Abstract:泛素（ubiquitin）是真核生物中高度保守的一种由76个氨基酸组成的蛋白质，其与底物蛋白的赖氨酸残基共价结合的过程称作泛素化。泛素化作为一种功能多样的翻译后修饰，几乎参与所有细胞生命活动，其调控异常与肿瘤等重大疾病密切相关［1-3］。泛素化主要由泛素活化酶（E1）、泛素结合酶（E2）、泛素连接酶（E3）和去泛素化酶（DUB）介导的多酶级联反应实现，参与该过程调控的酶有700多种，总数超过磷酸化修饰途径的酶类［4］。泛素可与底物共价结合，形成单泛素化和多聚泛素化修饰。泛素分子中的7个赖氨酸残基（K6、K11、K27、K29、K33、K48和 K63）和 N 末端的甲硫氨酸（M1）残基，均可以形成泛素链，由此形成8种类型的泛素链，部分链型之间还可形成杂合链［5］。底物蛋白除了经典的赖氨酸外，丝氨酸、苏氨酸和半胱氨酸残基也可被泛素化［6-7］，这赋予了泛素化修饰高度的复杂性和特异性。不同类型泛素链和泛素修饰位点对应了不同的“书写器”、“阅读器”和“擦除器”，高度动态可逆地调控泛素密码的生物学功能［8］。泛素化机器作为疾病药物靶点的研究也受到广泛关注，蛋白酶体抑制剂硼替佐米于2003年被FDA批准，是治疗多发性骨髓瘤的一线药物。目前已有6种靶向蛋白酶体的药物获批上市，12种进入临床。更为重要的是，基于泛素-蛋白酶体降解系统开发的靶向蛋白降解的嵌合体（PROTAC）技术，使靶向“难成药靶点”成为可能，已经成为制药行业研究的热点和前沿问题。因此，揭示泛素化修饰机制及其生理和病理功能，对于发展新型的疾病诊疗技术具有重要意义。本期《生物化学与生物物理进展》以“泛素化修饰调控与疾病”为主题，共刊出13篇文章，包括10篇综述论文和3篇研究报告，主要涵盖3个方面的研究进展，分别是：泛素化修饰调控蛋白质稳态的机制、泛素化修饰与疾病治疗、泛素化修饰的识别和靶向技术。
首先，泛素化修饰是蛋白质稳态调控的重要方式之一，泛素-蛋白酶体系统（UPS）是真核细胞内蛋白质降解的主要方式之一。泛素连接酶E3决定了底物识别的特异性，去泛素化酶DUB特异性剪切和加工泛素链，促进泛素分子的循环利用，蛋白酶体负责细胞内大多数蛋白质的降解。但它们如何有机协作控制底物蛋白的命运一直是领域内研究的热点。“泛素化修饰调控蛋白质稳态的机制”部分包括5篇综述和2篇研究报告：针对磷酸化与泛素化修饰的相互作用，季然等总结了泛素蛋白已知的磷酸化修饰位点，梳理了泛素蛋白T12、S57、S65等位点的磷酸化修饰对其生物物理特性带来的改变；姚怡辰等综述了磷酸化和泛素化修饰在TORC1信号通路中的重要作用，为药物靶点的发现提供新思路。针对泛素连接酶的和去泛素化酶的重要作用，周璐等梳理了动植物中COP1泛素连接酶介导的信号转导与蛋白质稳态调控机制；黄彬等探索了线性泛素化连接酶LUBAC介导RabGEF1的线性泛素化修饰；张新等揭示了去泛素化酶USP10通过稳定泛素连接酶Smurf1抑制TGF-β/BMP信号通路的作用，为进一步理解泛素化修饰机制提供了认识；应淑敏等总结了过氧化物酶体的稳态维持与膜接触位点的研究进展，戴炜等综述了原核生物中Pup-蛋白酶体系统的作用机制和生物学功能，并对该系统在生物技术研发中的应用前景进行了展望。
其次，蛋白质泛素化修饰异常是肿瘤等重大疾病发生发展的重要因素，理解泛素化修饰控制和参与疾病进程的功能，对发展疾病治疗策略至关重要。“泛素化修饰与疾病治疗”部分包括4篇综述：曹心怡等探讨了SPOP对前列腺癌（PCa）分层治疗的重要意义，提出在治疗早期对PCa患者进行基因检测，根据分子分型确定不同个性化的分层治疗方案，这可能对改善PCa的预后状况产生有利影响；彭韵桦等分析了靶向蛋白质泛素化修饰在PCa治疗中的机遇与挑战；郑雅文等综述了蛋白质拟素化和去拟素化在肺癌发生发展中的作用，详细介绍了拟素化调节肺癌细胞的生长、存活和肺癌微环境以及免疫反应的功能和机制，进一步探讨了靶向拟素化对肺癌临床治疗的价值。针对神经退行性疾病，贾凤菊等以E3泛素连接酶和DUBs为切入点，综述了蛋白质泛素化和去泛素化修饰参与多巴胺能神经元损伤机制的最新研究进展。
最后，泛素化机器作为疾病药物靶点的研究受到广泛关注，发展基于泛素化机器的靶向技术，将为发展新的疾病治疗药物奠定基础。“泛素化修饰的识别和靶向技术”部分包括1篇综述和1篇研究报告：泛素链的分型检测及动态追踪一直是泛素研究技术上的难点，化学探针作为一类可用于探测活性酶的工具，近期被发展并愈来愈多的用于研究泛素化过程的微观机制，梁家伟等总结了针对泛素化与去泛素化酶的化学探针及其应用，为认识并发展泛素特异的化学探针提供了基础；刘明秋等建立了一种联合Nanoluc技术和荧光分析的PROTAC筛选策略，为PROTAC的开发应用提供新的技术策略。
以上论文选题和内容属于当前泛素化修饰研究领域的前沿进展，希望这些作者的专业视野或研究结果能够为深入理解和认识泛素化修饰在疾病发生发展中的作用和精准靶向提供新的思路和有益参考。

