Down-expression of GOLM1 enhances the chemo-sensitivity of cervical cancer to methotrexate through modulation of the MMP13/EMT axis.

The highly refractory nature of cervical cancer to chemotherapeutic drugs and its epithelial-to-mesenchymal transition (EMT) are the key reasons contributing to the poor prognosis of this disease. Golgi Membrane Protein 1 (GOLM1), a protein involved in the trafficking of proteins through the Golgi apparatus, has been shown to be oncogenic in a variety of human cancers. Herein, we found GOLM1 was markedly up-regulated in cervical cancer and GOLM1 down-expression enhanced the anti-tumor effect of methotrexate. By performing mechanistic studies using both in vitro and in vivo models, we found that GOLM1 could target matrix metallopeptidase 13 (MMP13), a member of the MMPs, and regulate the EMT process. Moreover, altered EMT progression compromised the chemotherapy-enhancing effects of GOLM1 knock-down. Finally, we found significantly higher levels of GOLM1 and MMP13 in cervical cancer tissues compared with adjacent noncancerous tissues, and this was also associated with poor cervical cancer patients' prognosis. Taken together, our results suggest that the GOLM1/MMP13/EMT axis is an important factor involved in regulating methotrexate in cervical cancer, and highlights the potential of novel GOLM1-based clinical modalities as a therapeutic approach in cervical cancer patients.

Understanding the Structure, Multimerization, Subcellular Localization and mC Selectivity of a Genomic Mutator and Anti-HIV Factor APOBEC3H.

APOBEC3H (A3H) is a member of the APOBEC3 subfamily of DNA cytosine deaminases that are important for innate immune defense and have been implicated in cancer biogenesis. To understand the structural basis for A3H biochemical function, we determined a high-resolution structure of human A3H and performed extensive biochemical analysis. The 2.49 Å crystal structure reveals a uniquely long C-terminal helix 6 (h6), a disrupted ß5 strand of the canonical five-stranded ß-sheet core, and a long loop 1 around the Zn-active center. Mutation of a loop 7 residue, W115, disrupted the RNA-mediated dimerization of A3H yielding an RNA-free monomeric form that still possessed nucleic acid binding and deaminase activity. A3H expressed in HEK293T cells showed RNA dependent HMW complex formation and RNase A-dependent deaminase activity. A3H has a highly positively charged surface surrounding the Zn-active center, and multiple positively charged residues within this charged surface play an important role in the RNA-mediated HMW formation and deaminase inhibition. Furthermore, these positively charged residues affect subcellular localization of A3H between the nucleus and cytosol. Finally, we have identified multiple residues of loop 1 and 7 that contribute to the overall deaminase activity and the methylcytosine selectivity.

Molecular Interactions of a DNA Modifying Enzyme APOBEC3F Catalytic Domain with a Single-Stranded DNA.

The single-stranded DNA (ssDNA) cytidine deaminase APOBEC3F (A3F) deaminates cytosine (C) to uracil (U) and is a known restriction factor of HIV-1. Its C-terminal catalytic domain (CD2) alone is capable of binding single-stranded nucleic acids and is important for deamination. However, little is known about how the CD2 interacts with ssDNA. Here we report a crystal structure of A3F-CD2 in complex with a 10-nucleotide ssDNA composed of poly-thymine, which reveals a novel positively charged nucleic acid binding site distal to the active center that plays a key role in substrate DNA binding and catalytic activity. Lysine and tyrosine residues within this binding site interact with the ssDNA, and mutating these residues dramatically impairs both ssDNA binding and catalytic activity. This binding site is not conserved in APOBEC3G (A3G), which may explain differences in ssDNA-binding characteristics between A3F-CD2 and A3G-CD2. In addition, we observed an alternative Zn-coordination conformation around the active center. These findings reveal the structural relationships between nucleic acid interactions and catalytic activity of A3F.

Effect on intensity of treadmill running on learning, memory and expressions of cell cycle-related proteins in rats with cerebral ischemia.

