Abstract
A distinct conformational transition from the α-helix-rich cellular prion protein (PrPC) into its β-sheet-rich pathological isoform (PrPSc) is the hallmark of prion diseases, a group of fatal transmissible encephalopathies that includes spontaneous and acquired forms. Recently, a PrPSc-like intermediate form characterized by the formation of insoluble aggregates and protease-resistant PrP species termed insoluble PrPC (iPrPC) has been identified in uninfected mammalian brains and cultured neuronal cells, providing new insights into the molecular mechanism(s) of these diseases. Here, we explore the molecular characteristics of the spontaneously formed iPrPC in cultured neuroblastoma cells expressing wild-type or mutant human PrP linked to two familial prion diseases. We observed that although PrP mutation at either residue 183 from Thr to Ala (PrPT183A) or at residue 198 from Phe to Ser (PrPF198S) affects glycosylation at both N-linked glycosylation sites, the T183A mutation that results in intracellular retention significantly increased the formation of iPrPC. Moreover, while autophagy is increased in F198S cells, it was significantly decreased in T183A cells. Our results indicate that iPrPC may be formed more readily in an intracellular compartment and that a significant increase in PrPT183A aggregation may be attributable to the inhibition of autophagy.
Highlights
The abnormal proteinase K (PK)-resistant prion protein (PrPSc) is the only known component of the infectious prions that are associated with a group of fatal neurodegenerative disorders called prion diseases or transmissible spongiform encephalopathies [1]
The molecular weight of PrPF198S decreased more than that of PrPT183A compared to wild type controls, the small degree of change in the molecular weight might not be detectable by Western blotting
Glycosylation at only N197, but not N181, was predicted in the PrPT183A mutation (0.7196, almost identical to that of PrPWt), while the first and second glycosylation sites were predicted for PrPF198S (0.6633 and 0.6988, respectively)
Summary
The abnormal proteinase K (PK)-resistant prion protein (PrPSc) is the only known component of the infectious prions that are associated with a group of fatal neurodegenerative disorders called prion diseases or transmissible spongiform encephalopathies [1]. While it has been well documented that PrPSc is derived from a PK-sensitive cellular PrPC in the central nervous system through an alpha-helix to beta-sheet structural transition, the specific molecular mechanism(s) behind the PrP conversion remain poorly understood [2]. Several plausible theories have emerged, including the prevailing seeding model [3], which explains this conversion with the use of PrPSc seeds that are introduced either by exogenous infection in diseases such as kuru, iatrogenic Creutzfeldt-Jakob disease (CJD) and variant CJD, or formed by endogenous PrPSc molecules including sporadic CJD and various familial prion diseases. The exact molecular nature of the endogenous PrPSc has not been elucidated. In 2006, we first identified small amounts of PKresistant PrP aggregates in uninfected brains of humans, cattle and hamsters [4]. Subsequent studies have revealed similar insoluble structures in a wide www.impactaging.com
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