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上海生科院发现炎症因子ATP调节P2X3受体上膜转运新机制

ATP门控离子通道P2X3受体选择性地表达于初级感觉神经元,对生理性和病理性痛觉调节至关重要。P2X3受体的合成、组装和转运对于其行使正常的功能是必需的。病理条件下大量释放的包括ATP在内的各种炎症因子从多方面调节着P2X3受体的功能。中国科学院上海生命科学研究院生物化学与细胞生物学研究所鲍岚课题组前期的研究工作发现ATP可以促进P2X3受体的内吞,进而形成信号内吞体,在初级感觉神经元轴突中逆向转运到胞体,活化转录因子CREB,调节神经元的兴奋性。然而,ATP对P2X3受体上膜转运的调节及机制并不十分清楚。

4月23日,Journal of Molecular Cell Biology在线发表鲍岚课题组的研究工作:ATP时程依赖性地促进了重组的以及背根节神经元内源性的P2X3受体的上膜转运,而同家族的P2X1和P2X2受体则没有此效应。ATP激活P2X3受体引起的钙离子内流,可以激活钙/钙调素依赖性蛋白激酶IIα (CaMKIIα),CaMKIIα通过一种三级结构依赖的模式,调节P2X3受体的上膜转运。P2X3受体的N端负责与CaMKIIα相互作用,而位于其C端的第388位苏氨酸(Thr388)可被CaMKIIα磷酸化。将Thr388突变成Val氨基酸模拟去磷酸化效应,消除了ATP依赖的P2X3受体的上膜转运。进一步研究发现,脂筏结构的组成蛋白caveolin-1能与P2X3受体相互作用,通过基因沉默抑制caveolin-1的表达或者消除P2X3受体结合caveolin-1的能力均可抑制ATP依赖的P2X3受体的上膜转运。CaMKIIα介导的Thr388磷酸化可以促进P2X3受体和caveolin-1的相互作用,增强P2X3受体上膜转运。此外,研究还发现ATP依赖的P2X3受体上膜运输可以促进与其组成异源三聚体的P2X2受体向细胞膜的转运,此过程同样依赖于P2X3受体Thr388的磷酸化。最终,细胞膜上由于Thr388磷酸化增加的P2X3受体促进了其介导的信号转导效应。该研究表明,P2X3受体的上膜运输受其配体的调控,需要 CaMKIIα和caveolin-1的协同作用,并可带动与其形成多聚体的P2X2受体的共转运。此项研究为痛觉传递中P2X3受体的功能调控提供了一种可能的机制。该项工作由助理研究员陈序谯博士等在鲍岚研究员的指导下完成。

该课题得到了国家自然科学基金和国家基础研究计划等项目的资助。

日期:2014年5月5日 - 来自[技术要闻]栏目
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Acidic pH inhibits ATP depletion-induced tubular cell apoptosis by blocking caspase-9 activation in apoptosome

【关键词】  cell

    Department of Cellular Biology and Anatomy, Center for Genomic Medicine, Medical College of Georgia, Augusta, Georgia
    Kresge Eye Institute, Wayne State University, Detroit, Michigan
    Medical Research Service, Department of Veterans Affairs Medical Center, Augusta, Georgia

    ABSTRACT

    Tubular cell apoptosis has been implicated in the development of ischemic renal failure. In in vitro models, ATP depletion-induced apoptosis of tubular cells is mediated by the intrinsic pathway involving Bax translocation, cytochrome c release, and caspase activation. While the apoptotic cascade has been delineated, much less is known about its regulation. The current study has examined the regulation of ATP depletion-induced tubular cell apoptosis by acidic pH, a common feature of tissue ischemia. Cultured renal tubular cells were subjected to 3 h of ATP depletion with azide and then recovered in full culture medium. The treatment led to apoptosis in 40% of cells. Apoptosis was significantly reduced, if the pH of ATP depletion buffer was lowered from 77.4 to 66.5. This was accompanied by the inhibition of caspase activation. However, acidic pH did not prevent Bax translocation and oligomerization in mitochondria. Cytochrome c release from mitochondria was not blocked either, suggesting that acidic pH inhibited apoptosis at the postmitochondrial level. To determine the postmitochondrial events that were blocked by acidic pH, we conducted in vitro reconstitution experiments. Exogenous cytochrome c, when added into isolated cell cytosol, induced caspase activation. Such activation was abrogated, when pH during the reconstitution was lowered to 6 or 6.5. Nevertheless, acidic pH did not prevent the recruitment and association of caspase-9 by Apaf-1, as shown by coimmunoprecipitation. Together, this study demonstrated the inhibition of tubular cell apoptosis following ATP depletion by acidic pH. A critical step blocked by acidic pH seems to be caspase-9 activation in apoptosome.

    ATP depletion; ischemia

    RECENT STUDIES HAVE PROVIDED compelling evidence for the involvement of apoptotic mechanisms in the development of ischemic renal cell injury (6, 11, 18, 30, 32). Morphologically, apoptotic cells were identified in renal tubules of ischemia-reperfused kidneys (20, 33). Biochemically, renal ischemia-reperfusion led to the activation of caspases and endonucleases, which are responsible for the disassembly of apoptotic cells (3, 19). In addition, regulation of apoptotic genes including the Bcl-2 family was shown in kidneys following ischemia (2, 17). Finally, several pharmacological agents appeared to ameliorate ischemic renal injury, at least in part, by diminishing apoptosis (10, 20, 21, 23, 35, 39, 41).

    Two major pathways of apoptosis have been delineated. In the extrinsic pathway, ligation of death receptors leads to the formation of a death-inducing signaling complex and the activation of caspase-8 (1). In the intrinsic pathway, cellular stress leads to mitochondrial disruption, releasing apoptogenic molecules including cytochrome c (7). Cytochrome c, after being released from mitochondria, binds Apaf-1 in the cytosol, recruiting caspase-9 to form the caspase activation complex called apoptosome. Both apoptotic pathways have been implicated in the development of ischemic renal injury and renal failure (9, 28).

    In in vitro models, our previous work suggested a critical role for the intrinsic pathway in tubular cell apoptosis following renal cell hypoxia or ATP depletion (14, 15, 31, 44). Under the experimental conditions, Bax, a proapoptotic Bcl-2 family protein, translocated to mitochondria and oligomerized in the outer membrane. Consequently, cytochrome c was released from mitochondria, followed by the activation of caspases in the cytosol and development of apoptotic morphology (14, 15, 31, 44). These observations have been confirmed and extended in related models of tubular cell apoptosis in vitro (12, 25) and in vivo in ischemia-reperfused cadaveric kidney allografts (9). Our recent work selected death-resistant tubular cells through repeated episodes of hypoxia (14). The cells upregulated the antiapoptotic protein Bcl-XL, which prevented Bax activation and cytochrome c release, resulting in the preservation of cell viability (14). These findings further support a role for the intrinsic pathway in tubular cell apoptosis.

