蛋白质泛素化和SUMO化修饰是重要的翻译后修饰,泛素或SUMO通过E1,E2,E3酶联级作用被添加到底物蛋白质的赖氨酸残基上,参与调控多种细胞过程。[1]寻找泛素及SUMO特异性的结合蛋白需要化学生物学工具,如光亲和探针和活性探针,它们可将蛋白-蛋白之间的弱相互作用转化为共价作用,从而进行pull-down实验和组学分析。在这里,我们化学合成了K11链型的di-SUMO光亲和探针和E2-Ub活性探针,并在体外验证了它们对结合蛋白或E3酶具有高效的铰链活性。对于di-SUMO光亲和探针,考虑到SUMO主要位于细胞核中,我们使用Hela细胞核裂解液和探针进行pull-down实验,通过组学分析寻找更多的SUMO链的结合蛋白。我们也对E2-Ub活性探针进行了组学分析,发现了多个HECT家族E3酶。

DNA损伤修复是生命体维持基因组遗传稳定的重要手段,对植株生长发育至关重要。研究DNA损伤修复的分子机制,为培育耐受紫外线辐射的植物、确保恶劣环境下的粮食增产提供理论基础。近年来的报道显示,作为一种泛素密码,K27链接类型泛素链参与DNA损伤修复过程调控。因此,研究K27链接类型泛素链的识别及调控机制,为培育新品种植株以及在恶劣环境下保证粮食产量具有重要意义。泛素化是一种重要的翻译后修饰,在真核生物中广泛存在。通过对底物蛋白的修饰,泛素化在植物生长发育调控中扮演了重要的调控角色。泛素分子之间可通过异肽键形成不同结构特点的泛素链,即泛素密码。泛素密码的不同生物学功能由其分子结构特征所决定。K27链接类型泛素链是参与DNA损伤修复的主要泛素密码。目前唯一报道的K27链接类型二泛素（K27 diub）的特异性识别蛋白是去泛素化酶UCHL3。UCHL3可通过对底物RAD51多聚泛素链的剪切参与DNA损伤修复过程。值得注意的是,迄今为止的研究显示,UCHL3在体外只能切割Ub-AMC,不能对任何类型的泛素链进行切割。UCHL3上75位丝氨酸(Ser75)发生的磷酸化,会使其在细胞内切割多聚泛素链的活性明显增强,但这种调控尚缺乏体外证据支持。本文围绕UCHL3识别K27泛素链,并参与DNA损伤修复调控,展开了UCHL3识别K27泛素链的分子机制研究。获得如下发现:1.利用ITC方法体外验证了UCHL3对K27 diub的特异性识别作用。2.通过解析UCHL3与K27二泛素复合物的晶体结构,首次发现UCHL3具有S1和S2两个泛素识别位点。S1位点识别近端泛素分子C末端。S2位点与远端泛素分子上经典的I44产生疏水相互作用,并辅以His153及Glu158为中心形成氢键网络。对UCHL3进行关键氨基酸点突变后,与K27 diub之间的相互作用力明显降低。3.通过结构比对,发现与UCHL3结合后K27 diub的构象更加伸展,呈现相对开放的状态。4.序列对比和系统发育树显示,磷酸化位点(Ser75)仅仅存在于UCHL3中,在其它UCH家族蛋白中中并不存在,暗示UCHL3的磷酸化调控在UCH家族中具有特异性。5.发现磷酸化位点(Ser75)在不同种属UCHL3中广泛存在,暗示UCHL3的磷酸化调控机制在进化上保守。值得注意的是,在模式生物拟南芥中磷酸化位点Ser突变为Glu,暗示UCHL3蛋白在拟南芥中可能是组成性活化的形式,无需其它激酶的磷酸化修饰便可切割泛素链。6.模拟磷酸化的UCHL3提高了对Ub-AMC的切割活性,但依旧无法对二泛素进行生理水平上的活性切割。上述工作首次阐明了UCHL3识别K27泛素链的分子机制,揭示了UCHL3磷酸化调控的独特性,为相应农业生产的分子调控奠定了基础。

