Quantitative proteomics and phosphoproteomics reveal insights into mechanisms of ocnus function in Drosophila testis development.
Quantitative proteomics and phosphoproteomics reveal insights into mechanisms of ocnus function in Drosophila testis development.
复制标题
DOI：
10.1186/s12864-023-09386-2
复制
发表时间：
2023-05-26
期刊：
影响因子：
4.4
通讯作者：
中科院分区：
文献类型：
--
作者：
睾丸是动物中唯一支持精子产生的器官，具有最多的蛋白质和组织特异性蛋白质。在我们以前的研究中，我们发现敲除睾丸特异性基因ocnus（ocn）后，果蝇的睾丸变小，没有生殖细胞。然而，果蝇睾丸中ocn基因敲低的分子后果尚不清楚。在这项研究中，通过iTRAQ定量蛋白质组学测序，从果蝇睾丸中鉴定出606种蛋白质，这些蛋白质在果蝇睾丸中的ocn敲低后表达发生了显著且至少1.5倍的变化，其中85种上调，521种下调。在差异表达蛋白中，除了与精子发生有关的蛋白外，其他蛋白广泛影响前体代谢物和能量的产生、代谢过程和线粒体转运等生物学过程。蛋白质-蛋白质相互作用（PPI）分析的DEP表明，几个激酶和/或磷酸酶与OCN相互作用。转录组再分析显示，在DEP中出现了150个差异表达基因（DEG），它们在ocn敲低后的表达变化趋势是一致的。许多常见的下调DEG和DEPs在D.黑腹菌定量RT-PCR（qRT-PCR）证实，在果蝇睾丸中，ocn基因敲低后，DEG和DEPs中的12个基因表达显著下调。此外，还鉴定了153个差异表达磷蛋白（DEPP），包括72个上调和94个下调的磷酸化蛋白（13个磷蛋白由于具有多个磷酸化位点而同时出现在上调和下调组中）。除了与精子发生相关的DEPP外，其他DEPP在基于肌动蛋白的过程、蛋白质折叠和中胚层发育中富集。一些DEPs和DEPPs参与Notch、JAK/STAT和细胞死亡途径。考虑到ocn基因敲除对组织发育和睾丸细胞组成的强烈影响，在ocn基因敲除果蝇中蛋白质丰度的差异可能不一定是由于ocn失活导致的基因调控差异的直接结果。然而，我们的研究结果表明，ocn的表达是果蝇睾丸发育所必需的，其下调干扰了与细胞存活和分化相关的关键信号通路。这些DEPs和DEPP的鉴定可能为未来研究动物（包括人类）的雄性生殖机制提供重要的候选集。在线版本包含补充材料，可通过10.1186/s12864-023-09386-2获取。
Testis is the only organ supporting sperm production and with the largest number of proteins and tissue-specific proteins in animals. In our previous studies, we have found that knockdown of ocnus (ocn), a testis-specific gene, resulted in much smaller testis with no germ cells in Drosophila melanogaster. However, the molecular consequences of ocn knockdown in fly testes are unknown. In this study, through iTRAQ quantitative proteomics sequencing, 606 proteins were identified from fly abdomens as having a significant and at least a 1.5-fold change in expression after ocn knockdown in fly testes, of which 85 were up-regulated and 521 were down-regulated. Among the differential expressed proteins (DEPs), apart from those proteins involved in spermatogenesis, the others extensively affected biological processes of generation of precursor metabolites and energy, metabolic process, and mitochondrial transport. Protein-protein interaction (PPI) analyses of DEPs showed that several kinases and/or phosphatases interacted with Ocn. Re-analyses of the transcriptome revealed 150 differential expressed genes (DEGs) appeared in the DEPs, and their changing trends in expressions after ocn knockdown were consistent. Many common down-regulated DEGs and DEPs were testis-specific or highly expressed in the testis of D. melanogaster. Quantitative RT-PCR (qRT-PCR) confirmed 12 genes appeared in both DEGs and DEPs were significantly down-regulated after ocn knockdown in fly testes. Furthermore, 153 differentially expressed phosphoproteins (DEPPs), including 72 up-regulated and 94 down-regulated phosphorylated proteins were also identified (13 phosphoproteins appeared in both up- and down-regulated groups due to having multiple phosphorylation sites). In addition to those DEPPs associated with spermatogenesis, the other DEPPs were enriched in actin filament-based process, protein folding, and mesoderm development. Some DEPs and DEPPs were involved in Notch, JAK/STAT, and cell death pathways. Given the drastic effect of the ocn knockdown on tissue development and testis cells composition, the differences in protein abundance in the ocn knockdown flies might not necessarily be the direct result of differential gene regulation due to the inactivation of ocn. Nevertheless, our results suggest that the expression of ocn is essential for Drosophila testis development and that its down-regulation disturbs key signaling pathways related to cell survival and differentiation. These DEPs and DEPPs identified may provide significant candidate set for future studies on the mechanism of male reproduction of animals, including humans. The online version contains supplementary material available at 10.1186/s12864-023-09386-2.
登录
查看更多内容
影响因子：
56.9
作者：
Furlong, EEM;Andersen, EC;Scott, MP
通讯作者：
Scott, MP
影响因子：
4.8
作者：
Demarco, Rafael S.;Eikenes, Asmund H.;Haglund, Kaisa;Jones, D. Leanne
通讯作者：
Jones, D. Leanne
影响因子：
3.3
作者：
Deracinois B;Flahaut C;Duban-Deweer S;Karamanos Y
通讯作者：
Karamanos Y
影响因子：
5.8
作者：
Cheng, Alice;Grant, Charles E.;Bailey, Timothy L.
通讯作者：
Bailey, Timothy L.
影响因子：
21.3
作者：
Demarco, Rafael Senos;Uyemura, Bradley S.;Jones, D. Leanne
通讯作者：
Jones, D. Leanne