Kim Hyun Jin, Lee Daekyun, Lee Choong-Hwan, Chung Kwang Chul, Kim Jongsun, Paik Seung R
School of Chemical and Biological Engineering, College of Engineering, Seoul National University, Seoul 151-744, Republic of Korea.
Arch Biochem Biophys. 2006 Nov 1;455(1):40-7. doi: 10.1016/j.abb.2006.08.019. Epub 2006 Sep 11.
Alpha-synuclein is a pathological component of Parkinson's disease by participating in Lewy body formation. Imbalance in protein turnover could result in the abnormal protein aggregation responsible for eventual neuronal cell death. This in vitro digestion study showed that both m-calpain and 20S proteasome preferentially hydrolyzed the N-terminal half of alpha-synuclein, which made the hydrophobic NAC and following acidic C-terminal region resistant against the proteolyses. Since the acidic C-terminal region contains the PEST segment-a protein degradation signal enriched with amino acids of proline (P), glutamate (E), serine (S), and threonine (T)-, the PEST segment has not been processed or even required for the proteolyses. Alpha-synuclein would be recognized primarily by m-calpain since the common substrate was processed by m-calpain five times more effectively than 20S proteasome with k(cat)/K(m) of 1.64 x 10(4)M(-1)s(-1) and 0.32 x 10(4) M(-1)s(-1), respectively. The N-terminally truncated protease-resistant C-terminal fragment of alpha-syn61-140 was demonstrated to stimulate the 20S proteasome-mediated breakdown of alpha-synuclein and its mutant forms of Ala53Thr and Ala30Pro. The stimulation for Ala53Thr, however, was noticeably less efficient than those for the other proteins, which might support the previous observation of the prolonged intracellular life span of Ala53Thr by 1.5-fold compared to that of wild-type form. We have hypothesized that the N-terminally truncated C-terminal fragment derived from the abundant alpha-synuclein through intracellular proteolyses could be involved in various physiological or pathological effects which might be related to the formation of abnormal protein aggregation and subsequent neuronal degeneration by influencing the intracellular protein turnover or directly participating in the aggregate formation.
α-突触核蛋白通过参与路易小体的形成，成为帕金森病的一个病理成分。蛋白质周转失衡可能导致异常蛋白质聚集，最终导致神经元细胞死亡。这项体外消化研究表明，m-钙蛋白酶和20S蛋白酶体均优先水解α-突触核蛋白的N端一半，这使得疏水性的NAC及其后的酸性C端区域对蛋白水解具有抗性。由于酸性C端区域包含富含脯氨酸（P）、谷氨酸（E）、丝氨酸（S）和苏氨酸（T）的氨基酸的PEST片段——一种蛋白质降解信号，因此该PEST片段未被处理，甚至在蛋白水解过程中也不需要。α-突触核蛋白主要被m-钙蛋白酶识别，因为常见底物被m-钙蛋白酶处理的效率比20S蛋白酶体高五倍，其催化常数与米氏常数之比分别为1.64×10⁴M⁻¹s⁻¹和0.32×10⁴M⁻¹s⁻¹。α-突触核蛋白61-140的N端截短的抗蛋白酶C端片段被证明可刺激20S蛋白酶体介导的α-突触核蛋白及其Ala53Thr和Ala30Pro突变体形式的分解。然而，对Ala53Thr的刺激效率明显低于对其他蛋白质的刺激效率，这可能支持了之前的观察结果，即Ala53Thr的细胞内寿命比野生型延长了1.5倍。我们推测，通过细胞内蛋白水解从丰富的α-突触核蛋白衍生而来的N端截短的C端片段可能参与各种生理或病理效应，这些效应可能与异常蛋白质聚集的形成以及随后的神经元变性有关，通过影响细胞内蛋白质周转或直接参与聚集体形成。
