Department of Biochemistry, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, USA.
Department of Biochemistry, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, USA; Department of Neuroscience, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, West Virginia, USA; WVU Rockefeller Neuroscience Institute, Morgantown, West Virginia, USA; WVU Cancer Institute, Morgantown, West Virginia, USA.
J Biol Chem. 2022 Aug;298(8):102140. doi: 10.1016/j.jbc.2022.102140. Epub 2022 Jun 14.
Proteasome activator 28γ (PA28γ/REGγ) is a member of the 11S family of proteasomal regulators that is constitutively expressed in the nucleus and implicated in various diseases, including certain cancers and systemic lupus erythematosus. Despite years of investigation, how PA28γ functions to stimulate proteasomal protein degradation remains unclear. Alternative hypotheses have been proposed for the molecular mechanism of PA28γ, including the following: (1) substrate selection, (2) allosteric upregulation of the trypsin-like (T-L) site, (3) allosteric inhibition of the chymotrypsin-like (CT-L) and caspase-like (C-L) sites, (4) conversion of the CT-L or C-L sites to new T-L sites, and (5) gate opening alone or in combination with a previous hypothesis. Here, by mechanistically decoupling gating effects from active site effects, we unambiguously demonstrate that WT PA28γ allosterically activates the T-L site. We show PA28γ binding increases the Kcat/Km by 13-fold for T-L peptide substrates while having little-to-no effect on hydrolysis kinetics for CT-L or C-L substrates. Furthermore, mutagenesis and domain swaps of PA28γ reveal that it does not select for T-L peptide substrates through either the substrate entry pore or the distal intrinsically disordered region. We also show that a previously reported point mutation can functionally switch PA28γ from a T-L activating to a gate-opening activator in a mutually exclusive fashion. Finally, using cryogenic electron microscopy, we visualized the PA28γ-proteasome complex at 4.3 Å and confirmed its expected quaternary structure. The results of this study provide unambiguous evidence that PA28γ can function by binding the 20S proteasome to allosterically activate the T-L proteolytic site.
蛋白酶体激活因子 28γ（PA28γ/REGγ）是 11S 家族蛋白酶体调节因子的成员，在细胞核中组成性表达，并与各种疾病有关，包括某些癌症和系统性红斑狼疮。尽管经过多年的研究，PA28γ 如何刺激蛋白酶体蛋白降解的功能仍不清楚。已经提出了 PA28γ 的分子机制的替代假设，包括以下几种：（1）底物选择，（2）对胰蛋白酶样（T-L）位点的变构上调，（3）对糜蛋白酶样（CT-L）和半胱天冬酶样（C-L）位点的变构抑制，（4）将 CT-L 或 C-L 位点转换为新的 T-L 位点，以及（5）门控打开单独或与之前的假设结合。在这里，通过将门控效应与活性位点效应机械地分离，我们明确证明 WT PA28γ 变构激活了 T-L 位点。我们表明，PA28γ 结合将 T-L 肽底物的 kcat/Km 提高了 13 倍，而对 CT-L 或 C-L 底物的水解动力学几乎没有影响。此外，PA28γ 的突变和结构域交换表明，它既不通过底物进入孔也不通过远端无规卷曲区域选择 T-L 肽底物。我们还表明，先前报道的点突变可以以相互排斥的方式将 PA28γ 从 T-L 激活剂功能转换为门控激活剂。最后，使用低温电子显微镜，我们在 4.3 Å 下可视化了 PA28γ-蛋白酶体复合物，并证实了其预期的四级结构。这项研究的结果提供了明确的证据，证明 PA28γ 可以通过与 20S 蛋白酶体结合来变构激活 T-L 蛋白水解位点。
