Lon是一種ATP依賴型的蛋白酶,在分類上被歸類在AAA+ superfamily，此蛋白酶不僅在原核生物和真核生物中都呈現高度的保留性。Lon的蛋白分解活性目前已被確認在針對蛋白質的品量控制和代謝調控上皆扮演著非常重要的角色，因此，當此蛋白酶要執行其功能時，如何正確無誤地辦認其結合性蛋白或需要被分解的受質，在生物系統的交互作用上是非常重要的一個環節，這種精準的蛋白分解過程是屬於不可逆的轉錄後調控，由此可知，選擇其適當的分子目標蛋白尤其重要，因為錯誤的多胜肽鏈分解會嚴重導致細胞傷害甚至致死。
在這篇研究報告中，我們建立了一個以蛋白質體學為基礎的實驗策略來捕捉受質，藉此鑑定出大腸桿菌中Lon蛋白酶的潛在性受質，其主要生物性材料使用BW野生株大腸桿菌 (BW25113, BW)、lon基因剃除的大腸桿菌株(JW0429, JW)、以及能表現His6融合性LonS679D點突變蛋白的建構性質體 (其特性為保有ATPase活性卻失去蛋白酶活性)。因此我們將純化好的His6融合性LonS679D點突變蛋白以達到飽和的方式鍵結在鎳離子螯合層析管柱上，藉此捕捉來自BW或JW大腸桿菌細胞均質液內的Lon受質或結合性蛋白質，之後再把鍵結在LonS679D上的蛋白洗出，最後以溶液內胜肽水解法分解成短胜肽鏈，隨後經由串聯式液態層析質譜儀 (LC-MS/MS)進行分析。藉此散彈槍蛋白質體分析法以及比較來自BW或JW大腸桿菌的這些被分析鑑定到的蛋白質，此方法能夠讓我們去區分屬於Lon的分解性受質或單純只是結合性蛋白，結果我們總共找到了38個可能會被Lon分解的受質以及6個結合性蛋白質。然而，為了更進一步證實此分析結果的可信度，基於現有的研究文獻為基準，我們挑選了10個最有可能的潛在性受質，包含了XerC/D、UspG、Fis、BssR、ArnA、BasR、FabR、MukE和CbpA，藉由同時進行胞內和胞外蛋白水解分析法，結果我們確認出XerC/D、Fis、BssR、和ArnA可能直接或間接透過Lon依賴型的方式來執行蛋白質水解，但是關於這些受質被水解的生理意義在生物體內仍須更待進一步的研究。
總結我們所用的這項捕捉受質實驗，目前已經能成功驗證並且找到現階段研究皆屬未知的全新Lon分解受質，另外，在未來我們可以使用這種失去蛋白酶活性的Lon來當餌，以便在不同生長條件下幫助我們找出更多關於Lon蛋白酶的生物意義。

ATP-dependent Lon protease, which belongs to the AAA+ superfamily, is highly conserved in prokaryotes and eukaryotic organelles. Lon is important for not only protein quality control but also regulation of metabolic processes via proteolysis. The recognition of the correct partners or substrates is critical for almost all biological transactions. For irreversible posttranslational regulation like protein degradation, choosing the appropriate molecular targets is especially important because cleavage of the wrong polypeptides could be damaging or even fatal.
In this study, we utilized a proteomic-based substrate trapping strategy to identify potential substrates of E. coli Lon (EcLon). Using BW25113 (BW; a wild type strain), JW0429 (JW; a lon knockout strain), and one construct expressed His6-fused EcLonS679D (loss of protease activity but remain ATPase activity) as materials, we thereby saturated Ni2+-NTA beads with EcLonS679D to pull down the trapped substrates or associated proteins from lysates of BW and JW. The eluants were then applied to in-solution digestion for subsequent analysis via LC-MS/MS. Using shotgun proteomic approach and comparison of each pool of identified proteins from BW and JW to distinguish Lon’s substrates from associated proteins, we totally identified 38 proteins which might be subjected to Lon-mediated proteolysis, and 6 proteins might associate with Lon protease. To validate the credibility of the results, 10 potential candidates were selected, including XerC/D, UspG, Fis, BssR, ArnA, BasR, FabR, MukE, and CbpA, based on the criteria of previous studies. By both in vitro and in vivo proteolytic assays, we were able to confirm that XerC/D, Fis, BssR, and ArnA are subjected to Lon-dependent degradation through a direct or indirect manner. However, the physiological purposes for these degradation processes still remain further investigation.
Our trapping method has proven successfully validate previously unknown substrates of Lon. In the future, using protease-deficient Lon as bait under different growth conditions might help reveal even more of this versatile protease.