OBJECTIVE: We discussed the intensity of treadmill running on learning, memory and expression of cell cycle-related proteins in rats with cerebral ischemia. METHOD: Eighty healthy male SD rats were randomly divided into normal group, model group, intensity I group and intensity II group, with 20 rats in each group. The four-vessel occlusion method of Pulsinelli (4-VO) was used to induce global cerebral ischemia. Brain neuronal morphology was observed by hematoxylin-eosin (HE) staining at 3h, 6h, 24h and 48h after modeling, respectively. Hippocampal expressions of cyclin A and cyclin E were detected by immunohistochemistry. At 48h after modeling, the learning and memory performance of rats was tested by water maze experiment. RESULT: Compared with the normal group, the other three groups had a significant reduction in surviving neurons, prolonging of escape latency and decreased number of passes over the former position of the platform (P<0.05). The number of surviving neurons and the number of passes over the former position of the platform were obviously lower in the model group than in intensity I group (P<0.05), but significantly higher compared with intensity II group (P<0.05). Escape latency of the model group was obviously prolonged as compared with intensity I group (P<0.05), but much shorter than that of intensity II group (P<0.05). Compared with the normal group, the expressions of cyclin A and cyclin E were significantly upregulated at different time points after modeling (P<0.05). The expression of the model group was higher than that of intensity I group, but lower than that of intensity II group (P<0.05). CONCLUSION: Moderate intensity of treadmill running can help protect brain neurons and improve learning and memory performance of rats with global cerebral ischemia. But high intensity of treadmill running has a negative impact, possibly through the regulation of cell cycle-related proteins in ischemia/reperfusion injury.

mTOR/autophagy pathway in the hippocampus of rats suffering intermittent hypoxia preconditioning and global cerebral ischemia-reperfusion.

We explored the role of mTOR/autophagy pathway in the aggravation of cerebral ischemia-reperfusion nerve injury caused by intermittent hypoxia. Eighty male wistar rats were divided into four groups by the random number method: sham operation group (SO group, n=20), cerebral ischemia-reperfusion group (I/R group, n=20), intermittent hypoxia and cerebral ischemia-reperfusion group (IH+I/R group, n=20), intermittent hypoxia and cerebral ischemia-reperfusion group plus mTOR inhibitor group (inhibitor group, n=20).The results showed that compared with the SO group, HE staining showed structural damage of neurons at each time point, the immunohistochemical assay showed an increasing number of mTOR and beclin1 immune-positive cells (P<0.05) and RT-PCR showed enhanced expression of mTOR and beclin1 protein in the I/R group (P<0.05). Compared with the I/R group, HE staining showed exacerbating structural damage of neurons at each time point, the immunohistochemical assay showed an increasing number of mTOR and beclin1 immune-positive cells (P<0.05) and RT-PCR showed enhanced expression of mTOR and beclin1 protein in the IH+I/R group (P<0.05). Compared with the IH+I/R group, HE staining showed remissive structural damage of neurons at each time point, the immunohistochemical assay showed a decreasing number of mTOR immune-positive cells and a rising number of beclin1immune-positive cells (P<0.05) and RT-PCR showed weakened expression of mTOR protein and enhanced expression of beclin1 protein in the inhibitor group (P<0.05). Thence, the present study indicated that intermittent hypoxia preconditioning can aggravate the nerve injury of the global cerebral ischemia-reperfusion model, and the mechanism is associated with the activation of mTOR/autophagy pathway.

The structure of SV40 large T hexameric helicase in complex with AT-rich origin DNA.

