IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.
Centre for Molecular and Structural Biomedicine, CBME/IBB, LA, Portugal ; Current address: Biophysics Section, Department of Life Sciences, Imperial College, London, UK.
Comput Struct Biotechnol J. 2013 Sep 10;7:e201304006. doi: 10.5936/csbj.201304006. eCollection 2013.
Protein degradation is essential for maintaining cellular homeostasis. The proteasome is the central enzyme responsible for non-lysosomal protein degradation in eukaryotic cells. Although proteasome assembly is not yet completely understood, a number of cofactors required for proper assembly and maturation have been identified. Ump is a short-lived maturation factor required for the efficient biogenesis of the 20S proteasome. Upon the association of the two precursor complexes, Ump is encased and is rapidly degraded after the proteolytic sites in the interior of the nascent proteasome are activated. In order to further understand the mechanisms behind proteasomal maturation, we expressed and purified yeast Ump in E. coli for biophysical and structural analysis. We show that recombinant Ump is purified as a mixture of different oligomeric species and that oligomerization is mediated by intermolecular disulfide bond formation involving the only cysteine residue present in the protein. Furthermore, a combination of bioinformatic, biochemical and structural analysis revealed that Ump shows characteristics of an intrinsically disordered protein, which might become structured only upon interaction with the proteasome subunits.
蛋白质降解对于维持细胞内稳态至关重要。蛋白酶体是真核细胞中非溶酶体蛋白降解的核心酶。尽管蛋白酶体的组装尚未完全了解，但已经鉴定出许多用于正确组装和成熟的辅助因子。Ump 是一种短寿命的成熟因子，对于 20S 蛋白酶体的有效生物发生是必需的。在前体复合物结合后，Ump 被包裹起来，并在新生蛋白酶体内部的蛋白水解位点被激活后迅速降解。为了进一步了解蛋白酶体成熟的机制，我们在大肠杆菌中表达和纯化了酵母 Ump 进行生物物理和结构分析。我们表明，重组 Ump 作为不同寡聚体的混合物被纯化，并且寡聚化是通过涉及蛋白质中唯一半胱氨酸残基的分子间二硫键形成来介导的。此外，生物信息学、生化和结构分析的组合表明，Ump 表现出固有无序蛋白的特征，其可能仅在与蛋白酶体亚基相互作用时才变得有结构。
