N端螺旋結構域的陰電性變化如何影響細胞自噬蛋白LC3

What Negative Charges on NHD Can Impart to LC3

王旭祺(Hsu-Chi Wang)

指導教授 ： 楊維元

國立臺灣大學/生命科學院/生化科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.00398

摘要

細胞自噬普遍存在於真核生物細胞中。細胞會藉由細胞自噬的過程來清除較次要的有機顆粒、受損的胞器，甚至胞內的病原體，以換取必須能量、進行養份循環、完成免疫反應等。細胞自噬時，將被降解的物質首先會由一種稱作吞噬泡的雙層膜構造的擴展所包圍而後封閉，稱為自噬體。自噬體接著與胞內的溶酶體融合成自噬溶酶體，經由溶酶體的水解酶消化自噬體的內容物，並釋出降解後的產物回細胞質。細胞自噬已知與多種人類的病理過程相關，如神經退化、癌症及老化。
LC3是一種類似泛素的細胞自噬蛋白，連接並修飾著擴展過程中的吞噬泡表面。LC3經由一套類似E1-E3的酵素系統，與吞噬泡上的磷脂醯乙醇胺連結。LC3被認為藉由其自身的多聚化，能促使吞噬泡間膜的連結與半融合。先前的研究顯示LC3由兩個結構域構成：N端螺旋結構域(NHD)和C端的類泛素結構域(ULD)。當LC3與自噬泡上的脂質連結，其NHD可能會減少與ULD間的接觸面，而成一個「開啟」的形式，使得LC3得以暴露ULD的部分表面區域，誘發LC3行多聚化。
2010年的兩項研究顯示小鼠的蛋白激酶A與大鼠的蛋白激酶C各別能磷酸化LC3之第12號絲胺酸與第6號蘇胺酸。這暗示著可能存在一套未知的LC3調控模式，特別是藉由改變NHD在LC3上的動態。
本研究使用仿磷酸化的人類LC3突變型，T6D (將第6號蘇胺酸突變為天門冬氨酸)及T12D (將第12號蘇胺酸突變為天門冬氨酸)，來與野生型的LC3和重組的ULD比較，利用圓二色光譜技術(CD)，作為觀察LC3二級構造之集體行為的工具；以測試當NHD上存在帶額外負電荷的官能基時， LC3的動態或穩定性是否發生顯著改變，進而影響LC3的活性。
結果顯示相較於ULD，不論野生型或仿磷酸化的LC3，在波長222奈米附近皆具有較強且相似的CD訊號；α螺旋結構的存在會大幅提高此處的訊號，故當此結構消失或變性時，CD222訊號會大幅下降。利用在不同濃度的尿素與鹽酸胍下產生的CD222，來建立各種LC3的變性曲線，結果發現仿磷酸化的LC3顯著較野生型容易變性；若以ULD的變性曲線作為參考，LC3變性或去摺疊之過程的前期，可能部分發生在其NHD的α螺旋結構，而早於其ULD出現大規模的變性。

關鍵字

LC3 ； 細胞自噬 ； 圓二色光譜 ； 變性曲線 ； 仿磷酸化

並列摘要

Autophagy is ubiquitous in all eukaryotic cells. It is crucial for cells to clear less necessary organic particles, damaged organelles, or intracellular pathogens for emergent energy needs, nutrient recycling, immune response, etc. In autophagy, cellular components to be degraded are first surrounded by a double membrane structure called phagophore; this membrane gradually expands and is sealed into a close structure called autophagosome, which subsequently fuses with lysosome, becoming autolysosome; the content within the autolysosome is then digested with hydrolytic enzymes, and freed from the structure into cytosol. Autophagy has been linked to numerous human pathological processes, such as neurodegeneration, cancer, and aging.
LC3, a ubiquitin-like autophagic protein, is decorated on the phagophore during membrane expansion. In an E1-E3-like enzyme system, LC3 is conjugated to the phosphatidylethanolamine (PE) on the phagophore. LC3 is suggested to promote membrane tethering and hemifusion between phagophores through its multimerization. Previous studies showed that LC3 consists of two domains: the N-terminal helical domain (NHD) and the C-terminal ubiquitin-like domain (ULD). During lipidation, the NHD may dissociate from the ULD, adopting an “open” form, and exposing a region on the ULD surface to induce LC3 multimerization.
In 2010, two studies showed that, both in vitro and in vivo, protein kinase A (PKA) and protein kinase C (PKC) could phosphorylate LC3’s Ser12 and Thr6, respectively. These imply a novel mode of LC3 regulation, possibly through changing its NHD dynamics.
In this study, the phosphomimetic human LC3s, T6D and T12D, were used for comparison with the LC3 wild type and the ULD, to investigate whether the additional negative charges on the NHD influence their dynamics, or stabilities, and hence the activities of LC3. The technique circular dichroism (CD) was utilized for examining the collective behavior of their secondary structures.
The results showed that compared with the ULD, the wild type, T6D, and T12D all had much stronger and similar signals around the wavelength 222 nm, which largely arises from the presence of α-helices, and decreases drastically in their loss or denaturation. Further examining the urea and the guanidine hydrochloride denaturation curves by CD222 of these LC3 variants found that there are significant differences between the wild type and the phosphomimetic mutants, showing the later more readily denatured. Compared with the denaturation curve of the ULD, the observed early unfolding of the LC3 could be, at least partially, accounted by the NHD, which might begin massive denaturation before the ULD does.

並列關鍵字

LC3 ； autophagy ； circular dichroism ； denaturation curve ； phosphomimetic

參考文獻

1. Meléndez, A. et al. Autophagy genes are essential for dauer development and life-span extension in C. elegans. Science 301, 1387-1391 (2003).

2. Levine, B. & Klionsky, D. J. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev. Cell 6, 463-477 (2004).

3. Nakagawa, I. et al. Autophagy defends cells against invading group A Streptococcus. Science 306, 1037-1040 (2004).

4. Yang, Z. & Klionsky, D. J. Eaten alive: a history of macroautophagy. Nat. Cell Biol. 12, 814-822 (2010).

5. Nakatogawa, H., Ichimura, Y. & Ohsumi, Y. Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion. Cell 130, 165-178 (2007).