DNA replication is a fundamental biological process. The initial step in eukaryotic DNA replication is the assembly of the pre-initiation complex, including the formation of two head-to-head hexameric helicases around the replication origin. How these hexameric helicases interact with their origin dsDNA remains unknown. Here, we report the co-crystal structure of the SV40 Large-T Antigen (LT) hexameric helicase bound to its origin dsDNA. The structure shows that the six subunits form a near-planar ring that interacts with the origin, so that each subunit makes unique contacts with the DNA. The origin dsDNA inside the narrower AAA+ domain channel shows partial melting due to the compression of the two phosphate backbones, forcing Watson-Crick base-pairs within the duplex to flip outward. This structure provides the first snapshot of a hexameric helicase binding to origin dsDNA, and suggests a possible mechanism of origin melting by LT during SV40 replication in eukaryotic cells.

Crystal structures of APOBEC3G N-domain alone and its complex with DNA.

APOBEC3G (A3G) is a potent restriction factor of HIV-1. The N-terminal domain of A3G (A3G-CD1) is responsible for oligomerization and nucleic acid binding, both of which are essential for anti-HIV activity. As a countermeasure, HIV-1 viral infectivity factor (Vif) binds A3G-CD1 to mediate A3G degradation. The structural basis for the functions of A3G-CD1 remains elusive. Here, we report the crystal structures of a primate A3G-CD1 (rA3G-CD1) alone and in complex with single-stranded DNA (ssDNA). rA3G-CD1 shares a conserved core structure with the previously determined catalytic APOBECs, but displays unique features for surface charge, dimerization and nucleic acid binding. Its co-crystal structure with ssDNA reveals how the conformations of loops and residues surrounding the Zn-coordinated centre (Zn-centre) change upon DNA binding. The dimerization interface of rA3G-CD1 is important for oligomerization, nucleic acid binding and Vif-mediated degradation. These findings elucidate the molecular basis of antiviral mechanism and HIV-Vif targeting of A3G.

Complex between α-bungarotoxin and an α7 nicotinic receptor ligand-binding domain chimaera.

To identify high-affinity interactions between long-chain α-neurotoxins and nicotinic receptors, we determined the crystal structure of the complex between α-btx (α-bungarotoxin) and a pentameric ligand-binding domain constructed from the human α7 AChR (acetylcholine receptor) and AChBP (acetylcholine-binding protein). The complex buries ~2000 Ų (1 Å=0.1 nm) of surface area, within which Arg³6 and Phe³² from finger II of α-btx form a π-cation stack that aligns edge-to-face with the conserved Tyr¹84 from loop-C of α7, while Asp³° of α-btx forms a hydrogen bond with the hydroxy group of Tyr¹84. These inter-residue interactions diverge from those in a 4.2 Å structure of α-ctx (α-cobratoxin) bound to AChBP, but are similar to those in a 1.94 Å structure of α-btx bound to the monomeric α1 extracellular domain, although compared with the monomer-bound complex, the α-btx backbone exhibits a large shift relative to the protein surface. Mutational analyses show that replacing Tyr¹84 with a threonine residue abolishes high-affinity α-btx binding, whereas replacing with a phenylalanine residue maintains high affinity. Comparison of the α-btx complex with that coupled to the agonist epibatidine reveals structural rearrangements within the binding pocket and throughout each subunit. The overall findings highlight structural principles by which α-neurotoxins interact with nicotinic receptors.

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors.

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr¹84 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr¹84 depends on local residues, we generated mutations in an α7/5HT(3A) (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured ¹²5I-labelled α-btx binding. The results show that mutations of individual residues near Tyr¹84 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurements show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr¹84 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr¹84 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr¹84 and local residues contributes to high-affinity subtype-selective α-btx binding.

Ligand-binding domain of an α7-nicotinic receptor chimera and its complex with agonist.

