Research Article: Characterization of spontaneously generated prion-like conformers in cultured cells

Date Published: October 9, 2011

Publisher: Impact Journals LLC

Author(s): Roger S. Zou, Hisashi Fujioka, Jian-Ping Guo, Xiangzhu Xiao, Miyuki Shimoji, Crystal Kong, Cecilia Chen, Megan Tasnadi, Chesinta Voma, Jue Yuan, Mohammed Moudjou, Hubert Laude, Robert B. Petersen, Wen-Quan Zou.



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.

Partial Text

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. However, the exact molecular nature of the endogenous PrPSc has not been elucidated.

The two N-linked glycosylation sites located at residue 181, Asn-Ile-Thr residues 181-183, and residue 197, Asn-Phe-Thr residues 197-199 [21] have been believed to play a crucial role in the stabilization of prion protein conformation. The naturally occurring mutations at residue 183, Thr to Ala (PrPT183A), or at residue 198, Phe to Ser (PrPF198S), falling in the two consensus sites are linked to two distinct familial prion diseases [22, 23]. Elimination of each of the two sites or two sites together by mutagenesis of hamster PrP in CV1 cells induced the intracellular accumulation of mutant proteins [24]. Lehman and Harris observed that mouse PrP mutated at T182 alone or at both T182 and T198 in CHO cells failed to reach the cell surface but those mutated at T198 did. Moreover, all three mutant PrP acquired PrPSc-like physicochemical properties reminiscent of PrPSc while to a limited extent PrPWt did so only when synthesized in the presence of inhibitor tunicamycin [25]. Using M17 cells expressing human PrPN181G or PrPT183A, Capellari et al. observed that PrPN181G, but not PrPT183A, reached the cell surface even though both mutations eliminated glycosylation at the first site [10]. This indicates that the Thr to Ala mutation itself, not the elimination of the first glycosylation site, altered the physical properties of the mutant protein [10]. Although the F198S mutation falls within the second glycosylation site, Asn-Phe-Thr residues 197-199, PrPF198S slightly increased the efficiency of glycosylation at the first glycosylation site (N181), and greatly increased the efficiency of glycosylation at the second site (N197) in cultured cells [11].





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