1 Introduction 1
1-1 AAA+ superfamily and Lon Protease 1
1-2 Structure and functionality of Lon protease 4
Figure 1. Domain organization of Lon (LonA) 4
1-3 General principles for AAA+ proteases to discriminate their substrates 6
1-4 Recognition and degradation of cytoplasmic proteins by Lon 7
Table 1. Known substrates of E. coli Lon protease 12
1-5 Candidate Substrates of EcLon 14
1-6 Aim of this Study 14
2 Materials and Methods 15
2-1 Bacterial strains and plasmids 15
2-2 Identification of Potential Substrates of EcLon 15
2-3 In-solution digestion and desalting 17
2-4 Liquid chromatography - mass spectrometric analysis 17
2-5 Cloning of EcLon, and its potential substrates 19
Table 2. Oligonucleotides used in this study 21
2-6 Protein Expression and Purification 24
2-7 In-gel digestion 24
2-8 Identification of the cleavage sites 25
2-9 LC-ESI-MS/MS Analyses 26
2-10 In vitro degradation assays 27
2-11 In vivo proteolytic assays 28
2-12 Preparation of protein extracts and immunodetection 28
2-13 Circular dichroism spectra of Fis 29
3 Results and Discussion 30
3-1 Validation of EcLonS679D as trap for substrate identification 30
3-2 Identification of EcLonS679D-trapped Proteins 31
Figure 2. Principle of the EcLonS679D-based substrate identification approach 33
Figure 4. Workflow of identification potential substrates of EcLon 35
Figure 5. Scheme of the identified proteins in BW25113 and JW0429 39
3-3 Validation of the Potential Substrates in EcLon 40
Figure 6. in vitro and in vivo putative substrates validation 41
Table 5. Summary of in vitro and in vivo degradation of 10 selected candidates 42
3-3.1 Validation of five novel substrates 43
3-3.1.1 Fis 43
Figure 7. Fis is subjected to Lon-dependent proteolysis both in vitro and in vivo 46
Figure 8. Cleavage sites of Fis and respective positions on tertiary structure. 47
3-3.1.2 BssR and ArnA 48
Figure 9. Interaction between BssR and ArnA 52
Figure 10. Validation of BssR and ArnA in vitro and in vivo 53
Figure 11. Cleavage sites of BssR 54
3-3.1.3 XerC/D 55
Figure 12. XerC was subjected to Lon-mediated proteolysis in vivo 58
Figure 13. Position of His6-tag influenced the tendency for degradation in XerD 59
Figure 14. Cleavage sites of XerD 60
Figure 15. Respective positions of cleavage sites in XerD on tertiary structure. 61
Figure 16. Possible regulation mechanisms for XerC/D proteolysis 62
3-3.1.4 UspG and its truncated form (UspG△136) 63
Figure 17. Validation of UspG in vitro and in vivo 65
Figure 18. UspG△136 might be subjected to Lon-mediated proteolysis in vivo 66
3-3.2 Not all proteins co-purified with LonS679D are protease substrates 67
3-3.2.1 FabR 67
3-3.2.2 MukE 68
3-3.2.3 BasR 68
3-3.2.4 CbpA 69
Figure 19. Validation of FabR in vivo 70
Figure 20. Validation of MukE in vitro and in vivo 71
Figure 21. Validation of BasR in vitro and in vivo 72
Figure 22. Validation of CbpA in vitro and in vivo 73

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