    While the apoptotic events at the mitochondrial and postmitochondrial levels have been delineated, much less is known about their regulation by cytosolic factors. Nevertheless, alterations of cellular pH have been demonstrated during apoptosis (22, 38). Importantly, in different experimental models, the pH changes seem essential for the initiation and progression of apoptosis. For example, in staurosporine-induced apoptosis of HeLa cells, there was a rise of intracellular pH, whereas tumor necrosis factor--induced apoptosis was accompanied by a drop in pH. In both models, prevention of pH changes inhibited apoptosis by blocking critical mitochondrial events including Bax activation and cytochrome c release (38). Recent studies by Segal and colleagues (4, 34) further demonstrated that alkaline pH inhibited caspase activation in an in vitro system by blocking apoptosome maturation. In the current study, we determined the effects of acidic pH on ATP depletion-induced apoptosis in renal tubular cells. We were particularly interested in acidic pH, because acidosis is a common feature of ischemic injury in organs including the brain, heart, and kidneys (5, 24, 36). We show that acidic pH inhibits tubular cell apoptosis following ATP depletion. However, mitochondrial events of apoptosis including Bax activation and cytochrome c release are not affected. A critical step that is blocked by acidic pH seems to be caspase-9 activation in apoptosome.

    MATERIALS AND METHODS

    Materials. Rat kidney proximal tubular epithelial cells (RPTC) were originally obtained from Dr. U. Hopfer at Case Western Reserve University, Cleveland, OH. The cells were cultured as described previously (45). HK-2 cells were purchased from ATCC (Manassas, VA) and cultured according to the instruction. DEVD.AFC and free AFC were purchased from Enzyme Systems Products (Dublin, CA). Dithiobisn (succinimidyl propionate) (DSP) was purchased from Pierce (Rockford, IL). The rabbit polyclonal antibody specific to the active form of caspase-3 was a gift from Dr. A. Srinivasan at Idun Pharmaceuticals (La Jolla, CA). Other antibodies were purchased from the following sources: polyclonal antibody to lamin B from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal antibody to cytochrome c from BD Pharmingen (San Diego, CA); monoclonal antibody to Bax from NeoMarkers (Fremont, CA); monoclonal antibody to caspase-9 from R&D Systems (Minneapolis, MN); polyclonal antibody to Apaf-1 from Cell Signaling Technologies (Beverly, MA); and all secondary antibodies from Jackson ImmunoResearch (West Grove, PA). Other reagents were purchased from Sigma (St. Louis, MO).

    ATP depletion. ATP depletion was conducted as detailed recently (15, 43, 44). Briefly, cells were incubated with 10 mM azide for 3 h in glucose-free Krebs-Ringer bicarbonate solution (composition in mM: 115 NaCl, 3.5 KCl, 25 NaHCO3, 1 KH2PO4, 1.25 CaCl2, and 1 MgSO4; gassed with 5% CO2). To control the pH of the solution, 25 mM HEPES at various pH levels was also included. The pH of the solution was monitored before, during, and after ATP depletion. After ATP depletion, groups of cells were transferred to full culture medium for recovery.

    Morphological examination of apoptosis. Apoptosis was examined by morphological criteria as described previously (14, 41, 44). Cell morphology was monitored by phase contrast microscopy. Nuclear morphology was examined by fluorescence microscopy after Hoechst 33342 staining. Typical apoptotic cells showed cellular shrinkage, formation of apoptotic bodies, nuclear condensation, and fragmentation.

    Measurement of caspase activity. Caspase activity was measured enzymatically using the fluorogenic peptide substrate DEVD.AFC (13, 14, 41, 44). Briefly, cells were extracted with 1% Triton X-100. The lysates of 25 μg protein were added to enzymatic reactions containing 50 μM DEVD.AFC. After 60 min of reaction, fluorescence at excitation 360 nm/emission 530 nm was monitored by a GENios plate-reader (Tecan US, Research Triangle Park, NC). For each measurement, a standard curve was constructed using free AFC. Based on the standard curve, the fluorescence reading from each enzymatic reaction was converted into the nanomolar amount of liberated AFC to indicate caspase activity.

    Immunofluorescence of cytochrome c, Bax, and active caspase-3. Cells were grown on collagen-coated glass coverslips for immunofluorescence as described in our previous work (14, 16, 44). For immunofluorescence of Bax and active caspase-3, cells were fixed with 4% paraformaldehyde. For immunofluorescence of cytochrome c, cells were fixed with a modified Zamboni's fixative containing picric acid and 4% paraformaldehyde. The fixed cells were incubated with 2% normal goat serum for blocking and then exposed to specific primary antibodies (rabbit polyclonal anti-active caspase-3, mouse monoclonal anti-Bax, or mouse monoclonal anti-cytochrome c). Finally, the cells were incubated with Cy3-labeled goat anti-rabbit, Cy3-labeled goat anti-mouse, or FITC-labeled goat anti-mouse secondary antibodies. Signals were examined by fluorescence microscopy using Cy3 or FITC channel.

    Cellular fractionation. To analyze the subcellular redistributions of Bax and cytochrome c during apoptosis, cells were fractionated into cytosolic fraction and the membrane-bound organellar fraction. The fractionation was facilitated by using low concentrations of digitonin, as described in our previous work (14, 16, 31, 41). Digitonin at low concentrations selectively permeabilizes the plasma membrane, without solubilizing mitochondria. The feasibility of digitonin extraction in our experiments was further supported by two observations: first, digitonin did not extract or solubilize cytochrome c from mitochondria in normal control cells, where cytochrome c resided in the intermembrane space of the organelles (see Fig. 3A); second, COX IV (an integral mitochondrial protein) was not detected in digitonin-soluble fraction, suggesting that contamination of this fraction by mitochondria was indeed minimal (data not shown). Cellular fractionation by digitonin was also utilized by other investigators to examine protein translocations during apoptosis (29, 42). Briefly, cells were incubated with 0.05% digitonin in isotonic sucrose buffer (in mM: 250 sucrose, 10 HEPES, 10 KCl, 1.5 MgCl2, 1 EDTA, and 1 EGTA; pH 7.1) for 2 min at room temperature. The released cytosol was collected. The digitonin-insoluble part was further extracted with 2% SDS to collect the membrane-bound organellar fraction. Bax and cytochrome c redistribution during apoptosis mainly takes place between the cytosol and mitochondria, thus immunoblot analysis of the organellar fraction is expected to reveal mitochondrial content of the molecules.

    Immunoblot analysis. Electrophoresis and immunoblot analysis of proteins were performed in a NuPAGE Gel System. After being blocked with 2% BSA, the blots were incubated with specific primary antibodies overnight at 4°C. The blots were then exposed to the horseradish peroxidase-conjugated secondary antibody, and antigens on the blots were revealed using the enhanced chemiluminescence (ECL) kit (Pierce).

