鏌撹壊璐ㄦ槸鐪熸牳鐢熺墿閬椾紶淇℃伅鍌ㄥ瓨鐨勮浇浣擄紝瀹冧互鏍稿皬浣撲负缁撴瀯鍗曞厓锛屽嚑涔庝竴鍒囩敓鍛芥椿鍔ㄩ兘绂讳笉寮？牳灏忎綋缁勮泲鐧戒笂缈昏瘧鍚庝慨楗扮殑璋冩帶銆傜粍铔嬬櫧鐨勬硾绱犲寲淇グ绫诲瀷澶氬厓锛屽湪澶氱鐢熷懡杩囩▼鎵紨鍏抽敭瑙掕壊锛屼絾绉戝瀹跺瀹冧滑琛屼娇鐢熺墿鍔熻兘鐨勫寲瀛︽満鍒朵粛鐭ヤ箣鐢氬皯锛屽叾涓噸瑕佸師鍥犳槸缂哄皯鑾峰彇璇ョ被澶嶆潅铔嬬櫧鏍峰搧鐨勬妧鏈墜娈点？鏈枃宸ヤ綔鏍稿績鏄熀浜庡鑲界～閰殑鑷劧鍖栧杩炴帴鏉ヨ璁℃柊鐨勬硾绱犲寲缁勮泲鐧藉寲瀛﹀悎鎴愮瓥鐣ワ紝骞跺埄鐢ㄥ悎鎴愮殑铔嬬櫧鏍峰搧杩涜鐩稿簲鐨勭敓鐗╁闂鎺㈢┒銆？鍓嶄汉鐮旂┒灏氭湭瑙ｆ瀽缁勮泲鐧紿3 K18/K23鍙屼綅鐐规硾绱犱慨楗板湪鏍稿皬浣撴按骞充綔鐢ㄤ簬DNA鐢插熀杞Щ閰禗NMT1鐨勫叿浣撴満鍒躲？鎴戜滑浠ヨ棰樼粍鍓嶆湡鍙戝睍鐨凜AET鍒嗗瓙浣滀负杩炴帴鑷傦紝鎴愬姛鍚堟垚浜嗗紓鑲介敭妯℃嫙缁撴瀯杩炴帴鐨凨18/K23鍙屼綅鐐规硾绱犲寲鍏ㄩ暱缁勮泲鐧紿3锛屽苟杩涗竴姝ョ粍瑁呬簡甯︽湁鍗婄敳鍩哄寲DNA鐨勬牳灏忎綋銆備互鏍稿皬浣撲负DNMT1璇嗗埆搴曠墿锛屾垜浠湪浣撳杩涜浜咲NMT1鐨勭粨鍚堝姏瀹為獙锛岃瘉鏄庝簡娉涚礌淇グ鑳芥樉钁楁彁鍗囨牳灏忎綋涓庤泲鐧界殑缁撳悎鑳藉姏銆傝繘涓？鎴戜滑鍒╃敤鍖栧浜よ仈璐ㄨ氨鎵嬫鍒濇鎺㈢储浜咲NMT1涓庢硾绱犲寲鏍稿皬浣撶殑缁撳悎鐣岄潰锛屼负浠婂悗娣卞叆鐨勭粨鏋勬満鍒剁爺绌舵彁渚涗簡鍙傜収銆？浣撳唴缁勮泲鐧紿3 K18/K23鍙屼綅鐐规硾绱犱慨楗版按骞冲彈鍘绘硾绱犲寲閰禪SP7璋冩帶锛屼絾鐜颁粖鏈湁鏈夋晥璇佹嵁璇存槑杩欑鍘绘硾绱犲寲閰跺叿鏈変綅鐐圭壒寮傛？銆傛垜浠璁′簡鍩轰簬閰告晱鎰熻緟鍩轰粙瀵艰繛鎺ョ殑鏂扮瓥鐣ュ悎鎴愪簡寮傝偨閿繛鎺ョ殑H3 K18/K23鍙屼綅鐐规硾绱犱慨楗扮粍铔嬬櫧銆傚悓鏃舵垜浠悎鎴愪簡澶氱鍏朵粬浣嶇偣娉涚礌鍖栫粍铔嬬櫧锛屽苟杩涗竴姝ョ粍瑁呮垚鏍稿皬浣擄紝瀵筓SP7鐨勬硾绱犳按瑙ｅ姛鑳芥？璐ㄨ繘琛屼簡鐢熺墿鍖栧灞傞潰鐨勬帰绌躲？缁撴灉涓嶄粎璇存槑浜哢SP7瀵笻3 K18/K23娉涚礌淇グ鍦ㄦ牳灏忎綋姘村钩鍏锋湁浣嶇偣閫夋嫨鎬э紝骞朵笖瀹冨浜庡弻浣嶇偣娉涚礌淇グ鐨勬按瑙ｆ椿鎬ф樉钁楅珮浜庡崟浣嶇偣锛屽苟涓斾骇鐢熻繖绉嶅樊寮傜殑鍘熷洜涓嶆槸USP7瀵筀18/K23鐨勯？鎷╂？銆？ 缁勮泲鐧戒笂鍙︿竴缁勨？鈥擧2A C绔疜125/K127/K129涓磋繎浣嶇偣娉涚礌鍖栦慨楗颁篃鍏锋湁閲嶈鐢熺悊鍔熻兘銆備絾鍓嶆湡宸ヤ綔鍙戠幇鐜版湁鏂规硶鏃犳硶鏈夋晥鍚堟垚鐩稿簲铔嬬櫧鏍峰搧锛屽洜姝ゆ垜浠嫙閲囩敤杩戞湡鍙戝睍鐨凪PyE杈呭熀銆傚湪璇佹槑鍏朵粙瀵煎浣嶇偣寮傝偨閿瀯寤烘晥鐜囦紭浜庣幇鏈夋柟娉曞悗锛屾垜浠繘涓？璁捐绛栫暐鎴愬姛瀹炵幇浜咹2A C绔复杩戝弻浣嶇偣浠ュ強鍗曚綅鐐规硾绱犱慨楗板強缁勮泲鐧界殑鍚堟垚銆傚湪姝ゅ熀纭？笂鎴戜滑缁勮浜嗘牳灏忎綋骞跺璇ヤ綅鐐圭殑鐗瑰紓鎬у幓娉涚礌鍖栭叾USP48杩涜浜嗘椿鎬ц〃寰侊紝骞堕？杩囩敓鍖栧疄楠岃瘉鏄庝簡USP48瀵瑰弻浣嶇偣娉涚礌淇グ鐨勬按瑙ｆ椿鎬ф樉钁楅珮浜庡崟浣嶇偣锛屽苟涓旇繖绉嶅樊寮備笉鏉ユ簮浜庢硾绱犲瘑搴﹀閰朵笌搴曠墿缁撳悎鍔涚殑褰卞搷銆傛澶栨垜浠埄鐢ㄥ寲瀛︿氦鑱旇川璋辨墜娈靛涓よ？鐨勭粨鍚堢晫闈㈣繘琛屼簡鎺㈢储锛岀矖鐣ユ瀯寤轰簡USP48鍦ㄦ牳灏忎綋涓婅浣垮姛鑳芥椂鐨勬ā鍨嬨？
Chromosome, with nucleosomes as its basic unit, carries all of the genetic material in eukaryotes, and almost every cellular process is regulated by the post-translational modification of histones in nucleosome core particle (NCP). Ubiquitination of histone plays a key role in those life processes, however scientists鈥？current knowledge to their functioning mechanisms is only the tip of the iceberg, attributing to the lack of efficient tools to synthesize such proteins. This paper is mainly focused on developing new protein synthesis strategies on the use of peptide thioester-based natural chemical ligation, and utilize the synthetic proteins to conduct researches on corresponding biological problems.H3 K18/K23 ubiquitination is referred to recruit DNMT1 for cellular maintenance DNA methylation, but none has looked into the detailed molecular mechanism of the recruitment on an NCP-level. We utilize CAET, a molecule we developed, as the linker to successfully