Tryptophan residue 32 in human Cu-Zn superoxide dismutase modulates prion-like propagation and strain selection

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) associated with familial amyotrophic lateral sclerosis cause the protein to aggregate via a prion-like process in which soluble molecules are recruited to aggregates by conformational templating. These misfolded SOD1 proteins can propagate aggregation-inducing conformations across cellular membranes. Prior studies demonstrated that mutation of a Trp (W) residue at position 32 to Ser (S) suppresses the propagation of misfolded conformations between cells, whereas other studies have shown that mutation of Trp 32 to Phe (F), or Cys 111 to Ser, can act in cis to attenuate aggregation of mutant SOD1. By expressing mutant SOD1 fused with yellow fluorescent protein (YFP), we compared the relative ability of these mutations to modulate the formation of inclusions by ALS-mutant SOD1 (G93A and G85R). Only mutation of Trp 32 to Ser persistently reduced the formation of the amorphous inclusions that form in these cells, consistent with the idea that a Ser at position 32 inhibits templated propagation of aggregation prone conformations. To further test this idea, we produced aggregated fibrils of recombinant SOD1-W32S in vitro and injected them into the spinal cords of newborn mice expressing G85R-SOD1: YFP. The injected mice developed an earlier onset paralysis with a frequency similar to mice injected with WT SOD1 fibrils, generating a strain of misfolded SOD1 that produced highly fibrillar inclusion pathology. These findings suggest that the effect of Trp 32 in modulating the propagation of misfolded SOD1 conformations may be dependent upon the “strain” of the conformer that is propagating.

与家族性肌萎缩侧索硬化症相关的铜锌超氧化物歧化酶1（Cu/Zn superoxide dismutase 1, SOD1）突变，可通过朊病毒样过程引发该蛋白聚集：可溶性分子通过构象模板化机制被招募至聚集体中。此类错误折叠的SOD1蛋白可通过细胞膜传播诱导聚集的构象。既往研究显示，将32位色氨酸（Trp, W）突变为丝氨酸（Ser, S）可抑制细胞间错误折叠构象的传播；另有研究表明，将32位色氨酸突变为苯丙氨酸（Phe, F），或111位半胱氨酸突变为丝氨酸，可在顺式作用下减弱突变型SOD1的聚集。本研究通过表达与黄色荧光蛋白（yellow fluorescent protein, YFP）融合的突变型SOD1，对比了上述突变对肌萎缩侧索硬化症相关突变型SOD1（G93A与G85R）形成包涵体的相对调控能力。仅32位色氨酸突变为丝氨酸，可持续减少此类细胞中形成的无定形包涵体，这与“32位丝氨酸可抑制聚集倾向性构象的模板化传播”这一假说相符。为进一步验证该假说，我们体外制备了重组SOD1-W32S的聚集纤维，并将其注射至表达G85R-SOD1:YFP的新生小鼠脊髓中。注射后的小鼠出现了更早的瘫痪发作，其发病频率与注射野生型SOD1纤维的小鼠相近，由此产生了一种可引发高度纤维状包涵体病理的错误折叠SOD1构象株。本研究结果提示，32位色氨酸对错误折叠SOD1构象传播的调控作用，可能取决于所传播构象的“株型”。