Abstract:TORC1 is a highly conserved kinase complex in eukaryotes that regulates cellular metabolism and growth by sensing signals such as nutrients, growth factors, and energy levels. Dysregulation of the TORC1 signaling pathway has been associated with metabolic disorders, neurodegenerative diseases, cancer, and aging. In this review, we compare the structure and function of TORC1 in yeast and mammalian cells. TORC1 in yeast consists of Tor1/Tor2, Kog1, Lst8, and Tco89, while mTORC1 in mammalian consists of mTOR (homolog of yeast TOR1/2), RAPTOR (homolog of yeast Kog1), mLST8 (homolog of yeast Lst8), PRAS40 and DEPTOR. We then review the critical roles of phosphorylation and ubiquitination in transducing external signals to TORC1 and regulating the downstream signaling pathways. Yeast TORC1 phosphorylates and activates Sch9 and Ypk3 to promote protein translation. Yeast TORC1 also regulates stress response, nitrogen metabolism, and autophagy by phosphorylating and inhibiting Tap42, Atg13, and Npr1. Mammalian AMPK, CK1α, Rheb, and AKT can phosphorylate mTORC1, thereby regulating the activity of mTORC1. mTORC1 regulates protein and lipid metabolism by phosphorylating downstream effector proteins, including S6K1, 4E-BP1, and LIPIN1. mTORC1 also inhibits autophagy by phosphorylating ULK1, TFEB, and ATG13. In addition, E3 ubiquitin ligases, including RNF167, RNF186, SCF, etc., either cause protein degradation or promote protein interactions through different forms of polyubiquitination, thus precisely modulating the mTORC1 signaling pathways. The crosstalk between phosphorylation and ubiquitination involved in the mTORC1 signaling pathway is also summarized. An in-depth understanding of the effects of phosphorylation and ubiquitination on the TORC1 signaling pathway can provide new insights for drug target discovery.

