單胞微藻Chlamydomonas reinhardtii SUMO之生化性質分析

SUMO (small ubiquitin-like modifier) 在1996年被第一次發現，當時它被當成是附在RanGAP1 (Ran GTPase-activating protein 1) 上的一段多肽。後來更多的研究顯示SUMO可以修飾細胞中許多的蛋白質，進而影響真核細胞中各種重要的基因表現與訊息傳導等路徑。然而，對植物系統之SUMO化的研究仍然不足，尤其是單細胞綠藻。因此本研究擬建立單胞微藻Chlamydomonas reinhardtii的SUMO化系統來進行初步的研究。經由選殖出單胞微藻三種SUMO (CrSUMO96、CrSUMO97與CrSUMO148) 的DNA序列，將其表現純化並利用哺乳類SUMO系統中的 E1 (SAE1/SAE2 or Aos1/Uba2) 與E2 (Ubc9) 來進行其生化性質的鑑定。實驗結果顯示不管是在胞外或在大腸桿菌內進行 SUMO化測試，C-端為di-glycine motif的CrSUMO96或CrSUMO97皆會形成polySUMO chain；而C-端不是以di-glycine motif結尾時則沒有此現象發生，證明露出C端di-glycine motif為進行SUMO化的必要條件。而在SUMO protease的活性測試中也可以看到，SENP1 (human SUMO protease) 可以對CrSUMO96與CrSUMO97進行processing，使CrSUMO96與CrSUMO97形成可以進行SUMO化修飾且C-端為di-glycine motif的型態，並發現SENP1對CrSUMO97 processing的活性比CrSUMO96來的強。而CrSUMO148在其C端具有四個序列重複的di-glycine motif，這個特色並不出現於其他物種的SUMO，在經過SENP1作用後，不管是何種長度的CrSUMO148都只會形成CrSUMO1481-83，即只含有一個di-glycine motif的型態。而經由建構不同長度的CrSUMO148所進行之胞外SUMO化測試發現，也只有CrSUMO1481-83才真正具有進行SUMO化的功能。此外本研究也進行了三種CrSUMO 的desumoylation活性測試。將三種CrSUMO的poly-chain與SENP1進行反應。發現所有的polySUMO chain在經SENP1作用下皆幾乎完全形成只有monoSUMO存在的型態。

關鍵字

類泛素小蛋白質修飾物；類泛素小蛋白質修飾物蛋白酶雙胱胺酸基序；人類類泛素小蛋白質修飾物活化酶小鼠類泛素小蛋白質修飾物活化酶類泛素小蛋白質修飾物銜接酶

並列摘要

SUMO (small ubiquitin-like modifier) was originally discovered in 1996, as a peptide associating with RanGAP1 (Ran GTPase-activating protein 1). More studies subsequently found that SUMO can modify many proteins and influence many cellular processes of gene expression and signal transduction in eukaryotes. However, investigation of sumoylation on plant systems, especially on unicellular green algae, remains little. Thus, this work was aimed to study the SUMO system in Chlamydomonas reinhardtii and also characterize their biochemical properties. Three genes encoding for SUMO proteins from Chlamydomonas reinhardtii have been identified and named CrSUMO96, CrSUMO97 and CrSUMO148. Using the E1 activating enzyme (SAE1/SAE2 or Aos1/Uba2) and E2 conjugating enzyme (Ubc9) of the animal SUMO system, the present data reveal that CrSUMO96 and CrSUMO97 with an exposed di-glycine motif at the C-terminus can form polymeric chains, whereas the polymeric chain was not observed by using the full length of CrSUMO96 or CrSUMO97 as the assay substrate. It clearly demonstrates that the exposed C-terminal di-glycine end is required for sumoylation. In SUMO processing activity assay, human SENP1 showed greater processing activity toward CrSUMO97 than CrSUMO96. Interestingly, CrSUMO148 have four repeated di-glycine motif at the C-terminus, which are not found in other SUMO proteins. SENP1 specifically digests CrSUMO148 at the first di-glycine motif to generate CrSUMO1481-83. Furthermore, only CrSUMO1481-83 can form polymeric chain in the in vitro sumoylation assay. The deconjugation activity of SENP1 towards poly-SUMO chains showed that all polyCrSUMO chains can be completely deconjugated to form SUMO monomers.

並列關鍵字

SUMO ； SENP1 ； di-glycine motif ； SAE1/SAE2 ； Aos1/Uba2 ； Ubc9

參考文獻

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單胞微藻Chlamydomonas reinhardtii SUMO之生化性質分析

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