Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that af...Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that affect the microtubule (MT) stabilization, and the recruitment and activation of coat and cargo proteins, including ADP-ribosylation factors (Arfs) and protein kinase D (PKD). Here, we report on the factors implicated in the regulation of MMP-9 secretion by salivary gland acinar cells in response to P. gingivalis LPS, and the effect of hormone, ghrelin. We show that the LPS-elicited induction in MMP-9 secretion is associated with the increase in α-tubulin acetylation and the enhancement in MT stabilization, while the modulatory effect of ghrelin is reflected in a decrease in α-tubulin acetylation. Further, the effect of the LPS occurs in concert with up-regulation in Arf-guanine nucleotide exchange factor (GEF)-mediated Arf1 activation and the TGN recruitment of PKD2, while ghrelin exerts the modulatory effect on Arf-GEF activation. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. The findings demonstrate that MT stabilization along with Arf-GEF-mediated Arf1/PKD2 activation play a major role in P. gingivalis LPS-induced up-regulation in salivary gland acinar cell MMP-9 secretion, and point the modulatory mode of action by ghrelin.展开更多
目的通过观察按法干预对激痛点骨骼肌细胞骨架α微管蛋白(α-tubulin)和微管相关蛋白4(microtubule-associated protein 4,MAP-4)的影响,探讨按法的舒筋解结作用。方法40只SPF级雄性大鼠随机分为空白组10只和激痛点造模大鼠30只,采用钝...目的通过观察按法干预对激痛点骨骼肌细胞骨架α微管蛋白(α-tubulin)和微管相关蛋白4(microtubule-associated protein 4,MAP-4)的影响,探讨按法的舒筋解结作用。方法40只SPF级雄性大鼠随机分为空白组10只和激痛点造模大鼠30只,采用钝性打击结合离心运动的方法建立大鼠激痛点模型,模型评价后将符合标准的20只大鼠随机分为模型组和按法组,每组10只。空白组和模型组不予按法干预,仅正常观察,按法组以自制按法刺激器干预14 d。干预结束后在激痛点局部取材,用Western blot和免疫荧光双染法检测α-tubulin和MAP-4。结果α-tubulin和MAP-4在肌细胞和细胞外基质均有一定的共表达,空白组α-tubulin和MAP-4主要分布在肌细胞膜与细胞质,分布连续性较好,模型组α-tubulin分布连续性和完整性欠佳,在肌细胞内表达减少,在细胞外基质表达增加,MAP-4在肌细胞和细胞外基质表达均增加,两者在按法组有一定改善。与空白组相比,模型组α-tubulin表达下降、MAP-4表达升高(P<0.05);与模型组相比,按法组α-tubulin表达升高、MAP-4表达下降(P<0.05)。结论按法的舒筋解结作用可能与抑制微管蛋白解聚并促进其合成有关。展开更多
MIcroglia/macrophage-mediated erythrophagocytosis plays a crucial role in hematoma clearance after intracerebral hemorrhage.Dynamic cytoskeletal changes accompany phagocytosis.However,whether and how these changes are...MIcroglia/macrophage-mediated erythrophagocytosis plays a crucial role in hematoma clearance after intracerebral hemorrhage.Dynamic cytoskeletal changes accompany phagocytosis.However,whether and how these changes are associated with microglia/macrophage-mediated erythrophagocytosis remain unclear.In this study,we investigated the function of acetylatedα-tubulin,a stabilized microtubule form,in microglia/macrophage erythrophagocytosis after intracerebral hemorrhage both in vitro and in vivo.We first assessed the function of acetylatedα-tubulin in erythrophagocytosis using primary DiO GFP-labeled red blood cells co-cultured with the BV2 microglia or RAW264.7 macrophage cell lines.Acetylatedα-tubulin expression was significantly decreased in BV2 and RAW264.7 cells during erythrophagocytosis.Moreover,silencingα-tubulin acetyltransferase 1(ATAT1),a newly discoveredα-tubulin acetyltransferase,decreased Ac-α-tub levels and enhanced the erythrophagocytosis by BV2 and RAW264.7 cells.Consistent with these findings,in ATAT1-/-mice,we observed increased ionized calcium binding adapter molecule 1(Iba1)and Perls-positive microglia/macrophage phagocytes of red blood cells in peri-hematoma and reduced hematoma volume in mice with intracerebral hemorrhage.Additionally,knocking out ATAT1 alleviated neuronal apoptosis and pro-inflammatory cytokines and increased anti-inflammatory cytokines around the hematoma,ultimately improving neurological recovery of mice after intracerebral hemorrhage.These findings suggest that ATAT1 deficiency accelerates erythrophagocytosis by microglia/macrophages and hematoma absorption after intracerebral hemorrhage.These results provide novel insights into the mechanisms of hematoma clearance and suggest ATAT1 as a potential target for the treatment of intracerebral hemorrhage.展开更多