    In vitro reconstitution of caspase activation. Reconstitution of caspase activation by adding exogenous cytochrome c to isolated cytosol was conducted as described in our previous work (13, 14, 16). Briefly, cell cytosol was extracted with 0.05% digitonin and concentrated to 45 mg protein/ml with 3-kDa cutoff microconcentrators. For reconstitution, 1 μl of 0.5 mg/ml cytochrome c, 1 μl of 10 mM dATP, and 0.5 μl of 200 mM MgCl2 were added to 7.5 μl of cell cytosol containing 25 μg protein. After 1 h of incubation at 30°C, the reconstitution mixture was transferred to caspase assay buffer containing DEVD.AFC to determine caspase activity.

    Analysis of Bax oligomerization. DSP, a homobifunctional amine-reactive cross-linker, was utilized to analyze Bax oligomerization, as described in our previous work (14, 27). Briefly, cells were cross-linked with 1 mM DSP in phosphate-buffered saline for 30 min at room temperature under constant mixing. The cells were then fractionated with 0.05% digitonin to collect membrane fraction. Finally, the membrane fraction was subjected to electrophoresis and immunoblot analysis under nonreducing conditions.

    Coimmunoprecipitation of caspase-9 and Apaf-1. Coimmunoprecipitation was conducted by a method modified from our previous work (14, 41, 47). Briefly, cell lysates were precleared by incubation with 1 μg of normal mouse serum and 30 μl of protein A/G agarose (Santa Cruz Biotechnology). The precleared samples were subsequently incubated for 2 h with 1 μg of caspase-9 immunoprecipitation antibody and 30 μl of protein A/G agarose. Immunoprecipitates were collected by centrifugation and dissolved in 2% SDS sample buffer for immunoblot analysis of caspase-9 and Apaf-1.

    Statistics. Data were expressed as means ± SD (n  3). Statistical differences between various groups were determined by multiple comparisons, which were conducted by Tukey's posttests following ANOVA using the GraphPad Prism software. P < 0.05 was considered significantly different.

    RESULTS

    Suppression of ATP depletion-induced apoptosis by acidic pH. ATP depletion is a primary cause of ischemic renal cell injury in vivo and has been used in in vitro models for mechanistic studies (26). Our recent work examined the apoptotic mechanisms that are activated by ATP depletion in cultured renal tubular cells (1315, 27, 31, 43). Using this well-characterized apoptotic model, we examined the effects of acidic pH in the current study. RPTC, a rat proximal tubular cell line, was subjected to ATP depletion by incubation with azide in glucose free medium. Subsequently, the cells were returned to full culture medium for recovery. As shown in Fig. 1A, significant amounts of cells developed apoptotic morphology following the treatment, showing cellular shrinkage and the formation of apoptotic bodies. Hoechst33342 staining further revealed nuclear condensation and fragmentation in these cells. Importantly, when the pH of the ATP depletion medium was decreased from 7.4 to 6.5, apoptosis was drastically reduced. For quantification, we counted the cells that showed typical apoptotic morphology (Fig. 1B). The basal level of apoptosis in control RPTC cells was 2%. After 3 h of ATP depletion at pH 7.4 and 1 h of recovery, 43% of cells became apoptotic. The percentage of apoptosis was slightly lower, when the pH of ATP depletion buffer was decreased to 7.0. However, a further decrease in pH to 6.5 or 6.0 induced a drastic reduction in apoptosis. Under these conditions, the rates of apoptosis were below 10% (Fig. 1B).

    Inhibition of caspase activation during ATP depletion by acidic pH. To identify the apoptotic events that were blocked by acidic pH, we first examined caspase activation. The results are shown in Fig. 2. By enzymatic assays, we detected significant caspase activation during ATP depletion at pH 7.4 and 7.0. When the pH of ATP depletion buffer was lowered to 6.5 or 6.0, caspase activation was attenuated (Fig. 2A). The inhibitory effects of acidic pH were further indicated by immunoblot analysis of lamin B, an endogenous caspase substrate. As shown in Fig. 2B, lamin B of 72 kDa was cleaved into a characteristic fragment of 46 kDa following ATP depletion at pH 7.0 and 7.4 (lanes 4 and 5), but not at pH 6.0 or 6.5 (lanes 2 and 3). We also examined caspase activation in situ in intact cells by immunofluorescence of active caspase-3. As shown in Fig. 2C, many cells displayed immunofluorescence of active caspase-3 following ATP depletion at pH 7.4, while significantly fewer positive cells were shown in the group that experienced ATP depletion at pH 6.5. Together, the results suggest that acidic pH suppressed caspase activation and apoptosis following ATP depletion in renal tubular cells.

    Effects of acidic pH on cytochrome c during ATP depletion. Previous work by this and other laboratories demonstrated a role for the intrinsic mitochondrial pathway in tubular cell apoptosis following ATP depletion (1214, 25, 31). A critical event of this pathway is the release of cytochrome c from mitochondria into cytosol. Thus we determined whether acidic pH inhibited cytochrome c release during ATP depletion. We initially analyzed cytochrome c release by immunoblotting following cellular fractionation. The results are shown in Fig. 3A. As expected, cytochrome c in control cells was detected in the mitochondrial fraction (lane 1). After ATP depletion, cytochrome c was lost from the mitochondrial fraction and appeared in the cytosolic fraction, indicating its release. Importantly, regardless of the pH variations from 7.4 to 6.0, cytochrome c release persisted and was comparable (lanes 2-5). The release of cytochrome c was confirmed by immunofluorescence analysis (Fig. 3B). Clearly, ATP depletion with azide induced cytochrome c release in many cells, resulting in a diffuse cytosolic staining. We subsequently counted the cells that released cytochrome c in representative fields. Consistent with the immunoblot results, no significant differences were shown between the groups at various pH levels (Fig. 3C). Together, the results suggest that acidic pH blocked apoptosis and caspase activation without affecting mitochondrial release of cytochrome c.

    It was noticed that cytochrome c staining in some azide-treated cells was weaker than that of control cells (Fig. 3B). Interestingly, recent work by Zager et al. (48) showed that, in isolated mouse proximal tubules, cytochrome c after mitochondrial release traversed plasma membranes into the extracellular space. To determine whether cytochrome c was lost from azide-treated cells, we examined and compared cytochrome c in control and azide-treated cells by immunoblot. Cells were incubated for 3 h in glucose-free buffer in the absence or presence of azide. Whole cell lysates were then collected from these two groups of cells for immunoblot analysis. The results of samples collected from three separate experiments are shown in Fig. 3D (top blot). Densitometry of the blots showed that cytochrome c in the azide-treated group was 14.8 ± 33.8% lower than in the control group; however, the difference was not statistically significant (P > 0.05). We also collected and concentrated the incubation medium for cytochrome c immunoblot and did not detect significant signal (Fig. 3D, bottom blot). Apparently, cytochrome c leakage from azide-treated cells was not as extensive as that shown in other experimental models. The lower immunostaining signal was likely caused by the diffusion/dilution of cytochrome c within the cells after being released from mitochondria. In addition, some of the cells might be out of focus during microscopic recording.

