Institute of Cytology of the Russian Academy of Sciences, 4 Tikhoretsky Ave., 194064 Saint-Petersburg, Russia.
Institute of Nanobiotechnologies, Peter the Great St. Petersburg Polytechnic University, 29 Polytechnicheskaya Str., 195251 Saint-Petersburg, Russia.
Molecules. 2020 Jul 31;25(15):3504. doi: 10.3390/molecules25153504.
The ubiquitin-proteasome system (UPS) is one of the major protein degradation pathways in eukaryotic cells. Abnormal functioning of this system has been observed in cancer and neurological diseases. The 20S proteasomes, essential components of the UPS, are present not only within the cells but also in the extracellular space, and their concentration in blood plasma has been found to be elevated and dependent upon the disease state, being of prognostic significance in patients suffering from cancer, liver diseases, and autoimmune diseases. However, functions of extracellular proteasomes and mechanisms of their release by cells remain largely unknown. The main mechanism of proteasome activity regulation is provided by modulation of their composition and post-translational modifications (PTMs). Moreover, diverse PTMs of proteins are known to participate in the loading of specific elements into extracellular vesicles. Since previous studies have revealed that the transport of extracellular proteasomes may occur via extracellular vesicles, we have set out to explore the PTMs of extracellular proteasomes in comparison to cellular counterparts. In this work, cellular and extracellular proteasomes were affinity purified and separated by SDS-PAGE for subsequent trypsinization and matrix-assisted laser desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) analysis. In total, we could identify 64 and 55 PTM sites in extracellular and cellular proteasomes, respectively, including phosphorylation, ubiquitination, acetylation, and succinylation. We observed novel sites of acetylation at K238 and K192 of the proteasome subunits β2 and β3, respectively, that are specific for extracellular proteasomes. Moreover, cellular proteasomes show specific acetylation at K227 of α2 and ubiquitination at K201 of β3. Interestingly, succinylation of β6 at the residue K228 seems not to be present exclusively in extracellular proteasomes, whereas both extracellular and cellular proteasomes may also be acetylated at this site. The same situation takes place at K201 of the β3 subunit where ubiquitination is seemingly specific for cellular proteasomes. Moreover, crosstalk between acetylation, ubiquitination, and succinylation has been observed in the subunit α3 of both proteasome populations. These data will serve as a basis for further studies, aimed at dissection of the roles of extracellular proteasome-specific PTMs in terms of the function of these proteasomes and mechanism of their transport into extracellular space.
泛素-蛋白酶体系统（UPS）是真核细胞中主要的蛋白质降解途径之一。该系统的异常功能已在癌症和神经退行性疾病中观察到。20S 蛋白酶体是 UPS 的重要组成部分，不仅存在于细胞内，也存在于细胞外空间，其在血浆中的浓度升高且与疾病状态相关，在癌症、肝脏疾病和自身免疫性疾病患者中具有预后意义。然而，细胞外蛋白酶体的功能及其释放机制在很大程度上仍然未知。蛋白酶体活性的主要调节机制是通过调节其组成和翻译后修饰（PTMs）来提供的。此外，已知多种蛋白质的 PTM 参与将特定元件加载到细胞外囊泡中。由于先前的研究表明细胞外蛋白酶体的运输可能通过细胞外囊泡发生，因此我们着手探索细胞外蛋白酶体与细胞内蛋白酶体相比的 PTMs。在这项工作中，我们通过 SDS-PAGE 亲和纯化和分离细胞内和细胞外蛋白酶体，然后进行胰蛋白酶消化和基质辅助激光解吸/电离（MALDI）傅里叶变换离子回旋共振（FT-ICR）质谱（MS）分析。总共，我们可以在细胞外和细胞内蛋白酶体中分别鉴定出 64 个和 55 个 PTM 位点，包括磷酸化、泛素化、乙酰化和琥珀酰化。我们观察到蛋白酶体亚基 β2 和 β3 上 K238 和 K192 处的新乙酰化位点，这些位点是细胞外蛋白酶体所特有的。此外，细胞蛋白酶体在 α2 的 K227 处显示出特异性乙酰化，在 β3 的 K201 处显示出泛素化。有趣的是，β6 残基 K228 上的琥珀酰化似乎不仅存在于细胞外蛋白酶体中，而且细胞外和细胞内蛋白酶体也可能在此位点被乙酰化。β3 亚基上的 K201 也存在同样的情况，其中泛素化似乎是细胞内蛋白酶体所特有的。此外，在两种蛋白酶体群体的亚基 α3 中观察到乙酰化、泛素化和琥珀酰化之间的串扰。这些数据将为进一步的研究提供基础，旨在剖析细胞外蛋白酶体特异性 PTMs 在这些蛋白酶体功能及其进入细胞外空间的运输机制中的作用。