Abstract:Peroxisome is a kind of organelle conserved in eukaryotes, which is involved in many biochemical metabolic processes, including β-oxidation of fatty acids, production and degradation of reactive oxygen species, etc. Peroxisome biogenesis has growth and division model and de novo biogenesis model, which involves the import of peroxisome matrix and membrane proteins. Under normal physiological conditions, the proliferation and degradation of peroxisomes are balanced. While the matrix protein and membrane proteins in the peroxisome are misfolded and excessively accumulated, or the peroxisome is under environmental stress, such as high reactive oxygen species (ROS) concentration was exhibited, the peroxisomes homeostasis will be unbalanced. In order to maintain homeostasis in the biogenesis process and environmental stress, the peroxisome through division and degradation for quality control. What’s more, peroxisome has evolved multiple degradation pathways, including pexophagy, the receptor accumulation and degradation in the absence of recycling (RADAR) depending on ubiquitin-proteasome system (UPS) and so on. These pathways of peroxisomal degradation are significant for maintaining the integrity of cell structure and function. As the metabolic hub of eukaryotic cells, peroxisomes exchange substances and transmits signals with other organelles through peroxisomal membrane contact sites (MCSs), such as mitochondria, endoplasmic reticulum, lysosome and so on. These peroxisomal MCSs play a vital role in metabolic functions and homeostasis regulation, including lipid metabolism, peroxisome division, autophagy and other biological processes. In recent years, the maintenance of peroxisome homeostasis and MCSs have become research hotspots at home and abroad. The quantity change and spatio-temporal distribution of peroxisome are regularly dynamic to maintain the organism’s normal life activities, while the homeostasis imbalance of peroxisome will result in serious physiological dysfunction of cells. In humans, more and more diseases have been confirmed to be related to the imbalance of peroxisome homeostasis or mutations of peroxisome membrane contact sites, including cancer, diabetes, Alzheimer’s disease and Parkinson’s disease. In plant pathogenic fungi, recent studies have proved that the key genes of peroxisome biogenesis play an important role in pathogenicity, such as of rice blast fungus. This paper reviews the recent advances in the mechanism of peroxisome homeostasis and MCSs.

Abstract:COP1 is a RING E3 ligase first identified in plants, where it is instrumental in establishing photomorphogenesis. A complex regulatory network centered around COP1 has since been demonstrated to constitute a major signal transduction axis mediating light/darkness or temperature response, whereby changes in COP1 activity link light sensor modules (cryptochrome, phytochrome, UVR8, etc.) to core transcription factors (HY5, PIF, etc.) to reprogram cellular metabolism and growth response. While the photoreception network does not serve to orchestrate energy production in animals, mammalian COP1 is still critical in regulating metabolism and cell growth during tumorigenesis. In this review, we summarize currently known COP1 functions in animals v.s. plants. It can be seen that the stimuli sensed by mammalian COP1 and the signal transduction pathway there of remain largely unknown. Sunlight acts as both major energy source and an upstream signal of COP1 in plants. Similarly, glucose, the major energy source for animals, maybe highly correlated with COP1 upstream signaling. In both organisms, COP1 may respond to stimuli by altered localization, activity or assembly into super-E3 complex with CRL4, which remains to be further understood. Nonetheless, pharmacological studies on animal COP1 points to therapeutic directions, while also providing tools for further research on this enigmatic E3. COP1 is of vital importance in the regulation of cellular life process. Its has diverse function and mechanism through evolution, which needs to be further explored.

Abstract:The Pup-proteasome system (PPS) is a prokaryotic post-translational protein degradation pathway, in which prokaryotic ubiquitin-like protein (Pup) is activated by deamidase of Pup (Dop) and covalently linked to target proteins by proteasome accessory factor A (Paf A), and then the Pup-labeled target proteins are degraded by proteasome. PPS in mycobacteria is involved in the reactions to a various stress conditions, such as oxidative stress, nutritional deficiency, heat stress, and DNA damage, and participates in the regulation of metal ion homeostasis, toxin-antitoxin system, and host immune resistance. It has been reported that PPS is closely related to the retention and pathogenicity of Mycobacterium tuberculosis, therefore the PPS components, PafA, Dop and proteasome, have emerged as new targets for anti-tuberculosis drug development. Several small molecule inhibitors targeting PPS have been identified as the potential novel anti-tuberculosis drugs. In addition, PafA-catalyzed protein pupylation has been applied in biotechnology research, and developed into a new proximity labeling method, named pupylation-based interaction tagging (PUP-IT), which has been successfully used in the study of protein-protein interactions. This article reviews the research progress in the mechanism of action and biological functions of PPS, as well as the research progress in PPS inhibitors and PPS biotechnology applications.