The α(7) acetylcholine receptor (AChR) mediates pre- and postsynaptic neurotransmission in the central nervous system and is a potential therapeutic target in neurodegenerative, neuropsychiatric and inflammatory disorders. We determined the crystal structure of the extracellular domain of a receptor chimera constructed from the human α(7) AChR and Lymnaea stagnalis acetylcholine binding protein (AChBP), which shares 64% sequence identity and 71% similarity with native α(7). We also determined the structure with bound epibatidine, a potent AChR agonist. Comparison of the structures revealed molecular rearrangements and interactions that mediate agonist recognition and early steps in signal transduction in α(7) AChRs. The structures further revealed a ring of negative charge within the central vestibule, poised to contribute to cation selectivity. Structure-guided mutational studies disclosed distinctive contributions to agonist recognition and signal transduction in α(7) AChRs. The structures provide a realistic template for structure-aided drug design and for defining structure-function relationships of α(7) AChRs.

[Effect of sleep deprivation on p38MAPK phosphorylation in the rat hippocampus].

OBJECTIVE: To investigate the effect of sleep deprivation (SD) on the expression of p38Mitogen-activated protein kinase (p38MAPK) phosphorylation in the rat hippocampus. METHODS: Male Sprague-Dawley rats (n=60) were divided randomly into control and sleep deprivation groups. The sleep deprivation models were established with the modified multiple platform methods. At 1 d, 3 d, 5 d, and 7 d after sleep deprivations, changes of neuron morphous in the hippocampal region of the rats were observed by HE staining. The expression of p38MAPK phosphorylation was detected by immunohistochemistry and Western blot. The learning-memory function was tested with Morris water maze and 4-PTT dry path maze. RESULTS: More obvious neuronal morphous damages, increased p38MAPK phosphorylation cells and p38MAPK phosphorylation expression, and decreased learning-memory function were found in the rats subject to sleep deprivation than those in the control. The changes were enhanced with the length of sleep deprivation. CONCLUSION: p38MAPK can be activated by sleep deprivation, which mediates the process of neuronal injury.

Protective effects of edaravone on diffuse brain injury in rats.

BACKGROUND: Edaravone can alleviate brain injury and improve neurological functions and symptoms. This study aimed to investigate the effect of edaravone on the p38Mitogen-activated protein kinases/Caspase-3 (p38MAPK /Caspase-3) pathway after diffuse brain injury (DBI) in rats. METHODS: DBI models were established according to the description of Marmarou's method. A total of 250 rats were divided (random number) into four groups: control group (CG, n=45), model group (MG, n=77), low-dose edaravone group (n=67, dosage 5 mg/kg) and high-dose edaravone group (n=61, dosage 10 mg/kg). After 1, 6, 24, 48, and 72 hours after injury, brain tissues were collected. The changes of neuron morphous in the hippocampal region were observed through Nissl staining. The expression levels of phosphorylated p38MAPK and caspase-3 were detected by immunohistochemistry and Western blotting respectively. Learning and memory function were tested with Morris water maze from the 3rd to 7th day after injury. RESULTS: Some neurons had histopathologic changes of necrosis and apoptosis in the model group compared with the control group. The phosphorylated p38MAPK expressions increased at 1, 6, 4, and 48 hours (P<0.05), but no significant difference was observed at 72 hours (0.54±0.19 vs. 0.40±0.14, P>0.05). Caspase-3 expressions increased at 6, 24, 48, and 72 hours respectively (P<0.05), but there was no significant difference at 1 hour (0.59±0.29 vs. 0.40±0.17, P>0.05). From the 3rd to 6th day during the Morris water maze test, the latency to find the platform was significantly prolonged (P<0.05) and times of rats crossing the platform was decreased on the 7th day (2.28±1.18 vs. 8.20±1.52, P<0.05). The phosphorylated p38MAPK expressions decreased at 6, 24 and 48 hours respectively in the low dose edaravone group compared with the model group (P<0.05), whereas no significant difference was seen at 1 hour (1.66±0.80 vs. 1.85±0.86, P>0.05). Caspase-3 expression decreased at 6, 24, 48, and 72 hours (P<0.05). The latency to find the platform was significantly shortened (P<0.05), and times of rats crossing the platform increased (4.17±1.15 vs. 2.28±1.18, P<0.05). The above mentioned parameters changed more significantly in the high-dose edaravone group than in the low-dose edaravone group. CONCLUSION: Edaravone can alleviate brain tissue damage after DBI, inhibit p38MAP signal activation after early injury, reduce the expression of caspase-3, and promote the recovery of neurological function in the late period.