    Effects of acidic pH on Bax activation during ATP depletion. Our previous work suggested a critical role for Bax in mitochondrial disruption and cytochrome c release during ATP depletion of renal tubular cells (1416, 27, 31). Bax was activated in ATP-depleted cells and translocated to mitochondria, where it oligomerized and presumably formed pathological pores on the outer membrane (1416, 27, 31). Thus to further identify the apoptotic events that were blocked by acidic pH, we analyzed Bax activation. We initially examined Bax translocation by immunoblotting following cellular fractionation. As shown in Fig. 4A, in control cells, the majority of Bax was detected in the cytosolic fraction (lane 1). After 3 h of ATP depletion, significant amounts of Bax moved to the membrane-bound fraction enriched with mitochondria (lanes 2-5). Of note, Bax accumulation in the membrane fraction was similar at various pH levels. Bax translocation to mitochondria during ATP depletion was confirmed by immunofluorescence (Fig. 4B). Particularly, there was a population of cells that had intense Bax signal in mitochondria, showing a perinuclear organellar staining. To quantify the cells with intense mitochondrial Bax, we counted these cells in representative fields; there was no difference between the groups that were ATP depleted at various pH levels (Fig. 4C). We further analyzed the oligomerization status of Bax (Fig. 4D). To preserve Bax oligomers, cells were subjected to chemical cross-linking by DSP. Cross-linked samples were then analyzed by immunoblotting. Clearly, Bax oligomerized in mitochondrial membranes, irrespective of the pH values during ATP depletion (Fig. 4D: lanes 1, 3, 5, 7). Of note, treatment of the cross-linked samples with DTT led to the dissociation of Bax oligomers (lanes 2, 4, 6, 8). This was due to the cleavage of the disulfide bond in DSP by the reducing agent. These results, together with the cytochrome c data, indicated that apoptotic events at the mitochondrial level were not abrogated by acidic pH.

    Acidic pH blocks cytochrome c-stimulated caspase activation in isolated cytosol. Our analyses of Bax and cytochrome c suggest that acidic pH blocked apoptosis at the postmitochondrial level, downstream of cytochrome c release. Thus, to further identify the pH-sensitive event(s), we set up an in vitro system by adding exogenous cytochrome c to isolated cytosol. In this set of experiments, cytosol was isolated from control cells without ATP depletion and was free of cytochrome c (not shown). Exogenous cytochrome c was then added to the cytosol at various pH levels to induce caspase activation. As shown in Fig. 5A, cytochrome c stimulated caspase activation in isolated cytosol at pH 7.0 and 7.4, but not at acidic pH 6.0 or 6.5 (bar graph). Importantly, caspase-9 was processed into active forms following cytochrome c stimulation at pH 7.0 and 7.4 (blot: lanes 6 and 8), while the processing was markedly suppressed by lower pH (blot: lanes 2 and 4). The results were confirmed by using cytosol that was isolated from HK-2 cells, a human tubular cell line. As shown in Fig. 5B, both caspase activation and caspase-9 processing following cytochrome c stimulation were inhibited by acidic pH.

    Acidic pH inhibits caspase-9 activation in apoptosome without blocking Apaf-1/caspase-9 association. In the intrinsic pathway of apoptosis, an immediate step following cytochrome c release is the binding of cytochrome c to Apaf-1, leading to the exposure of the CARD domain and the recruitment of caspase-9 to form a cytochrome c-Apaf-1-caspase-9 complex called apoptosome. Subsequently, caspase-9 is activated in apoptosome by a mechanism of proximity (7). Because cytochrome c-induced caspase-9 activation in isolated cytosol was inhibited by acidic pH (Fig. 5), we hypothesized that acidic pH might prevent the conformational changes in Apaf-1 that were needed for the recruitment and association of caspase-9. Recent work screened a panel of antibodies and identified a monoclonal antibody of human caspase-9 that is suitable for immunoprecipitation (40). Thus we determined the effects of buffer pH on the caspase-9/Apaf-1 association by coimmunoprecipitation (Fig. 6). Cytosol was isolated from HK2 cells and incubated at pH 7.4 or 6.5 in the absence or presence of cytochrome c. At the end of incubation, a portion of the samples was directly analyzed for caspase-9 and Apaf-1 by immunoblotting (lanes 1-4). The other portion was subjected to immunoprecipitation with the caspase-9-specific monoclonal antibody. Immunoprecipitates were then analyzed for caspase-9 and Apaf-1 by immunoblotting (lanes 5-8). As shown in Fig. 6, lanes 1-4, comparable amounts of caspase-9 and Apaf-1 were detected under various experimental conditions before immunoprecipitation. Consistent with previous observations, cytochrome c induced the processing of caspase-9 into its active fragments at pH 7.4 (lane 2) but not at pH 6.5 (lane 4). Following immunoprecipitation, significant amounts of caspase-9 were precipitated, including the intact and processed forms (lanes 5-8: bottom). In the absence of cytochrome c, limited amounts of Apaf-1 were coprecipitated along with caspase-9 (lanes 5 and 7: top). After cytochrome c stimulation, the coprecipitation or association between Apaf-1 and caspase-9 was markedly induced (lanes 6 and 8: top). Importantly, the association was not prevented by lowering the pH from 7.4 (lane 6) to 6.5 (lane 8). By densitometry of blots from four separate experiments, the amount of Apaf-1 that bound caspase-9/cytochrome c under pH 6.5 was similar to that bound under pH 7.4 (107 ± 36, if the signal of the pH 7.4 samples was arbitrarily set as 100). Collectively, the results indicate that acidic pH did not inhibit the recruitment of caspase-9 by Apaf-1 to form apoptosome; however, caspase-9 activation in this protein complex was suppressed.

    DISCUSSION

    Acidosis is a common feature of tissue ischemia (5, 24, 36). Under ischemia, cells within the affected tissues are forced to anaerobic glycolysis due to oxygen deprivation, leading to the accumulation of metabolic byproducts such as lactic acid, resulting in significant decreases in cellular pH. Acidosis is associated with ischemia of the brain, heart, liver, kidneys, and other organs (5, 24, 36). For example, in the brain, pH falls to 6.5 or lower following severe ischemic injury (36). Despite the recognition of acidosis in ischemic tissues, whether it is injurious or protective has been quite controversial. Acidosis is proposed to be a key detrimental factor for ischemic tissue damage (36). In addition to many earlier studies, the latest work by Xiong et al. (46) provided strong support for an injurious role of acidic pH. They showed that cells and animals lacking the acid-activated Ca2+-permeable channel became resistant to ischemic injury in the brain (46). On the other hand, cytoprotective effects of mild acidosis have been demonstrated in vitro in different types of cells (5, 24). For example, in freshly isolated renal tubules, hypoxic injury was prevented, when the pH of the incubation buffer was decreased from 7.4 to 6.9, although the underlying mechanism was elusive (8). The current study specifically examined the effects of acidic pH on ATP depletion-induced tubular cell apoptosis, a process implicated in ischemic renal injury.