Abstract:Ubiquitylation, also termed ubiquitination, is one of the most important post-translational modifications in eukaryotic cells. It is a process by which a small signaling protein, called ubiquitin composed of 76 amino acids, is conjugated to protein substrates via an E1-E2-E3 enzymatic cascade. Ubiquitin can be attached to lysine, serine, threonine and cysteine residues of its substrates. Ubiquitin itself contains seven lysine residues, therefore, ubiquitylation can form various polyubiquitin chains to produce complex ubiquitin codes. Ubiquitylation can alter the fates of ubiquitylated proteins including kinase activation, alteration of protein localization and proteolysis via the 26S proteasome and is involved in nearly every aspect of biological activities in eukaryotic cells. Recent studies indicated that more complicated post-translational modifications can also be found on ubiquitin including sumoylation, ubiquitylation, phosphorylation and acetylation. These modifications largely increase the complexity of ubiquitin signals. Ser65 of ubiquitin is the first characterized phosphorylation site whose biological functions have been extensively studied in human cells. It has been shown that Ser65 phosphorylation by PINK1 kinase is critical for the activation of Parkin ubiquitin ligase during mitophagy induction. The researches on Ser65 phosphorylation of ubiquitin boosted the studies on biological significance of the rest phosphorylation sites of ubiquitin. Now it is clear that phosphorylation of Ser57 residue is involved in endocytosis and stress responses, including oxidative stress in yeast, whereas phosphorylation of Thr12 and Thr66 residues plays important roles in DNA damage response. In the case of Ser57 residue, members of the AMPK-related kinases phosphorylate it, however, mechanisms by which Ser57 phosphorylation regulates endocytosis or oxidative stress response are still unclear. Also, no experimental evidences are available in mammalian system yet. One interesting fact is that many AMPK-related kinases contain a ubiquitin-associated domain (UBA), although some studies suggested these UBA domains do not possess any binding capabilities to polyubiquitin chains. However, some of these kinases could phosphorylate the Ser57 residues on the M1 polyubiquitin chain, implying these UBA domains do recognize certain polyubiquitin chains. In this review article, we summarized the post-translational modification sites of ubiquitin, especially phosphorylation sites and highlighted the biological functions of Ser65, Ser57, Thr12, Thr66 phospho-ubiquitin proteins. We also discussed alternations of biophysical properties brought by the phosphorylation of ubiquitin. Finally, we proposed a few future research directions related to the phosphorylated ubiquitin.

Abstract:Objective To verify that Rab guanine nucleotide exchange factor 1 (RabGEFl) is a novel substrate of linear ubiquitin.Methods Human RabGEFl gene was cloned into pEF6/Myc-His C vector. The interaction between RabGEFl and HOIP was verified by Co-IP experiment. The interaction domain between RabGEFl and HOIP was analyzed by GST-pulldown. The interaction and subcellular localization were verified by immunofluorescence. The linear ubiquitination of RabGEFl was detected by in vivo ubiquitination experiment. The NTA-His ubiquitination assay further confirmed that RabGEF1 can undergo linear ubiquitination modification. The ubiquitinated RabGEF1 proteinsamples were subjected to mass spectrometry to analyze the specific site modified by ubiquitin. The mutant plasmid of potential RabGEFl ubiquitination site was constructed according to the mass spectrometry results, and the ubiquitination site of RabGEFl was further verified with RabGEFl KR mutant plasmids in vivo ubiquitination experiment.Results RabGEFl interacts with HOIP via ZF-NEF domain. RabGEFl co-locates with HOIP in the cytoplasm. LUBAC mediated linear ubiquitination modification of RabGEFl depends on LUBAC enzyme activity, and the ubiquitination modification site of RabGEFl is K158.Conclusion RabGEFl is a novel substrate for linear ubiquitination modification, and the site of linear ubiquitination modification is K158.

Abstract:Objective To explore the regulated function and mechanisms of deubiquitinating enzyme USP10 regulation under physiological conditions.Methods GEO2R and Metascape analyze the differential gene expression and pathway enrichment in microarray (GSE198574) of Usp10^+/+ and Usp10^-/- neonatal kidney tissues. Western blot and immunohistochemistry are used to measure the expression levels of candidate transcription factors. Co-immunoprecipitation (Co-IP) and GST-pull down assays analyze the interaction between USP10 and the candidate molecules, and deubiquitination experiments verify the regulatory mechanisms of USP10 on target molecules. The expression of cell proliferation marker p21 and apoptosis marker Cleaved-caspase 3 are detected by Western blot. Meanwhile, CCK-8 and plate clonality assays analyze the regulatory functions of USP10 on cell proliferation.Results The TGF-β/BMP signaling pathway is activated in kidney tissues of Usp10^-/- neonatal mice. Physiological deficiency of Usp10 in mice leads to downregulation of Smad ubiquitin-related factor-1 (Smurf1) protein and upregulation of Smad1/5 without affecting their transcription levels. Mechanistically, USP10 interacts with Smurf1 and removes poly-ubiquitylation of Smurf1 which rely on its deubiquitination activity. USP10-deficient promotes the expression of cell cycle inhibitors p21, which is one of the transcriptional target gene of Smad1/5 and inhibits cell proliferation.Conclusion USP10 inhibits TGF-β/BMP signaling pathway by deubiquitinating and stabilizing Smurf1, thus maintains cell proliferation homeostasis.