Microtubules are highly dynamic cytoskeletal polymers of α/β-tubulin heterodimers that undergo multiple post-translational modifications essential for various cellular functions in eukaryotes. The lysine 40 （K40） ...Microtubules are highly dynamic cytoskeletal polymers of α/β-tubulin heterodimers that undergo multiple post-translational modifications essential for various cellular functions in eukaryotes. The lysine 40 （K40） is largely conserved in α-tubulins in many eukaryote species, and the post-translational modification by acetylation at K40 is critical for neuronal development in vertebrates. However, the biological function of K40 of α-tubulins in plants remains unexplored. In this study, we show in Arabidopsis thaliana that constitutive expression of mutated forms of α-tubulin6 （TUA6） at K40 （TUA6K40A or TUA6K40Q ）, in which K40 is replaced by alanine or glutamine, result in severely reduced plant size. Phenotypic characterization of the 35S：TUA6K40A transgenic plants revealed that both cell proliferation and cell expansion were affected. Cytological and biochemical analyses showed that the accumulation of α- and β-tubulin proteins was significantly reduced in the transgenic plants, and the cortical microtubule arrays were severely disrupted, indicating that K40 of the plant α-tubulin is critical in maintaining microtubule stability. We also constructed 35S：TUA6K40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine, and found that the 35S：TUA6K40R plants were phenotypically indistinguishable from the wild-type. Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated, these results suggest a structural importance for K40 of α-tubulins in cell division and expansion.展开更多
Microtubules (MTs) are regulated by a number of known posttranslational modifications (PTMs) on α/β-tubulin to fulfill diverse cellular functions. Here, we showed that SUMOylation is a novel PTM on α-tubulin in viv...Microtubules (MTs) are regulated by a number of known posttranslational modifications (PTMs) on α/β-tubulin to fulfill diverse cellular functions. Here, we showed that SUMOylation is a novel PTM on α-tubulin in vivo and in vitro. The SUMOylation on α-tubulin mainly occurred at Lys 96 (K96), K166, and K304 of soluble α-tubulin and could be removed by small ubiquitin-related modifier (SUMO)-specific peptidase 1. In vitro experiments showed that tubulin SUMOylation could reduce interprotofilament interaction, promote MT catastrophe, and impede MT polymerization. In cells, mutation of the SUMOylation sites on α-tubulin reduced catastrophe frequency and increased the proportion of polymerized α-tubulin, while upregulation of SUMOylation with fusion of SUMO1 reduced α-tubulin assembly into MTs. Additionally, overexpression of SUMOylation-deficient α-tubulin attenuated the neurite extension in Neuro-2a cells. Thus, SUMOylation on α-tubulin represents a new player in the regulation of MT properties.展开更多
文摘Matrix metalloproteinas-9 (MMP-9) is a glycosylated endopeptidase, and hence its processing between the endoplasmic reticulum (ER), Golgi and trans-Golgi (TGN) network remains under a strict control of factors that affect the microtubule (MT) stabilization, and the recruitment and activation of coat and cargo proteins, including ADP-ribosylation factors (Arfs) and protein kinase D (PKD). Here, we report on the factors implicated in the regulation of MMP-9 secretion by salivary gland acinar cells in response to P. gingivalis LPS, and the effect of hormone, ghrelin. We show that the LPS-elicited induction in MMP-9 secretion is associated with the increase in α-tubulin acetylation and the enhancement in MT stabilization, while the modulatory effect of ghrelin is reflected in a decrease in α-tubulin acetylation. Further, the effect of the LPS occurs in concert with up-regulation in Arf-guanine nucleotide exchange factor (GEF)-mediated Arf1 activation and the TGN recruitment of PKD2, while ghrelin exerts the modulatory effect on Arf-GEF activation. Moreover, we reveal that the LPS-induced up-regulation in MMP-9 secretion is reflected in a marked increase in PKCδ-mediated PKD2 phosphorylation on Ser, while the modulatory effect of ghrelin is manifested by the SFK-PTKs-dependent phosphorylation of PKD2 on Tyr. The findings demonstrate that MT stabilization along with Arf-GEF-mediated Arf1/PKD2 activation play a major role in P. gingivalis LPS-induced up-regulation in salivary gland acinar cell MMP-9 secretion, and point the modulatory mode of action by ghrelin.
文摘目的通过观察按法干预对激痛点骨骼肌细胞骨架α微管蛋白(α-tubulin)和微管相关蛋白4(microtubule-associated protein 4,MAP-4)的影响,探讨按法的舒筋解结作用。方法40只SPF级雄性大鼠随机分为空白组10只和激痛点造模大鼠30只,采用钝性打击结合离心运动的方法建立大鼠激痛点模型,模型评价后将符合标准的20只大鼠随机分为模型组和按法组,每组10只。空白组和模型组不予按法干预,仅正常观察,按法组以自制按法刺激器干预14 d。干预结束后在激痛点局部取材,用Western blot和免疫荧光双染法检测α-tubulin和MAP-4。结果α-tubulin和MAP-4在肌细胞和细胞外基质均有一定的共表达,空白组α-tubulin和MAP-4主要分布在肌细胞膜与细胞质,分布连续性较好,模型组α-tubulin分布连续性和完整性欠佳,在肌细胞内表达减少,在细胞外基质表达增加,MAP-4在肌细胞和细胞外基质表达均增加,两者在按法组有一定改善。与空白组相比,模型组α-tubulin表达下降、MAP-4表达升高(P<0.05);与模型组相比,按法组α-tubulin表达升高、MAP-4表达下降(P<0.05)。结论按法的舒筋解结作用可能与抑制微管蛋白解聚并促进其合成有关。
基金supported by Science and Technology Innovation Enhancement Project of Army Medical University(to LX).