    Our results show that tubular cell apoptosis as well as caspase activation were inhibited by acidic pH. Drastic inhibitory effects were shown, when the buffer pH was reduced from 7.0 to 6.5, suggesting a breakpoint within this range. Analysis of cytochrome c and Bax indicates clearly that upstream apoptotic events at the mitochondrial level were not suppressed by acidic pH. To identify the apoptotic events that were acidosis sensitive, we used an in vitro reconstitution system. When exogenous cytochrome c was added to isolated cell cytosol, caspase activation and caspase-9 processing were induced. Importantly, both events were attenuated by acidic pH. In the in vitro system, caspase-9 was expected to be the initiator caspase, which on activation would further process and activate downstream caspases. Thus, our results suggest that critical steps responsible for caspase-9 activation within the apoptosome were blocked by acidic pH. Nevertheless, cytochrome c-induced recruitment and association of caspase-9 by Apaf-1 were not inhibited by acidic pH. Taken together, it is concluded that acidic pH inhibited tubular cell apoptosis following ATP depletion by blocking caspase-9 activation in apoptosome.

    Although the pH of the incubation buffer was vigorously controlled and monitored, we did not measure the pH within the cells. Nevertheless, the specific feature of the experimental model might facilitate a quick equilibration of protons across the plasma membrane. In these experiments, cells were depleted of ATP within 30 min of azide incubation in glucose-free medium (data not shown). Without ATP, the cellular capacity of maintaining ion homeostasis via channels and pumps was expected to be compromised. As a result, passive diffusion and transport of protons were mainly driven by concentration gradients, leading to the equilibration of intracellular pH with extracellular space. Importantly, the inhibitory effects of acidic pH on apoptosis and caspase activation in intact cells were also reproduced in the in vitro reconstitution system, where the pH of isolated cytosol was directly monitored.

    It is noteworthy that acidic pH does not prevent or slow down ATP depletion under various experimental conditions (24). Similar results were shown in our experiments (not shown). Such a conclusion is also supported by the observation that upstream apoptotic events at the mitochondrial level were not suppressed by acidic pH.

    Alterations of cellular pH have been shown to be involved in apoptosis. In T-lymphocytes, withdrawal of IL-7 led to Bax activation and consequent apoptosis, which were preceded by a rise in intracellular pH (22). Moreover, the active conformation of Bax could be induced by pH of 7.8 or higher (22). More recently, Tafani et al. (38) demonstrated changes in intracellular pH of HeLa cells in different apoptotic models. Although staurosporine treatment induced a rise in pH, TNF induced a decrease in cellular pH. Interestingly, prevention of pH alterations in either direction suppressed Bax activation and apoptosis in both models (38). These results support an important role for cellular pH in the initiation and progression of apoptosis. However, in our experimental model of ATP depletion, Bax activation and consequent cytochrome c release do not seem to be pH dependent. First, as discussed above, in ATP-depleted cells, pH is expected to equilibrate with the extracellular space and variations would be very limited. Under this condition, Bax activation and cytochrome c release were shown under a physiological pH of 77.4. Second, when pH was decreased to 6.5 and 6, neither cytochrome c release nor Bax activation was affected. In these experiments, we analyzed Bax and cytochrome c by several approaches including immunoblotting and immunofluorescence and obtained consistent results. Thus we conclude that pH alterations may not be the primary cause of mitochondrial events of apoptosis during ATP depletion, including Bax activation and cytochrome c release. These observations, however, do not exclude a role of intracellular pH in Bax activation and cytochrome c release in other apoptotic models, as shown previously (22, 38).

    The fact that acidic pH inhibited apoptosis without blocking mitochondrial events suggests that the key pH-sensitive step is in the cytosol, downstream of cytochrome c release. This scenario is supported by the in vitro reconstitution experiments, showing that exogenous cytochrome c-stimulated caspase activation in isolated cell cytosol was suppressed, when the reconstitution pH was lowered from 77.4 to 66.5. Importantly, the processing of caspase-9, the initiator caspase in this system, was inhibited by lower pH, suggesting that a critical step blocked by acidic pH might be the activation of caspase-9 rather than downstream executioner caspases. In line with these observations, purified recombinant caspases including caspase-3 maintained significant enzymatic activity within a relatively broad pH range (37).

    In the in vitro reconstitution system, cytochrome c after being added to the cytosol is expected to bind the WD40 domain of Apaf-1, leading to conformational changes of the latter and the exposure of the CARD domain. An exposed CARD domain in Apaf-1 recruits caspase-9 to form a protein complex called apoptosome. Caspase-9 is activated in apoptosome probably by proximity-induced autocatalysis (7). Apparently, a critical step for caspase-9 activation is the recruitment and association of caspase-9 by the adaptor protein Apaf-1, which holds caspase-9 molecules together to reach a concentration that is required for their autocatalytic activation (7). Thus, to explain the inhibitory effects of acidic pH in our experimental models, we initially hypothesized that lower pH might affect the recruitment and association of caspase-9 by Apaf-1 on cytochrome c stimulation. However, this possibility was not supported by our subsequent experiments of coimmunoprecipitation. It is clear that cytochrome c-induced caspase-9/Apaf-1 association was not prevented by pH 66.5. Thus, under mild acidic pH, cytochrome c after being released into the cytosol was able to induce conformational changes in Apaf-1 to recruit caspase-9 and form apoptosome; however, the apoptosome is not functional and caspase-9 is not activated under these conditions.

    It remains unclear why the apoptosome formed under mild acidic pH does not function. Recently, well-controlled experiments by Beem et al. (4) investigated the mechanism of apoptosome formation using an in vitro system that was similar to that of our study. Their results led to a model of apoptosome maturation, starting with an initial 700-kDa complex, then dimerized into a 1,400-kDa intermediate, and finally transformed into a functional 700-kDa apoptosome. In this interesting model, caspase-9 is activated in the 1,400-kDa intermediate complex, while executioner caspases such as caspase-3 is proposed to be activated in the final 700-kDa complex. This model is supported by the observation that 150 mM KCl blocked the dimerization of the initial 700-kDa complex into the 1,400-kDa intermediate, while alkaline pH prevented the transformation of the 1,400-kDa intermediate into the final 700-kDa apoptosome (4). Should this occur in our system, it would not be far-fetched to speculate that acidic pH may inhibit the first step of apoptosome maturation, i.e., dimerization of the initial 700-kDa complex. Thus further investigations need to analyze the effects of acidic pH on the formation of various forms of apoptosome complexes.

    In conclusion, this study examined the effects of acidic pH on ATP depletion-induced apoptosis in renal tubular cells. While apoptosis and caspase activation were suppressed by acidic pH, apoptotic events at the mitochondrial level including Bax activation and cytochrome c release were not affected. A critical step that was blocked by acidic pH was shown to be caspase-9 activation in apoptosome. These observations may have implications in apoptotic regulation during ischemic renal injury, a condition associated with acidosis.

    GRANTS

    This work was supported in part by grants from National Institutes of Health, American Society of Nephrology and Department of Veterans Affairs.

    DISCLOSURES

    P. Ketsawatsomkron and Y. Sui are rotation students from the Biomedical Graduate Program at Medical College of Georgia, Augusta, GA.