[Effect of BuYangHuanWu recipe on cerebral microcirculation in gerbils with ischemia-reperfusion].

OBJECTIVE: To explore the protective mechanism of BuYangHuanWu recipe on neurofunction in gerbils with cerebral ischemia- reperfusion. METHODS: Gerbils (n = 48) were divided randomly into three groups: animal model group, BuYangHuanWu recipe group, and sham control group. The animal model of cerebral ischemia was established using bilateral common carotid artery occlusion followed by unclamp 45 min after occlusion. The microcirculation was observed with a Laser Doppler. The density of microvascular was measured using Tannic acid ferric chloride mordant dyeing. The BBB (blood brain barrier) permeability was assessed using evan's blue (EB) dye. The water content in brain tissues was tested with wet and dry method. The learning and memory function test was performed with a 4-PTT dry path maze. RESULTS: Compared with the animal model group, BuYangHuanWu recipe increased blood flow in the hippocampal region at 1 and 5 min after occlusion, inhibited hypoperfusion at 15 min after reperfusion, increased blood flow at 30, 60 and 120 min after reperfusion. Meanwhile, BuYangHuanWu recipe inhibited the increase of BBB permeability and water content in brain tissues after reperfusion (P < 0.05). BuYangHuanWu recipe also improved the scores of learning and memory function of the gerbils. CONCLUSION: BuYangHuanWu recipe protects the neurofunction in gerbils with ischemia-reperfusion through modulating cerebral microcirculation damages.

Octameric structure of the human bifunctional enzyme PAICS in purine biosynthesis.

Phosphoribosylaminoimidazole carboxylase/phosphoribosylaminoimidazole succinocarboxamide synthetase (PAICS) is an important bifunctional enzyme in de novo purine biosynthesis in vertebrate with both 5-aminoimidazole ribonucleotide carboxylase (AIRc) and 4-(N-succinylcarboxamide)-5-aminoimidazole ribonucleotide synthetase (SAICARs) activities. It becomes an attractive target for rational anticancer drug design, since rapidly dividing cancer cells rely heavily on the purine de novo pathway for synthesis of adenine and guanine, whereas normal cells favor the salvage pathway. Here, we report the crystal structure of human PAICS, the first in the entire PAICS family, at 2.8 A resolution. It revealed that eight PAICS subunits, each composed of distinct AIRc and SAICARs domains, assemble a compact homo-octamer with an octameric-carboxylase core and four symmetric periphery dimers formed by synthetase domains. Based on structural comparison and functional complementation analyses, the active sites of SAICARs and AIRc were identified, including a putative substrate CO(2)-binding site. Furthermore, four symmetry-related, separate tunnel systems in the PAICS octamer were found that connect the active sites of AIRc and SAICARs. This study illustrated the octameric nature of the bifunctional enzyme. Each carboxylase active site is formed by structural elements from three AIRc domains, demonstrating that the octamer structure is essential for the carboxylation activity. Furthermore, the existence of the tunnel system implies a mechanism of intermediate channeling and suggests that the quaternary structure arrangement is crucial for effectively executing the sequential reactions. In addition, this study provides essential structural information for designing PAICS-specific inhibitors for use in cancer chemotherapy.

Crystallization and preliminary crystallographic studies of human septin 1 with site-directed mutations.

Septin 1 is a member of an evolutionarily conserved family of GTP-binding and filament-forming proteins named septins, which function in diverse processes including cytokinasis, vesicle trafficking, apoptosis, remodelling of the cytoskeleton, infection, neurodegeneration and neoplasia. Human septin 1 has been expressed and purified, but suffers from severe aggregation. Studies have shown that septin 1 with site-directed mutations of five serine residues (Ser19, Ser206, Ser307, Ser312 and Ser315) has a much lower degree of aggregation and better structural homogeneity and that the mutations cause only slight perturbations in the secondary structure of septin 1. This septin 1 mutant was crystallized and diffraction data were collected to 2.5 A resolution. The space group is P422, with unit-cell parameters a = b = 106.028, c = 137.852 A.

