Department of Microbiology and Biotechnology, Daejeon University, Daejeon 300-716, Republic of Korea.
J Proteomics. 2013 Jul 11;87:40-52. doi: 10.1016/j.jprot.2013.05.009. Epub 2013 May 18.
FlbA is a regulator of G-protein signaling protein that plays a central role in attenuating heterotrimeric G-protein mediated vegetative growth signaling in Aspergillus. The deletion of flbA (∆flbA) in the opportunistic human pathogen Aspergillus fumigatus results in accelerated cell death and autolysis in submerged culture. To further investigate the effects of ∆flbA on intracellular protein levels we carried out 2-D proteome analyses of 2-day old submerged cultures of ∆flbA and wild type (WT) strains and observed 160 differentially expressed proteins. Via nano-LC-ESI-MS/MS analyses, we revealed the identity of 10 and 2 proteins exhibiting high and low level accumulation, respectively, in ∆flbA strain. Notably, the GliT protein is accumulated at about 1800-fold higher levels in ∆flbA than WT. Moreover, GliT is secreted at high levels from ∆flbA strain, whereas Sod1 (superoxide dismutase) is secreted at a higher level in WT. Northern blot analyses reveal that ∆flbA results in elevated accumulation of gliT mRNA. Consequently, ∆flbA strain exhibits enhanced tolerance to gliotoxin toxicity. Finally, ∆flbA strain displayed enhanced SOD activity and elevated resistance to menadione and paraquat. In summary, FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and of exogenous gliotoxin in A. fumigatus.
Regulator of G protein Signaling (RGS) proteins play crucial roles in fundamental biological processes in filamentous fungi. FlbA is the first studied filamentous fungal RGS protein, yet much remains to be understood about its roles in the opportunistic human pathogen Aspergillus fumigatus. In the present study, we examined the effects of the deletion of flbA using comprehensive analyses of the intra- and extracellular proteomes of A. fumigatus wild type and the flbA deletion mutant. Via MS analyses, we identified 10 proteins exhibiting high level accumulation in the flbA deletion mutant and 8 proteins differentially secreted in wild type and the flbA mutant. Based on proteomic analyses, we further examined the role of FlbA and found that FlbA down-regulates gliT expression and SOD activity. Our results proposed that FlbA-mediated signaling control negatively affects cellular responses associated with detoxification of reactive oxygen species and exogenous gliotoxin in A. fumigatus.
FlbA 是一种 G 蛋白信号转导蛋白的调节剂，在衰减曲霉属异源三聚体 G 蛋白介导的营养生长信号转导中起核心作用。机会性病原体烟曲霉中 flbA（∆flbA）的缺失导致浸没培养中的细胞死亡和自溶加速。为了进一步研究 ∆flbA 对细胞内蛋白质水平的影响，我们对 2 天龄的 ∆flbA 和野生型（WT）菌株的浸没培养物进行了 2D 蛋白质组分析，观察到 160 种差异表达的蛋白质。通过纳升 LC-ESI-MS/MS 分析，我们揭示了在 ∆flbA 菌株中高水平和低水平积累的 10 种和 2 种蛋白质的身份。值得注意的是，GliT 蛋白在 ∆flbA 中的积累水平比 WT 高约 1800 倍。此外，GliT 从 ∆flbA 菌株中大量分泌，而 Sod1（超氧化物歧化酶）在 WT 中分泌水平更高。Northern blot 分析显示，∆flbA 导致 gliT mRNA 的积累增加。因此，∆flbA 菌株表现出对Gliotoxin 毒性的增强耐受性。最后，∆flbA 菌株表现出增强的 SOD 活性和对 Menadione 和 Paraquat 的更高抗性。总之，FlbA 介导的信号转导控制对与曲霉属中活性氧和外源 Gliotoxin 解毒相关的细胞反应产生负面影响。
G 蛋白信号转导（RGS）蛋白在丝状真菌的基本生物学过程中起着至关重要的作用。FlbA 是研究的第一个丝状真菌 RGS 蛋白，但在机会性病原体烟曲霉中，其作用仍有许多有待了解。在本研究中，我们使用 A. fumigatus 野生型和 flbA 缺失突变体的细胞内和细胞外蛋白质组的综合分析来研究 flbA 缺失的影响。通过 MS 分析，我们鉴定了在 flbA 缺失突变体中高水平积累的 10 种蛋白质和在野生型和 flbA 突变体中差异分泌的 8 种蛋白质。基于蛋白质组学分析，我们进一步研究了 FlbA 的作用，发现 FlbA 下调 gliT 表达和 SOD 活性。我们的结果表明，FlbA 介导的信号转导控制对与曲霉属中活性氧和外源 Gliotoxin 解毒相关的细胞反应产生负面影响。