synthesize the K18/K23 dual-monoubiquitinated full-length histone H3 analog. Then we assemble the NCPs with hemi-methylated DNA to test affinities of DNMT1 for NCPs in vitro and find that ubiquitination can significantly improve the affinity between protein and its substrates. Furthermore, we use the chemical cross-linking mass spectrometry (CX-MS) to unveil the interaction sites between DNMT1 and ubiquitinated NCP, providing reference for structural biological research in the future.Level of H3 K18/K23 ubiquitination in vivo is regulated by DUB USP7, but there is no valid evidence that USP7 is site-specific. Given that the ubiquitinated histone analog cannot be identified and hydrolyzed by DUB, we design a new strategy on the use of an acid-sensitive auxiliary to prepare the H3 K18/K23 dual-monoubiquitination connected by an isopeptide bond. Meanwhile, we synthesize a variety of other ubiquitinated histones, further assembling into NCP, then we conduct biochemical assays of USP7‘s DUB properties. As a result, we confirm USP7 is specific for H3K18/K23 ubiquitination on an NCP-level. Also, it suggests that USP7 is capable of cleaving dual-monoubiquitination in significantly higher manner than monoubiquitination, which is not resulted from the selectivity between K18 and K23 of USP7.H2A C-terminal K125/K127/K129 ubiquitination also plays a vital role in numbers of physiological processes. However, our preliminary work suggests that using the existing methods could not chemically synthesize these proteins in vitro, so we intend to adopt the recently developed MPyE auxiliary. After proving its efficiency in multiple isopeptide bonds construction simultaneously, we further synthesize the C-terminal bi-site ubiquitination full-length histone H2As as well as single-site ones. We then assemble NCPs with this ubiquitinated histones and test the DUB activity of USP48, which is believed to be the site-specific DUB of H2A K125/K127/K129 ubiquitination. Our biochemical results show that DUB activity of USP48 on bi-site ubiquitination is significantly higher than that on single-site ubiquitination, and this is not caused by the ubiquitin density variation on histone. In addition, we use CX-MS to identify the binding interfaces between USP48 and ubiquitinated NCP, build a low-resolution model, and attempt to elucidate how USP48 works.
