Ghag Gaurav, Holler Christopher J, Taylor Georgia, Kukar Thomas L, Uversky Vladimir N, Rangachari Vijayaraghavan
Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, Mississippi, 39406.
Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia, 30322.
Protein Sci. 2017 Sep;26(9):1759-1772. doi: 10.1002/pro.3212. Epub 2017 Jun 22.
Granulins (GRNs) are a family of small (∼6 kDa) proteins generated by the proteolytic processing of their precursor, progranulin (PGRN), in many cell types. Both PGRN and GRNs are implicated in a plethora of biological functions, often in opposing roles to each other. Lately, GRNs have generated significant attention due to their implicated roles in neurodegenerative disorders. Despite their physiological and pathological significance, the structure-function relationships of GRNs are poorly defined. GRNs contain 12 conserved cysteines forming six intramolecular disulfide bonds, making them rather exceptional, even among a few proteins with high disulfide bond density. Solution NMR investigations in the past have revealed a unique structure containing putative interdigitated disulfide bonds for several GRNs, but GRN-3 was unsolvable due to its heterogeneity and disorder. In our previous report, we showed that abrogation of disulfide bonds in GRN-3 renders the protein completely disordered (Ghag et al., Prot Eng Des Sel 2016). In this study, we report the cellular expression and biophysical analysis of fully oxidized, native GRN-3. Our results indicate that both E. coli and human embryonic kidney (HEK) cells do not exclusively make GRN-3 with homogenous disulfide bonds, likely due to the high cysteine density within the protein. Biophysical analysis suggests that GRN-3 structure is dominated by irregular loops held together only by disulfide bonds, which induced remarkable thermal stability to the protein despite the lack of regular secondary structure. This unusual handshake between disulfide bonds and disorder within GRN-3 could suggest a unique adaptation of intrinsically disordered proteins towards structural stability.
颗粒蛋白（GRNs）是一类小分子（约6 kDa）蛋白质家族，由其前体前颗粒蛋白（PGRN）在多种细胞类型中经蛋白水解加工产生。PGRN和GRNs都参与了大量生物学功能，且二者作用往往相反。最近，GRNs因其在神经退行性疾病中的作用而备受关注。尽管它们具有生理和病理意义，但GRNs的结构-功能关系仍不清楚。GRNs含有12个保守的半胱氨酸，形成6个分子内二硫键，这使它们相当独特，即使在少数具有高二硫键密度的蛋白质中也是如此。过去的溶液核磁共振研究揭示了几种GRNs含有假定的相互交叉二硫键的独特结构，但GRN-3由于其异质性和无序性而无法解析。在我们之前的报告中，我们表明GRN-3中二硫键的消除使该蛋白完全无序（Ghag等人，《蛋白质工程与设计》，2016年）。在本研究中，我们报告了完全氧化的天然GRN-3的细胞表达和生物物理分析。我们的结果表明，大肠杆菌和人胚肾（HEK）细胞都不会只产生具有均匀二硫键的GRN-3，这可能是由于该蛋白中高半胱氨酸密度所致。生物物理分析表明，GRN-3的结构主要由仅通过二硫键维系在一起的不规则环组成，尽管缺乏规则的二级结构，但这些二硫键赋予了该蛋白显著的热稳定性。GRN-3中二硫键与无序之间这种不寻常的关系可能暗示了内在无序蛋白对结构稳定性的一种独特适应。