Abstract:Prostate cancer (PCa) has been the second most common cancer in men with the continuous development of the aging population, the increasing incidence and mortality rate of PCa, and complex occurrence and development mechanism of PCa. The overactivation of AR signaling pathway not only promotes the occurrence of castration-resistance PCa (CPRC), but also plays a key role in the drug resistance of PCa. Current strategies of PCa treatment are relatively limited and accompanied with several serious side-effects. Therefore, the continuous development of targeted therapy, we need to find new treatments to improve the efficacy. Numerous studies have demonstrated that the abnormal function of E3 ubiquitin ligase adaptor, speckle-type POZ protein (SPOP) has a close relationship with the occurrence and progression of PCa. Herein, this review will combine recent researches to describe the basic structure and function of SPOP, summarize the effect of SPOP on PCa, and explore the possibility of stratified treatment for PCa patients with SPOP mutations or not. For example,PCa patients with SPOP mutation can evidently enhance AR signal pathway, disrupt DNA damage response and immune response, leading to occurrence and progression of PCa. While, in the case of PCa patients with SPOP wildtype, can also occur the gene fusion, leading to imbalance of stress response protein, which may induce the occurrence and progression of PCa. Thus, it is essential that using genetic testing of patients in the early stage of treatment, which may indicate the stratified treatment for PCa patients, thus improving the prognosis of PCa efficiently.

Abstract:Prostate cancer is the fastest growing cancer among Chinese male population. Resistance to antiandrogen therapy is the leading cause of death in patients with prostate cancer. Therefore, solving the drug resistance conundrum is the key issue for translational research in prostate cancer. Mammalian cells utilize the ubiquitin-proteasome system to degrade targeted proteins. Consequently, key oncogenes in prostate cancer, such as upstream ubiquitination regulators (e.g., deubiquitinases) of androgen receptor (AR), are potential therapeutic targets. However, these enzymes have a broad spectrum of substrates and may be off target. Recently, the proteolysis-targeting chimeras (PROTAC) technology developed based on the ubiquitin-proteasome system is the most promising and revolutionary new anti-cancer drug discovery technology, enabling the degradation of target proteins by specific E3 ubiquitin ligases without affecting other substrates. Compared with traditional small molecule inhibitors, PROTAC hold great advantages in overcoming drug resistance as well as targeting undruggable targets. Currently, the PROTAC degraders targeting the AR has achieved success in phase II clinical trials. In the future, the new technology targeting protein ubiquitination and degradation pathway will bring new breakthroughs for the clinical treatment of prostate cancer.

Abstract:The mechanisms underlying of the specific loss of dopaminergic neurons and α-synuclein aggregation in the substantia nigra in Parkinson’s disease (PD) is still an enigma. Abnormal protein aggregation is largely caused by dysfunction of the ubiquitin-proteasome system (UPS). Protein ubiquitination is promoted by a cascade of ubiquitinating enzymes, and is reverse-regulated by deubiquitylases (DUBs). Abnormal process in ubiquitination and deubiquitination leads to abnormal protein aggregation and inclusion body formation, which will cause the neuronal damage. Recent studies have reported that protein ubiquitination and deubiquitination play an important role in the pathogenesis of PD. E3 ubiquitin ligases, which promote protein ubiquitination, are beneficial to α-synuclein clearance, promote the survival of dopaminergic neurons, and maintain mitochondrial function, etc. DUBs, which remove ubiquitin of substrate proteins, inhibit α-synuclein degradation, regulate mitochondrial function and iron metabolic homeostasis in neurons. In this review, we summarized the mechanism of protein ubiquitination and deubiquitination involved in dopaminergic neuronal injury through E3 ubiquitin ligase and DUBs.

