyl carrier protein (ACP) transacylase (MCAT) plays a key role in cell wall biosynthesis in Mycobacterium tuberculosis and other bacteria. The M. tuberculosis MCAT (MtMCAT) is encoded by the FabD gene and catalyzes the transacylation of malonate from malonyl-CoA to holo-ACP. Malonyl-ACP is the substrate in fatty acid biosynthesis and is a by-product of the transacylation reaction. This ability for fatty acid biosynthesis enables M. tuberculosis to survive in hostile environments, and thus understanding the mechanism of biosynthesis is important for the design of new anti-tuberculosis drugs. The 2.3 A crystal structure of MtMCAT reported here shows that its catalytic mechanism differs from those of ScMCAT and EcMCAT, whose structures have previously been determined. In MtMCAT, the C(beta)-O(gamma) bond of Ser91 turns upwards, resulting in a different orientation and thus an overall change of the active pocket compared to other known MCAT enzymes. We identify three new nucleophilic attack chains from the MtMCAT structure: His90-Ser91, Asn155-Wat6-Ser91 and Asn155-His90-Ser91. Enzyme activity assays show that His90A, Asn155A and His90A-Asn155A mutants all have substantially reduced MCAT activity, indicating that M. tuberculosis MCAT supports a unique means of proton transfer. Furthermore, His194 cannot form part of a His-Ser catalytic dyad and only stabilizes the substrate. This new discovery should provide a deeper insight into the catalytic mechanisms of MCATs.

Transfer of multiple loci of donor's genes to induce recipient tolerance in organ transplantation.

Donor organ rejection remains a significant problem. The present study aimed to assess whether transferring a donor's major histocompatibility complex (MHC) genes to the recipient could mitigate rejection in organ transplantation. Seven loci of MHC genes from donor mice were amplified and ligated into vectors; the vectors either contained one K locus, seven loci or were empty (control). The vectors were subsequently injected into the thymus of recipients (in heterotransplants, recipient rats received the vector containing one K locus), following which donor mouse hearts were transplanted. Following the transplantation of allograft and heterograft, electrocardiosignals were viable for a significantly longer duration in recipient mice and rats receiving the donor histocompatibility-2 complex (H-2)d genes compared with those in controls, and in mice that received seven vectors compared with those receiving one vector. Mixed lymphocyte cultures containing cells from these recipients proliferated significantly less compared with mixed lymphocyte cultures containing controls. Also, hearts from H-2d genes-treated recipients demonstrated less lymphocyte infiltration and necrosis compared with the control recipient. The present study concluded that allograft and heterograft rejection may be mitigated by introducing the donor's MHC into the recipient; transferring seven loci has been demonstrated to be more effective than transferring one locus.

Crystallization and preliminary crystallographic analysis of transgelin.

Transgelin was solubly expressed in E. coli. Crystals of transgelin have been grown at 291K using sodium formate or PEG-4000 as precipitants. X-ray diffraction by the crystal extends to 2.3 A resolution. The crystal belongs to the space group P2(1), with the unit cell parameters a=39.3, b=61.9, c=56.0 A and beta=90.2 degrees .

Protective effects of overexpression TCR Vbeta5.2-HSP70 and TCR Vbeta8.2-HSP70 against collagen-induced arthritis in rats.

Collagen-induced arthritis (CIA) is an animal model, which closely resembles human rheumatoid arthritis (RA) in pathogenesis and pathology. Evidence suggests that the inhibition of T lymphocytes or their functions can alleviate the progression of arthritis. So the administration of arthritogenic T cell receptor (TCR) variable region peptide or DNA vaccines encoding pathogenic TCR Vbeta variable region may provide useful information for designing specific immunotherapies against autoimmune diseases. Heat shock proteins (HSPs) have the function of raising antigenic immunogenicity and HSP70 has a protective effect against arthritis. We previously demonstrated the presence of pathogenic predominant T cell receptor Vbeta5.2 and Vbeta8.2 clonotypes in the joints of CIA rats. In this study, we constructed the recombinant eukaryotic expression vectors pTARGET-TCR Vbeta5.2/8.2-HSP70, and evaluated their protective effects on CIA rats. Protective effects were observed in CIA rats by injecting these recombinant DNA vaccines, which could alleviate arthritis index, decrease the levels of IFN-gamma and anti-CII antibody in serum, and increase the levels of IL-4. Pathological changes were not as serious as those observed in control CIA rats. The rat injected with two combined vaccines showed better protective effects than CIA rats administered with individual vaccine. These results showed that recombinant DNA vaccines pTARGET-TCR Vbeta5.2-HSP70 and pTARGET-TCR Vbeta8.2-HSP70 could significantly alleviate the arthritic symptoms of CIA rats, and better protective effects could be achieved if these two vaccines were used in combination.