Ubiquitination-dependent histone crosstalk plays critical roles in chromatin-associated processes and is highly associated with human diseases. Mechanism studies of the crosstalk have been of the central focus. Here our study on the crosstalk between H2BK34ub and Dot1L-catalyzed H3K79me suggests a novel mechanism of ubiquitination-induced nucleosome distortion to stimulate the activity of an enzyme. We determined the cryo-electron microscopy structures of Dot1L-H2BK34ub nucleosome complex and the H2BK34ub nucleosome alone. The structures reveal that H2BK34ub induces an almost identical orientation and binding pattern of Dot1L on nucleosome as H2BK12Oub, which positions Dot1L for the productive conformation through direct ubiquitin-enzyme contacts. However, H2BK34-anchored ubiquitin does not directly interact with Dot1L as occurs in the case of H2BK12Oub, but rather induces DNA and histone distortion around the modified site. Our findings establish the structural framework for understanding the H2BK34ub-H3K79me trans-crosstalk and highlight the diversity of mechanisms for histone ubiquitination to activate chromatin-modifying enzymes.

Long-chain scorpion toxin AaH-II isolated from Androctonus australis Hector can selectively inhibit mammalian voltage-gated sodium ion channel Na(v)1.7 responsible for pain sensation. Efficient chemical synthesis of AaH-II and its derivatives is beneficial to the study of the function and mechanism of Na(v)1.7 and the development of potential peptide inhibitors. Herein, we compared three different strategies, namely, direct solid-phase peptide synthesis, hydrazide-based two-segment native chemical ligation, and hydrazide-based three-segment native chemical ligation for the synthesis of AaH-II. The hydrazide-based two-segment native chemical ligation affords the target toxin with the optimal efficiency, which provides a practically robust procedure for the preparation of tool molecules derived from AaH-II to study the biological functions and modulation of Na(v)1.7. Our work highlights the importance of selecting suitable segment condensation approach in the chemical synthesis of protein toxins.

Ferroptosis is a novel characterized form of cell death featured with iron-dependent lipid peroxidation, which is distinct from any known programmed cell death in the biological processes and morphological characteristics. Recent evidence points out that ferroptosis is correlated with numerous metabolic pathways, including iron homeostasis, lipid metabolism, and redox homeostasis, associating with the occurrence and treatment of hematological malignancies, such as multiple myeloma, leukemia, and lymphoma. Nowadays, utilizing ferroptosis as the target to prevent and treat hematological malignancies has become an active and challenging topic of research, and the regulatory network and physiological function of ferroptosis also need to be further elucidated. This review will summarize the recent progress in the molecular regulation of ferroptosis and the physiological roles and therapeutic potential of ferroptosis as the target in hematological malignancies.

Linear (Met1-linked) ubiquitination is involved inflammatory and innate immune signaling. Previous studies have characterized enzymes regulating the addition and removal of this modification in mammalian systems. However, only a few plant-derived deubiquitinases targeting Met1-linked ubiquitin chains have been reported and their mechanism of action remains elusive. Here, using a dehydroalanine-bearing Met1-diubiquitin suicide probe, we discover OTUB1 from Oryza sativa (OsOTUB1) as a Met1-linked ubiquitin chain-targeting deubiquitinase. By solving crystal structures of apo OsOTUB1 and an OsOTUB1/Met1-diubiquitin complex, we find that Met1 activity is conferred by Met1-specific motifs in the S1' pocket of OsOTUB1. Large-scale sequence alignments and hydrolysis experiments provide evidence that these motifs are a general determinant of Met1 activity in the OTUB subfamily across species. Analysis of the species distribution of OTUBs capable of hydrolysing Met1-linked ubiquitin chains shows that this activity is conserved in green plants (Viridiplantae) and does not exist in metazoans, providing insights into the evolutionary differentiation between primitive plants and animals.Deubiquitinases (DUBs) targeting Met1-linked ubiquitin chains have important functions in mammals but are barely studied in plants. Here, the authors identify rice OTUB1 as a Met1-targeting DUB, characterize the structural determinants of this activity, and show that these features are conserved in green plants.

Ferroptosis is a novel characterized form of cell death featured with iron-dependent lipid peroxidation, which is distinct from any known programmed cell death in the biological processes and morphological characteristics. Recent evidence points out that ferroptosis is correlated with numerous metabolic pathways, including iron homeostasis, lipid metabolism, and redox homeostasis, associating with the occurrence and treatment of hematological malignancies, such as multiple myeloma, leukemia, and lymphoma. Nowadays, utilizing ferroptosis as the target to prevent and treat hematological malignancies has become an active and challenging topic of research, and the regulatory network and physiological function of ferroptosis also need to be further elucidated. This review will summarize the recent progress in the molecular regulation of ferroptosis and the physiological roles and therapeutic potential of ferroptosis as the target in hematological malignancies.