Abstract:Protein neddylation is a ubiquitylation-like post-translational modification, catalyzed by a cascade consisting of three enzymes: NEDD8-activating enzyme E1 (NAE), an NEDD8-conjugating enzyme E2 (UBE2M or UBE2F), and NEDD8 E3 ligases. Cullin family proteins are the physiological substrates of neddylation, and Cullin neddylation activates Cullin-RING ligases (CRLs). CRLs are the largest ubiquitin E3 ligase family and mediate the ubiquitylation of ~20% cellular proteins to regulate many biological processes, including cell cycle, DNA damage response, cell growth, metabolism, survival, autophagy, migration, and immune escape. In the process of deneddylation, NEDD8 is hydrolyzed and removed from the substrate protein by specific deneddylation enzymes, and is released into cells to maintain the dynamic balance of the neddylation/deneddylation. NEDD8 and NEDD8 enzymes are overexpressed or upregulated in a variety of cancers, leading to excessive activation of CRLs and subsequent degradation of many tumor suppressor proteins, thereby promoting the growth and survival of lung cancer cells, and lung tumorigenesis. Protein neddylation has been validated to be a promising target for anti-cancer therapy. Similarly, some deneddylation enzymes are highly expressed in lung cancer and their changes are also closely related to the growth and tumorigenesis of various malignant tumors, thus is a potential target for anti-cancer therapy. In this review, we mainly focus on the role of protein neddylation/deneddylation in lung cancer, how the expression of neddylation and deneddylation pathways is changed in lung cancer cells, and how they regulate the growth and survival of lung cancer cells, as well as the lung tumor microenvironment and the related inflammatory/immune response. A better understanding of the role of key components of neddylation/deneddylation pathways in the promotion of lung cancer cell growth and tumorigenesis and establishment of lung tumor microenvironment will provide a sound rationale for targeting this pathway for effective lung cancer therapy.

Abstract:Protein ubiquitination is one of the most important post-translational modifications in eukaryotes, which controls many key fate-decision processes in cells from division to death. Ubiquitin dynamics and homeostasis are strictly regulated by E1-E2-E3 cascade enzymatic system which add ubiquitin to substrate proteins through activating, conjugating and ligating steps, and reversely regulated by deubiquitinases that release conjugated ubiquitin from substrate proteins. In human, there are approximately 100 deubiquitinating enzymes, 2 E1 enzymes, 40 E2 enzymes and more than 600 E3 ligases, which form a balanced but complex system remain to be unraveled. Chemical probes targeting ubiquitination systems and deubiquitinases have emerged as a powerful technique to profile these important proteins and especially extensively extend our knowledge about the molecular mechanism of ubiquitination process. This review aims to summarize basic design principles and synthetic methods for the chemical probes as well as their biological applications. In the first part, we define the composition of chemical probes including the reporter tag, recognition module and reactive group, which can be classified further based on the reaction principles of different reactive groups. Then we summarize previously synthetic and ligation strategies of recognition modules by total chemical synthesis and semi-synthesis, and detailed methods for the incorporation of reactive groups. In the second part, we highlight various biological applications for these probes. For profiling deubiquitinases, different scaffolds such as mono-ubiquitin and di-ubiquitin were designed based on their mode of action, with the hand of these probes, great progress has been made in the study of the multi-layer regulation of deubiquitinases controlling chain length, cleavage direction, linkage type, and substrate specificity. Furthermore, we discuss recent work that has brought a giant leap in the identification and/or mechanistic characterization of E3 ligases illuminated by activity-based chemical probes. Exquisite structural designs help to capture E3 ligases in different catalytic states and novel ubiquitin transfer mechanisms were visualized by structural biology. We anticipate great expansion of knowledge in catalytic mechanism of E3 ligases from these chemical probes, facilitating the theory-driven drug discovery and, in particular, paving the way for highly attractive technologies such as proteolysis-targeting chimeras (PROTACs).

Abstract:Objective To screen efficient PROTAC from a bound of compounds designed to degrade a specific target, we established a stable and high-throughput screening system.Methods The two subunits of Nanoluc, HiBiT and LgBiT, was fused with mCherry-target-Halo and GFP, respectively. Colocalization of mCherry and GFP indicates the well-assembly of Nanoluc, and the activity of Nanoluc reflects the amount of target protein directly. Stable cell lines overexpressing HiBiT-mCherry-Target-Halo and GFP-LgBiT were constructed using lentivirus packaging systems. HaloPROTAC3,which recuits Halo tagged protein to Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex, was used to induce the degradation of Halo tag fused target protein. Western blot, Nanoluc activity analysis, and flow cytometry were used to evaluate the degradation efficiency of HaloPROTAC3.Results HaloPROTAC3 induce the degradation of HiBiT-mCherry-Target-Halo effectively in a time and concentrition dependent manner.Conclusion A novel strategy combined with Nanoluc and fluorescence analysis for PROTAC screening was developed. With HaloPROTAC3 as a positive control, the strategy provides a guarantee for the optimal development and application of PROTAC.