    ACKNOWLEDGMENTS

    We thank Dr. X.-M. Yin at the Department of Pathology, University of Pittsburgh School of Medicine, for critical reading of the manuscript. We also thank Dr. A. Srinivasan at Idun Pharmaceuticals (La Jolla, CA) for the antibody to active caspase-3.

    FOOTNOTES

    The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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日期:2013年9月26日 - 来自[2005年第288卷第9期]栏目

昆明动物所发现人体能量物质ATP抗微生物感染新功能

三磷酸腺苷(Adenosine triphosphate, ATP)是一种核苷酸(又叫腺苷三磷酸),作为细胞内能量传递的“分子通货”,储存和传递化学能。ATP已开发成为临床药物,包括口服和注射剂型,国家药典记载用于进行性肌萎缩、脑出血后遗症、心功能不全、心肌疾患及肝炎等的辅助治疗。感染是目前医院住院病人死亡的主要原因之一,据美国新英格兰医学杂志统计,仅在美国,每年败血症的发病率为75万人,其中死亡22.5万。随着传统抗生素的大量使用和滥用,在临床上出现了各种各样的耐药菌株,目前临床使用的抗生素对这些耐药菌已无疗效,成为目前特别是今后危害人类健康的重大威胁。

中国科学院昆明动物研究所动物模型与人类疾病机理重点实验室生物毒素与人类疾病课题组在张云研究员带领下,向阳博士等建立了临床耐药菌感染动物模型,揭示了ATP对于全身性和致死性耐药菌感染具有良好的预防和保护作用,进一步研究表明ATP抗微生物感染的药理学机制在于激活体内炎症小体, 促进细胞因子的释放和招募激活免疫细胞而达到清除感染微生物的作用。该研究成果揭示了ATP抗微生物感染的新功能,以及其作为药物用于预防和治疗感染性疾病新适应症的临床价值,提供了相关体内药效学和药理学的科学依据。

该研究成果已在线发表于美国公共科学图书馆杂志PLoS One。

该研究获得国家“973”计划项目以及国家基金委-云南省联合基金项目的资助。

日期:2013年5月28日 - 来自[技术要闻]栏目
循环ads

三磷酸腺苷与普罗帕酮转复阵发性室上性心动过速的临床观察

【摘要】  目的探讨三磷酸腺苷 (ATP )与普罗帕酮 (propafenone )转复阵发性室上性心动过速(PSVT)的临床疗效及安全性。方法2006年8 月—2011年 9月在本院诊断为PSVT的137例患者,随机分为ATP组(n=69)及普罗帕酮组(n=68),比较两者的转律成功率、转律时间、不良反应发生率等。 结果 ATP与普罗帕酮转复时间分别为 (0.85 ±0.30) min、(30.00 ±15.00 min) (P<0.01) ,转复成功率分别为 97.1 %、92.6 % (P>0.05);ATP组轻度不良反应发生率大于普罗帕酮组 (分别为 39.1 %、11.8%,P<0.01),普罗帕酮组严重不良反应率大于 ATP组 (分别为 7.4 %、1.4 %,P>0.05) 。结论ATP及普罗帕酮转律 PSVT具有较高的成功率。ATP转律有较多的轻度不良反应 ,但比普罗帕酮安全性高,起效快。

【关键词】  三磷酸腺苷;普罗帕酮;药物治疗;室上性心动过速

  Clinical observation of adenosine triphosphate and propafenone to treat paroxysmal supraventricular tachycardia

  XIE Xue-jian,WANG Yu-jing,LI Yang,et al.Department of Cardiology,the 323th Hospital of PLA,Xi'an 710054,China

  [Abstract]ObjectiveTo observe the clinical efficacy and safety of adenosine triphosphate and propafenone to paroxysmal supraventricular tachycardia.MethodsThere are one hundred and thirty-seven patients who were diagnosed paroxysmal supraventricular tachycardia in our hospital from August 2006 through September 2011.They were randomly separated into group ATP (n=69) and group propafenone (n=68).The achievement ratio,time of conversion,side effects of two drugs were compared.ResultsThe time of conversion in group ATP and group propafenone was 0.85±0.30min and 30.00±15.00min respectively (P<0.01).The achievement ratio was 97.1% in group ATP and 92.6% in group propafenone (P>0.05) .More mild side effects were recorded in group ATP (39.1% vs 11.8%,P<0.01),whereas more severe side effects were discovered in group propafenone (7.4% vs 1.4%,P>0.05).ConclusionTreat PSVT with ATP or propafenone can get a highly achievement ratio.Although ATP has more mild side effects in cardioversion ,it has more safety and less time than propafenone.

  [Key words]adenosine triphosphate; propafenone; drug therapy;supraventricular tachycardia

  阵发性室上性心动过速(paroxysmal supraventricular tachycardia,PSVT )是发自心房、房室交界处的快速而规则的异动心律,90%以上为房室结折返性心动过速和房室折返性心动过速,心率多在 150~250 次/min,心律绝对均齐,具有反复发作、突发突止的特点,如不及时纠正,少数病人可引起血流动力学改变,出现心绞痛、心功能不全、晕厥或休克等严重症状,需急诊处理[1]。其终止PSVT方法主要包括:刺激迷走神经、药物复律、食道调搏术、直流电复律、导管射频消融术等。但在临床中遇到PSVT正在发作的患者,应紧急处理 。为研究药物快速处理 PSVT 的临床治疗效果,笔者将 2006年8月1日— 2011年9月1日到本院就诊的137例 PSVT患者分为 ATP组 (n=69) 及普罗帕酮组(n=68) ,研究二者的复律成功率、复律时间及不良反应发生率等。

  1资料与方法

  1.1一般资料将 2006年8月1日— 2011年9月1日到本院就诊的 137 例临床诊断为 PSVT患者随机分为 ATP组及普罗帕酮组。ATP组69例,女30例,男39例;年龄25~70岁,平均40.2岁。普罗帕酮组68例,男34例,女34例;年龄27~72岁,平均43.1岁。纳入标准:(1)根据病史及心电图检查均确诊为阵发性室上性心动过速;(2)经常规单侧颈动脉窦按摩、Valsalva动作、诱导恶心等非药物治疗措施无效。两组患者病情、既往史和伴发病无统计学差异。

  1.2给药方法

  1.2.1三磷酸腺苷组首先从外周静脉快速注射( < 5s) ATP,其后在同处快速注射10ml 生理盐水。其起始剂量从10mg开始,如 PSVT未终止,5min后以10mg量递增,最大剂量为30mg。

  1.2.2普罗帕酮组普罗帕酮70 mg 用生理盐水20ml稀释后缓慢静脉推注 (>5min),20~30min如果不能转律 ,则以相同剂量方式给药,总量不超过210mg。

  1.3观察项目成功率、转律时间、轻度不良反应、严重不良反应。

  1.4不良反应 轻度不良反应:胸闷、颜面潮红、头痛、恶心、呕吐、咳嗽、胸痛等;严重不良反应:转律过程中及转律后出现持续心动过缓、房室传导阻滞、室颤、低血压、休克等需要进行药物治疗或心脏电学治疗。

