Dipartimento Scienze Chimiche, Università degli Studi di Catania, Viale A. Doria 6, 95125 Catania, Italy.
Istituto di Cristallografia-CNR Sede Secondaria di Catania, Via P. Gaifami 18, 95126 Catania, Italy.
Biomolecules. 2023 Apr 20;13(4):704. doi: 10.3390/biom13040704.
Many chronic diseases, including cancer and neurodegeneration, are linked to proteasome dysregulation. Proteasome activity, essential for maintaining proteostasis in a cell, is controlled by the gating mechanism and its underlying conformational transitions. Thus, developing effective methods to detect gate-related specific proteasome conformations could be a significant contribution to rational drug design. Since the structural analysis suggests that gate opening is associated with a decrease in the content of α-helices and β-sheets and an increase in random coil structures, we decided to explore the application of electronic circular dichroism (ECD) in the UV region to monitor the proteasome gating. A comparison of ECD spectra of wild type yeast 20S proteasome (predominantly closed) and an open-gate mutant (α3ΔN) revealed an increased intensity in the ECD band at 220 nm, which suggests increased contents of random coil and β-turn structures. This observation was further supported by evaluating ECD spectra of human 20S treated with low concentration of SDS, known as a gate-opening reagent. Next, to evaluate the power of ECD to probe a ligand-induced gate status, we treated the proteasome with H2T4, a tetracationic porphyrin that we showed previously to induce large-scale protein conformational changes upon binding to h20S. H2T4 caused a significant increase in the ECD band at 220 nm, interpreted as an induced opening of the 20S gate. In parallel, we imaged the gate-harboring alpha ring of the 20S with AFM, a technique that we used previously to visualize the predominantly closed gate in latent human or yeast 20S and the open gate in α3ΔN mutant. The results were convergent with the ECD data and showed a marked decrease in the content of closed-gate conformation in the H2T4-treated h20S. Our findings provide compelling support for the use of ECD measurements to conveniently monitor proteasome conformational changes related to gating phenomena. We predict that the observed association of spectroscopic and structural results will help with efficient design and characterization of exogenous proteasome regulators.
许多慢性疾病，包括癌症和神经退行性疾病，都与蛋白酶体的失调有关。蛋白酶体的活性对于维持细胞内的蛋白质平衡至关重要，它受到门控机制及其潜在构象转变的控制。因此，开发有效的方法来检测与门相关的特定蛋白酶体构象可能是对合理药物设计的重大贡献。由于结构分析表明，门的打开与α-螺旋和β-折叠含量的降低以及无规卷曲结构的增加有关，我们决定探索电子圆二色性（ECD）在 UV 区域的应用，以监测蛋白酶体的门控。野生型酵母 20S 蛋白酶体（主要关闭）和开门口突变体（α3ΔN）的 ECD 光谱比较显示，220nm 处 ECD 带的强度增加，这表明无规卷曲和β-转角结构的含量增加。这一观察结果进一步得到了用低浓度 SDS 处理人 20S 的 ECD 光谱评估的支持，SDS 是一种已知的门打开试剂。接下来，为了评估 ECD 探测配体诱导的门状态的能力，我们用 H2T4 处理蛋白酶体，H2T4 是一种四阳离子卟啉，我们之前的研究表明，它在与 h20S 结合时会引起大规模的蛋白质构象变化。H2T4 导致 220nm 处 ECD 带显著增加，解释为 20S 门的诱导打开。平行地，我们用原子力显微镜（AFM）对 20S 的α环进行成像，我们之前曾使用该技术来可视化潜伏的人或酵母 20S 的主要关闭门和α3ΔN 突变体的打开门。结果与 ECD 数据一致，表明在 H2T4 处理的 h20S 中，关闭门构象的含量明显减少。我们的发现为使用 ECD 测量方便地监测与门控现象相关的蛋白酶体构象变化提供了有力支持。我们预测，观察到的光谱和结构结果之间的关联将有助于外源性蛋白酶体调节剂的有效设计和表征。
