Cytotoxicity and morphology of prion protein (PrP) peptide aggregate

Abstract

ABSTRACT Prion disease is biochemically characterized by the structural change of the prion protein (PrP) from cellular prion protein (PrPC) to infectious scrapie prion protein (PrPSc). The structural change of the PrP is accomplished by increase in the content of hydrophobic beta sheet structure, which is the key to the formation of PrP aggregate. Human PrP peptide 106-126 (PrP106-126) is a hydrophobic, internal sequence prone to be aggregated. Hence, the biophysical and biochemical properties of PrP106-126 are similar to those of PrPSc. To study aggregation of PrP, we explored a variety of conditions for optimal formation of PrP106-126 aggregates using the Thioflavin-T (Th-T) binding assay in vitro. As a result, we found that the high concentrations of sodium chloride and the presence of glycine played an important role for the effective aggregation of PrP106-126. In the atomic force microscopy (AFM) analysis, aggregated PrP106-126 showed a large supramolecular structure in dimension of 4.5 ± 0.7 ㎛ length and 6.5 ± 0.5 nm height. This suggest that PrP106-126 aggregates generated under the condition used in this study were composed of several thousand (>5.000) PrP106-126 monomers. In MTT assay, no cytotoxicity was observed in CRBL cells incubated with PrP106-126 aggregates. In contrast, PrP106-126 aggregates were cytotoxic in N2a cells. Considering that CRBL cells are composed of both neuronal and glial cells and N2a cells are composed of neuronal cells only, the neuronal cell-specific cytotoxicity of PrP106-126 aggregates suggests a role of glial cells, which are related to the protection mechanisms. This study demonstrated that biochemical changes of PrP106-126 correlate with its biophysical properties, as well as its pathophysiological outcomes. This study provides a clue to understand the formation of the infectious prions found in the living body, morphology of PrPSc, and their cytotoxicity. Key word : prion, PrPC, PrPSc, PrP106-126, aggregation, cytotoxicity, morphology.|ABSTRACT IN KOREAN 프리온 질병은 세포 프리온 단백질 (PrPC)에서 감염성 스크래피 프리온 단백질 (PrPSc)로 프리온 단백질 (PrP)의 구조가 변화되는 생화학적 특징을 가진다. PrP의 3차원 구조 변화는 PrP 응집체 형성에서 중요한 역할을 하는 소수성 베타 시트 구조의 함량 증가에 의해 이루어진다. 사람의 프리온 단백질 펩타이드 106-126 (PrP106-126)은 소수성이며 응집화하는 경향이 있다. 또한PrP106-126은 PrPSc와 물리 화학적인 성질이 유사하며, PrPSc에서 응집체 형성에 필수적인 아미노산 서열 절편이다. PrP의 응집을 연구하기 위해, 우리는 시험관내에서 Th-T 결합 분석법을 사용하여 최적의 PrP106-126 응집체 형성을 위해 다양한 조건을 분석하였다. 그 결과, 우리는 높은 농도의 염화나트륨 및 글라이신 존재 시 PrP106-126의 효과적인 응집이 유도되는 것을 발견하였다. 이는 PrP 응집에 염 농도의 중요성을 제시한다. 원자간력 현미경 (AFM) 분석은 PrP106-126 응집체 형성의 최적 조건에서 형성된 응집체가 4.5 ± 0.7 ㎛ 길이와 6.5 ± 0.5 nm 높이의 구조를 보여주는 것을 확인하였다. 확인된 구조로 5,000개 이상의 PrP106-126 단량체가 하나의 응집체를 형성하는 것을 알수 있었다. MTT 분석으로 측정한 PrP106-126 응집체의 세포 독성은 신경세포와 신경교세포의 혼합 세포주인 CRBL 세포에서는 측정 불가하였으나, 신경세포주인 N2a 세포에서는 통계적으로 유의한 세포 독성이 확인되었다. 이러한 결과는 PrP106-126 응집체의 세포 독성이 신경 세포에 특이적이고 신경교세포는 보호기전과 관련 있음을 시사한다. 본 연구는 PrP 응집 조건에 따른 응집체 형성, 이들 응집체의 형태, 세포 독성의 상관관계를 밝혔으며, 생체에서 발견되는 프리온 감염체의 형성, 프리온 구성물질인 PrPSc의 형태 그리고 이들의 독성을 이해하는 기초자료를 제공할 것으로 사료된다.; ABSTRACT Prion disease is biochemically characterized by the structural change of the prion protein (PrP) from cellular prion protein (PrPC) to infectious scrapie prion protein (PrPSc). The structural change of the PrP is accomplished by increase in the content of hydrophobic beta sheet structure, which is the key to the formation of PrP aggregate. Human PrP peptide 106-126 (PrP106-126) is a hydrophobic, internal sequence prone to be aggregated. Hence, the biophysical and biochemical properties of PrP106-126 are similar to those of PrPSc. To study aggregation of PrP, we explored a variety of conditions for optimal formation of PrP106-126 aggregates using the Thioflavin-T (Th-T) binding assay in vitro. As a result, we found that the high concentrations of sodium chloride and the presence of glycine played an important role for the effective aggregation of PrP106-126. In the atomic force microscopy (AFM) analysis, aggregated PrP106-126 showed a large supramolecular structure in dimension of 4.5 ± 0.7 ㎛ length and 6.5 ± 0.5 nm height. This suggest that PrP106-126 aggregates generated under the condition used in this study were composed of several thousand (>5.000) PrP106-126 monomers. In MTT assay, no cytotoxicity was observed in CRBL cells incubated with PrP106-126 aggregates. In contrast, PrP106-126 aggregates were cytotoxic in N2a cells. Considering that CRBL cells are composed of both neuronal and glial cells and N2a cells are composed of neuronal cells only, the neuronal cell-specific cytotoxicity of PrP106-126 aggregates suggests a role of glial cells, which are related to the protection mechanisms. This study demonstrated that biochemical changes of PrP106-126 correlate with its biophysical properties, as well as its pathophysiological outcomes. This study provides a clue to understand the formation of the infectious prions found in the living body, morphology of PrPSc, and their cytotoxicity. Key word : prion, PrPC, PrPSc, PrP106-126, aggregation, cytotoxicity, morphology.