  1.5复律后观察转律后继续予以心电监护 1h ,如果患者未再发生 PSVT,生命体征平稳 ,则可以离院。如患者近期症状频繁发作 ,建议择期行心脏电生理检查及射频消融术。

  1.6统计学方法采用 SPSS15.0 统计软件 ,计量资料数据以x±s表示 ,两组转律时间比较采用t检验 ,计数资料用率表示采用χ2检验,以P<0.05 为差异有显著性。

  2结果

  ATP转律时间明显较普罗帕酮短,ATP组及普罗帕酮组转律时间分别为(0.85±0.30)min、(30.00±15.00)min( P<0.01) 。两组转复心律的成功率均比较高,两者之间统计学无明显差异。在不良反应上ATP组轻度不良反应高于普罗帕酮组,两组在统计学上有明显差异。严重不良反应,普罗帕酮组高于ATP组,两组有统计学上有明显差异。两组转律成功率、转律时间及不良反应具体见表1。表1两组成功率、转律时间、不良反应比较注:与普罗帕酮组比较,*P<0.01,#P>0.05

  3讨论

  心脏电生理研究表明,阵发性室上性心动过速(paroxysmal supraventricular tachycardia,PSVT ),90%以上为房室结折返性心动过速和房室折返性心动过速[2]。折返性心动过速的持续有赖于折返环路各部分组织传导时间的总和长于各组组织的有效不应期,当任何一部分组织传导时间的总和或某部分组织发生阻滞,均可终止折返发作。ATP进入体内迅速酶解为腺苷,其半衰期平均为6s[3],作用产生于其通过血液循环的当时,进入组织后可迅速降解,2min后作用完全消失。腺苷进入血液后特异性地与腺苷 A1 受体结合,使心肌细胞钾离子外流,抑制慢反应纤维的钙离子内流,进而阻滞或延缓房室结折返途径中的前向传导,从而终止房室结参与的折返型室上速,并有很强的拟迷走作用。由于ATP作用时间短 ,可以反复用药及更换其他抗心律失常药物等优点。普罗帕酮为Ic类广谱抗心律失常药物,直接阻断钠通道,延长动作电位及有效不应期,同时具有轻度受体阻滞及钙通道阻滞作用,同时抑制窦房结、心房、房室结、心室的自律性和传导性,并抑制房室旁道。 普罗帕酮对病态心肌有较强的影响室内传导及负性肌力作用[4] 。本研究表明ATP组成功率高于普罗帕酮组,但统计学上差异无显著性。在转律时间上,ATP组明显优于普罗帕酮组,二者在统计学上差异有显著性。轻度不良反应ATP组明显多于普罗帕酮组,但均为一过性,持续时间短。严重不良反应上普罗帕酮组多于ATP组,统计学上差异无显著性。ATP组有1例出现心脏停搏长于6s,患者自觉胸部有压迫感,给予紧急胸外按压,患者出现自主心律。因ATP半衰期短,作用时间短,即使出现严重心律失常,在心脏按压的辅助下均能很快消失。但对于有病窦综合征者、支气管哮喘者禁用,年龄>70岁以上老人慎用。对于孕妇ATP较普罗帕酮安全,且复律时间快。有1例孕妇,给予静推10mg ATP后,20s转复,患者有轻度的胸部憋闷。普罗帕酮组严重不良反应者有5例,其中3例为老年人,静推普罗帕酮35mg后出现心悸、大汗、胸部憋闷、视物模糊,有1例经给予胸外按压后缓解,有2例经给予多巴胺静滴、阿托品静滴后症状渐缓解。有2例出现胸部持续性憋闷,伴有头昏、头晕,心电图示,持续性交界性逸搏、高度房室传导阻滞,经给予阿托品静推、持续静滴异丙肾上腺素,患者上述症状渐缓解,心律恢复正常。后经彩超提示2例心脏均增大。一般认为,对心功能不全或心脏明显扩大者,不宜选用普罗帕酮。综上所述,ATP在转复成功率与普罗帕酮在统计学上无明显差异,但其转复的时间较普罗帕酮短,安全性好,严重副反应少等特点,适于作为临床中止PSVT的一线药物。

【参考文献】
    1王吉耀,瘳二元,胡品津,等.内科学.北京:人民卫生出版社,2005:221-222.

  2何金龙,陈蒙华.三磷酸腺苷应用于阵发性室上性心动过速的研究进展.临床荟萃,2007 ,22(9):681-683.

  3黄瑞健,李萌,孙培吾.生长激素预处理对大鼠心肌缺血再灌注后细胞凋亡与能量代谢的影响.中国医药,2008,3 (8) :449-451.

  4蒋文平.关于心律失常治疗的几个问题.中华心血管杂志,2006,34 (1):94.

日期:2013年2月27日 - 来自[2012年第10卷第11期]栏目

Nature子刊揭示细胞能量感应开关

斯克里普斯研究所(TSRI)的生物化学家们发现一条遗传序列可以改变宿主基因对细胞能量水平的反应。科学家们发现在细菌中这一特殊的能量感应开关可以成为新的一类强有力抗生素的靶点。如果人类基因也发现有相似的能量感应开关,它们或可用于治疗如2型糖尿病和心脏病等代谢相关疾病。研究结果在线发表在10月21日的《自然化学生物学》(Nature Chemical Biology)杂志上。

TSRI斯卡格化学生物学研究所成员、化学生理学和分子生物学系教授Martha J. Fedor说:“这一发现为我们理解生物学中最重要的一个过程——细胞如何感应并处理能量水平提供了新层面。”

燃料传感器

因为它出现在最早由一个基因DNA转录生成的核糖核酸(RNA)链上,这种类型的基因开关序列被称之为核糖开关(riboswitch)。不同于其他已知的核糖开关具有相对有限的功能,这一核糖开关是为所有活细胞供能并控制许多基因的基本分子燃料的一种感受器。

这一新发现的核糖开关可以检测一种称作三磷酸腺苷(ATP)的小分子。ATP是我们星球上所有已知生物体化学能量的标准单位。科学家们过去认为细胞只利用大型的和相对复杂的蛋白质来感应这些至关重要的能量分子,并相应调整细胞活动。并没有人在核糖开关中发现ATP传感器。核糖开关通常是通过阻断基因转录在更基础的水平上来改变细胞的活动。

此外,从前描述的核糖开关是一些影响有限代谢信号通路的相对简单的反馈传感器。其中大多数只感应和调整它们自身宿主基因的表达率。“这是第一个已知与整体代谢调控相关的核糖开关,”Fedor说。

近年来,Fedor小组发现一些迹象表明了有可能存在这样的核糖开关。许多可能具有核糖开关活性的RNA序列还从未被确定特征,一些核糖开关存在于与ATP密切相关的细菌感应分子中。Fedor与她实验室的一位研究生Peter Y. Watson由此开始着手寻找确实可能感应ATP的细菌核糖开关。