An attempt to increase the efficiency of protein crystal screening: a simplified screen and experiments.

Macromolecular crystallization remains a bottleneck in structure determination by X-ray diffraction. Based on the data reflecting success rates of crystallization conditions in different screens and the information derived from the BMCD and other related studies, a simplified screen has been designed to increase the success rate of traditional screening and to save samples, time and cost. The screen has been tested with six protein samples which had been crystallized before and its comparison with Crystal Screen (Hampton Research) was also performed with lysozyme crystallization. The experimental results show that for obtaining crystal leads, the success rate of the simplified screen is reasonably higher. In addition, it has been successful in crystallizing two new proteins from snake venom using the simplified screen that had failed with Crystal Screen. These results indicate that the simplified screen, which assembles and optimizes the efficient crystallization conditions from distinct screens, extends the region of crystallization and improves the success rate of screening. Based on information of newly published efficient crystallization conditions, the simplified screen could be developed and optimized continuously in the future.

2.6 A resolution crystal structure of the bacterioferritin from Azotobacter vinelandii.

The crystal structure of the bacterioferritin from Azotobacter vinelandii has been determined at 2.6 A resolution. Both the low occupancy of one iron ion in the dinuclear iron center and the deviation of its adjacent residue His130 from the center suggest migration of the iron ion from the dinuclear iron site to the inner nucleation site. The concerted movement of His130 and Glu47 may admit a dynamic gating mechanism for shift of the oxidized iron ion. Ba2+ binding to the fourfold channel implicates that the channel bears Fe2+ conductivity and selectivity to provide a route for iron access to the inner cavity during core formation.

[Topographical evaluation on decentration of orthokeratology lenses].

OBJECTIVE: To evaluate the degree and correlative factors of decentration of orthokeratology lenses and its effect on the visual function. METHODS: Two different kinds of orthokeratology lenses were fitted to 270 eyes of 135 patients [initial mean refractive error: (-3.98 +/- 1.51) D]. Humphery Instruments ATLAS 8.0 was used for the computer-assisted analysis of corneal differential topographical maps. The examination of corneal topography was proceeded on the patients before the fitting of orthokeratology lenses and 6-month later. The distance from center of optic zone to apex of the cornea was measured as the value of decentration of orthokeratology lenses. The factors influenced the value of decentration were analyzed, including the initial refraction error, astigmatism, keratometry values, corneal eccentricity, and the diameter of the lens. The complaints of patients were recorded. Questionnaires, involving the symptoms of monocular diplopia and glare, were used to evaluate the effects of decentration of orthokeratology lenses on the visual function. RESULTS: The mean distance of decentration was (0.49 +/- 0.34) mm after one night fitting, the mean distance of decentration after follow-up for 1 month, 3 months and 6 months was (0.57 +/- 0.41) mm, (0.55 +/- 0.48) mm and (0.59 +/- 0.39) mm, respectively. After one month, the distance of decentration was less than 0.5 mm in 51.1% eyes, 0.5 - 1.0 mm in 35.6% eyes and more than 1.0 mm in 13.3% eyes. The direction of decentration in eyes with more than 0.50 mm decentration was mainly in the temporal side (48.5%). Patients with greater initial astigmatism and smaller diameter of lens showed greater distance of decentration (P < 0.05). There was no statistically significant difference in the distance of decentration between two groups with different corneal eccentricities and keratometry values (P > 0.05). The distance of decentration was greater in patients with monocular diplopia and glare. CONCLUSIONS: The degree of decentration of orthokeratology depends on the degree of initial refractive error, astigmatism and the design of orthokeratology lenses. The degree of decentration can influence the visual function.