文摘MIcroglia/macrophage-mediated erythrophagocytosis plays a crucial role in hematoma clearance after intracerebral hemorrhage.Dynamic cytoskeletal changes accompany phagocytosis.However,whether and how these changes are associated with microglia/macrophage-mediated erythrophagocytosis remain unclear.In this study,we investigated the function of acetylatedα-tubulin,a stabilized microtubule form,in microglia/macrophage erythrophagocytosis after intracerebral hemorrhage both in vitro and in vivo.We first assessed the function of acetylatedα-tubulin in erythrophagocytosis using primary DiO GFP-labeled red blood cells co-cultured with the BV2 microglia or RAW264.7 macrophage cell lines.Acetylatedα-tubulin expression was significantly decreased in BV2 and RAW264.7 cells during erythrophagocytosis.Moreover,silencingα-tubulin acetyltransferase 1(ATAT1),a newly discoveredα-tubulin acetyltransferase,decreased Ac-α-tub levels and enhanced the erythrophagocytosis by BV2 and RAW264.7 cells.Consistent with these findings,in ATAT1-/-mice,we observed increased ionized calcium binding adapter molecule 1(Iba1)and Perls-positive microglia/macrophage phagocytes of red blood cells in peri-hematoma and reduced hematoma volume in mice with intracerebral hemorrhage.Additionally,knocking out ATAT1 alleviated neuronal apoptosis and pro-inflammatory cytokines and increased anti-inflammatory cytokines around the hematoma,ultimately improving neurological recovery of mice after intracerebral hemorrhage.These findings suggest that ATAT1 deficiency accelerates erythrophagocytosis by microglia/macrophages and hematoma absorption after intracerebral hemorrhage.These results provide novel insights into the mechanisms of hematoma clearance and suggest ATAT1 as a potential target for the treatment of intracerebral hemorrhage.
基金supported by grants from the Chinese National Scientific Foundation (30800601/31070163)the Chinese Academy of Sciences (KSCX2-EW-Q-1-04)the support of SA-SIBS Scholarship Program
文摘Microtubules are highly dynamic cytoskeletal polymers of α/β-tubulin heterodimers that undergo multiple post-translational modifications essential for various cellular functions in eukaryotes. The lysine 40 （K40） is largely conserved in α-tubulins in many eukaryote species, and the post-translational modification by acetylation at K40 is critical for neuronal development in vertebrates. However, the biological function of K40 of α-tubulins in plants remains unexplored. In this study, we show in Arabidopsis thaliana that constitutive expression of mutated forms of α-tubulin6 （TUA6） at K40 （TUA6K40A or TUA6K40Q ）, in which K40 is replaced by alanine or glutamine, result in severely reduced plant size. Phenotypic characterization of the 35S：TUA6K40A transgenic plants revealed that both cell proliferation and cell expansion were affected. Cytological and biochemical analyses showed that the accumulation of α- and β-tubulin proteins was significantly reduced in the transgenic plants, and the cortical microtubule arrays were severely disrupted, indicating that K40 of the plant α-tubulin is critical in maintaining microtubule stability. We also constructed 35S：TUA6K40R transgenic plants in which K40 of the engineered TUA6 protein is replaced by an arginine, and found that the 35S：TUA6K40R plants were phenotypically indistinguishable from the wild-type. Since lysine and arginine are similar in biochemical nature but arginine cannot be acetylated, these results suggest a structural importance for K40 of α-tubulins in cell division and expansion.
基金This work was supported by grants from the National Natural Science Foundation of China(31991194 and 31330046)the Strategic Priority Research Program of the Chinese Academy of Sciences(XDB19000000)Shanghai Science and Technology Committee(18JC1420301).
文摘Microtubules (MTs) are regulated by a number of known posttranslational modifications (PTMs) on α/β-tubulin to fulfill diverse cellular functions. Here, we showed that SUMOylation is a novel PTM on α-tubulin in vivo and in vitro. The SUMOylation on α-tubulin mainly occurred at Lys 96 (K96), K166, and K304 of soluble α-tubulin and could be removed by small ubiquitin-related modifier (SUMO)-specific peptidase 1. In vitro experiments showed that tubulin SUMOylation could reduce interprotofilament interaction, promote MT catastrophe, and impede MT polymerization. In cells, mutation of the SUMOylation sites on α-tubulin reduced catastrophe frequency and increased the proportion of polymerized α-tubulin, while upregulation of SUMOylation with fusion of SUMO1 reduced α-tubulin assembly into MTs. Additionally, overexpression of SUMOylation-deficient α-tubulin attenuated the neurite extension in Neuro-2a cells. Thus, SUMOylation on α-tubulin represents a new player in the regulation of MT properties.
