Densin-180蛋白之構築性質研究

Densin-180，又稱作LRRC7蛋白，為LAP (LRR and PDZ domain-containing)蛋白家族的成員之一，其共同特徵是胺基酸序列之N端具有Leucine-rich repeat (LRR)以及C端具有PSD95/Discs large/Zona occludens-1 (PDZ)這兩段特殊的區域。主要位在大腦中興奮性神經細胞的突觸後密質(postsynaptic density, PSD)，這類的細胞骨架蛋白可能參與神經細胞的形成、分化、訊息傳遞過程以及調控神經突觸的可塑性。在早期的研究中，認為densin-180蛋白是以穿膜的方式鑲嵌在細胞膜上，以執行神經突觸調控與細胞附著相關的功能。然而，近期的研究發現在海馬迴神經細胞的胞外biotin標定實驗中，無法成功的偵測到densin-180蛋白的訊號，此外，也有研究結果指出densin-180蛋白與LAP蛋白家族中另一個已被研究過的成員erbin蛋白之胺基酸序列比對結果相似度最高，因此認為densin-180應該和erbin這類的鷹架蛋白相似，是屬於位在細胞質中並且和細胞膜有聯繫(cytosolic membrane-associated)的蛋白。因此，本篇研究利用細胞生物以及生化實驗的方式，欲進一步的去探討densin-180蛋白在細胞內的位置。首先將densin-180蛋白之N端和C端分別接上GFP以及Flag-epitope，並且表現在heterologous expression系統中，之後進行免疫螢光染色實驗(immunostaining)。結果顯示只有在先以清潔劑(detergent)處理，使細胞膜穿孔(permeablized)的組別中可以偵測到螢光訊號。此外，利用專一辨識densin-180不同部位的三種抗體(Ab1、Ab2、Ab3)分別進行免疫螢光染色，發現結果與上述實驗相同，只會在預先以triton處理，使細胞膜穿孔的組別中才有螢光訊號。另外，利用西方墨點法分析，在細胞未破膜的狀態下，加入proteinase K處理的組別是否會影響densin-180蛋白的分子量大小。實驗結果發現，proteinase K並不會作用在未破膜組別的densin-180蛋白上，暗示著densin-180是一完全位於細胞質內並且可能和細胞膜有聯繫的蛋白，而不是以穿膜的方式鑲嵌在細胞膜上。此外，本論文亦利用穩定表達densin-180的細胞株，來探討是否能以核醣核酸干擾(RNA interference, RNAi)的方式降低其densin-180蛋白的表現量。希望透過此技術之應用，可提供未來研究densin-180蛋白所參與的訊息傳遞路徑以及找尋與其交互作用蛋白質一個新的方向，以進一步探索densin-180蛋白在神經突觸間所扮演的功能。

關鍵字

densin-180蛋白；突觸後緻密部

並列摘要

Densin-180, also known as Leucine-rich repeat-containing protein 7(LRRC7), is a member of the LAP protein family. The LAP proteins share the similar organization of leucine-rich repeat (LRR) and PSD95/Discs large/Zona occludens-1 (PDZ) domains, and are mostly enriched in postsynaptic density (PSD) of glutamatergic synapses. These scaffold proteins play a role in neuron formation and differentiation, and in the regulation of synaptic plasticity. Previous studies suggest that densin-180 is a trans-membrane protein that participates in specific adhesion function between pre-synaptic and post-synaptic membranes. However, several recent studies in cultured hippocampal neurons have shown that endogenous densin-180 is not accessible to extracellular biotin labeling. Furthermore, densin-180 shares the highest similarity to Erbin, another member of LAP protein family that has been known to be associated with the cytoplasmic side of plasma membrane via palmitoylation. These findings suggest that densin-180 may instead be a cytosolic or membrane-associated protein. Thus, in this study, we aim to re-investigate the membrane topology of densin-180. By applying GFP- and Flag-tagging at the N- and C-terminus, respectively, of densin-180 in the heterologous expression system, we found that both GFP- and Flag-epitope immunostaining signals were only available when membrane integrity of cell lines were destroyed by prior detergent treatments. We have also employed three antibodies (Ab1, Ab2, and Ab3) that recognize three different regions of densin-180 for immunocytochemistry studies. Similar to the results from epitope-tagging experiment, the immunostaining signals could only be detected when cells were permeablized by triton pre-treatment. In addition, we utilized Western Blotting analysis to determine if proteinase K treatment would affect the molecular weight of densin-180 protein in non-permeablized condition. We found out that densin-180 was resistant to proteinase K digestion. Take together, these results suggest that densin-180 is most likely a cytosolic or membrane-associated protein. In addition, we have applied RNA interference technique in a cell line stably expressing densin-180. By extending the scope of this densin-180 knock downtechnique, we aim to studying densin-180-interacting proteins, as well as understanding synaptic functions of densin-180.

並列關鍵字

densin-180 ； PSD

參考文獻

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Densin-180蛋白之構築性質研究

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