当场捕获

这一任务比看起来更具有挑战。Watson不能简单地将质疑的核糖开关暴露于ATP,看哪一个能最好地附着这一能量分子。ATP以高浓度存在于细胞中,它与已知蛋白质传感器的相互作用是必然短暂的、低亲和力的事件。与核糖开关的相互作用预计看起来是一样的。“这样的相互作用真的是太弱,用传统的方法无法检测,”Watson说。然而他发现的数据表明一个RNA与一个ATP样分子发生相互作用的方式使得可利用紫外线辐射当场捕获这一短暂的结合,在两个分子间构建出强有力的化学交联。

通过这种方式,他发现了一段称作ydaO 基序(motif)的明显的ATP结合RNA。Watson对ydaO进行结合绘制分析证实它结合了ATP,并精确确定了它的结合位置。将ydaO附着到一个“报告子”基因上,他在细菌细胞中发现当ATP水平正常时报告基因的表达处于低水平,当ATP水平下降时报告基因的表达水平大幅度升高——如果ydaO是一个真正的ATP感应核糖开关这正如预期。在一种测试B细菌的未改变细胞中,正常包含ydaO基序的基因以相同方式对应ATP水平改变而上升或下降。

这种ydaO基序存在于称作革兰氏阳性菌的大量细菌亚群中。在这些细菌物种中,迄今已在580种基因中发现了ydaO基序。“这些ydaO调控基因编码的蛋白质具有从细胞壁代谢到氨基酸运输等多种功能。控制这些不同过程的核糖开关会对如ATP这样的中心代谢产物做出反应,这是有道理的。”

新的可能性

这一研究发现具有基础科学重要性,因为它是第一个例子证实核糖开关结合ATP,它也是第一次知道具有如此广阔调控功能的核糖开关。“它透露了RNA开关参与一般代谢调控的可能性,”Fedor说。

ydaO基序充当了重要细菌基因的“off-开关”,这一事实使得它们成为了新抗生素的一个潜在的靶点。“用小分子ATP模拟药物击中这些核糖开关,使得它们无法开启促进细菌生长和生存的基因有可能是一种可行的方法,”Fedor.说。

她的实验室将继续在细菌和其他高等生物体中寻找其他的ATP感应核糖开关。一个人类的ATP感应核糖开关,如果用药物适当靶向,或许有可能改变细胞活性,以这种方式帮助治疗常见代谢疾病。2型糖尿病,目前累及全球几亿人群,已知是以细胞能ATP水平的不适当调控为特征。

日期:2012年10月25日 - 来自[细胞分子与蛋白质组]栏目
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膜蛋白ATP分解全程追踪

德国RUB大学的研究人员首次动态追踪了转运蛋白从能量储存分子ATP中得到所需动力的过程。他们通过时间分辨红外光谱技术,检测了细菌膜蛋白MsbA及其互作伙伴ATP的结构改变,文章发表在Journal of Biological Chemistry杂志上。

转运蛋白与多种疾病相关

ATP结合盒(ABC)转运蛋白是负责转运多种物质的细胞膜蛋白。这种膜转运过程的的动力来自于细胞内的通用能量储存分子ATP。ATP分子含有三个磷酸基团,如果其中一个磷酸基团分解就会释放出能量。转运蛋白在癌细胞对化疗药物的多药耐性中起着中心作用,并且与囊性纤维化CF等多种遗传疾病相关,因此在医疗研究中很重要。近年来,研究人员陆续揭开了一些这类转运蛋白的3D原子结构,并揭示了转运循环中的蛋白构象的改变。然而,人们还缺乏对能量携带者ATP分解实现多种物质跨生物膜转运的详细动态机制的了解。

蛋白控制ATP分解 Bochum研究人员在大肠杆菌脂肪转运蛋白MsbA(一种ABC转运蛋白)中首次动态追踪了ATP的水解过程。研究人员采用了傅里叶转换红外光谱技术,研究了MsbA的动力域,即该蛋白中ATP分解发生的位点。通过这一方法,研究人员能检测到蛋白质中纳秒级别的改变。该方法也能同时记录下蛋白与ATP相互作用的变化。

磷酸基团信号揭示ATP分解过程

傅里叶转换红外光谱技术数据分析的难点,是要有效将检测信号与特定分子或分子群对应起来。能够成功做到这一点,就能看到哪些分子在何时发生了结构改变。为此生物物理学家标记了ATP分子的磷酸基团,使它们在光谱分析中留下特征信号。研究人员以这种方式跟踪了ATP与转运蛋白结合的过程,并观察到一个磷酸基团分解并被释放到环境中。“我们的数据为研究ATP水解时蛋白质的运动提供了重要线索。为研究整个膜蛋白打下了基础,而这也是我们下一步研究的方向”,Hofmann教授说。

(生物通编辑:叶予)

日期:2012年7月17日 - 来自[细胞分子与蛋白质组]栏目

植物中的质子泵输

两种类型的质子泵输蛋白,即“液泡H+-ATP酶”和“H+-转位焦磷酸酶” (H+-PPases),共存于植物液泡膜上,将ATP和焦磷酸盐(分别)用作质子转位的能源。虽然“液泡H+-ATP酶”已被进行了非常广泛的研究,但H+-PPases的三维结构及其水解和质子转位反应的详细机制却不清楚。这篇论文报告了在一个“非可水解基质类似物”存在时的H+-PPases的晶体结构。一个不同寻常的质子转位通道由六个核心跨膜螺旋体形成。本文作者们提出了一个关于质子泵输和焦磷酸酶水解是怎样耦合在一起的模型。

日期:2012年4月24日 - 来自[技术要闻]栏目
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生化与细胞所发现ATP门控离子通道P2X3受体信号传导机制

ATP门控离子通道P2X3选择性地表达于初级感觉神经元,对生理性和病理性疼痛至关重要。传统的观点认为,位于神经末梢的P2X3受体激活后可以引起细胞外的钙离子内流进而引起动作电位的发放,而对于P2X3受体的长距离以及长时程的信号传递的方式及其机制并不十分清楚。
12月13日,Cell  Research  在线发表了中科院上海生命科学研究院生化与细胞所鲍岚研究组的研究工作——P2X3受体信号在初级感觉神经元的轴突中具有逆向转运的特性。研究证实,小GTP酶Rab5参与了P2X3受体进入内吞体的过程,Rab7则负责其长距离的逆向转运,P2X3受体的内吞和逆向转运都是受其配体ATP调控的。ATP激活的信号通路分子与内吞的P2X3受体一起进入到内吞体,形成了信号内吞体。神经元膜上的脂筏介导了P2X3受体的内吞和下游信号激活,信号内吞体进一步通过神经元轴突的逆向转运到胞体,调节胞体中转录因子CREB的磷酸化水平,同时影响神经元的兴奋性。
该研究不但证明了感觉神经元中P2X3受体能够逆向运输并以内吞体的形式传递信号,而且提供了一种门控离子通道新的信号传递机制。
该项工作由博士研究生陈序谯和王斌等完成。该工作得到了中国科学院、国家自然科学基金委、科技部蛋白质重大研究计划项目等的资助。
日期:2011年12月16日 - 来自[神经科]栏目
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