目的:我们旨在基于蛋白质序列将泛素特异性蛋白酶(USP)与去泛素化酶家族中的其他成员准确区分开来。此外,我们试图阐明USP26调节Krüppel样因子6(KLF6)的具体调控机制,并评估该调控对宫颈癌细胞增殖和迁移的后续影响.
方法:所有去泛素酶(DUB)序列分为USP和非USP。特征向量,包括188D,n-gram,和400D尺寸,从这些序列中提取,并通过Weka软件进行二元分类。接下来,还分析了30种人类USP,以鉴定保守的基序并确定进化关系。实验上,将90多个独特的DUB编码质粒转染到HeLa细胞系中,以评估KLF6蛋白水平的改变,并分离参与KLF6调节的特定DUB.随后的实验利用野生型(WT)USP26过表达和shRNA介导的USP26敲低两者来检查KLF6蛋白水平的变化。进行半衰期实验以评估USP26对KLF6蛋白稳定性的影响。免疫沉淀用于确认USP26-KLF6相互作用,和泛素化试验探讨USP26在KLF6去泛素化中的作用。进行另外的细胞测定以评估USP26对HeLa细胞增殖和迁移的影响。
结果:1.在188D的提取特征向量中,400D,和n-gram,所有12个分类器都表现出优异的性能。RandomForest分类器在此评估中表现出优异的性能。对30个人类USP的系统发育分析显示存在9个独特的基序,包含锌指和泛素特异性蛋白酶结构域。2.通过对去泛素酶文库的系统筛选,USP26被确定为与KLF6相关的唯一DUB。3.USP26正调节KLF6的蛋白质水平,如在293T和Hela细胞系中在shUSP26敲低后KLF6蛋白质表达的降低所证明。此外,半衰期实验表明USP26延长了KLF6的稳定性。4.免疫沉淀实验揭示了USP26和KLF6之间的强相互作用。值得注意的是,功能性相互作用结构域被定位到USP26的氨基酸285-913,而不是1-295区域.5.发现WTUSP26减弱KLF6的泛素化水平。然而,突变体USP26取消其去泛素化活性。6.功能性生物学实验表明,USP26的过表达抑制了HeLa细胞的增殖和迁移。相反,USP26的敲低被证明可以促进这些致癌特性。
结论:1.在蛋白质序列水平,USP家族的成员可以与非USP蛋白有效区分。此外,已经在人USP的序列中鉴定了特定的功能基序。2.去泛素化酶USP26已被证明靶向KLF6进行去泛素化,从而调节其稳定性。重要的是,USP26在调节宫颈癌细胞的增殖和迁移中起关键作用。
We aim to accurately distinguish ubiquitin-specific proteases (USPs) from other members within the deubiquitinating enzyme families based on protein sequences. Additionally, we seek to elucidate the specific regulatory mechanisms through which USP26 modulates Krüppel-like factor 6 (KLF6) and assess the subsequent effects of this regulation on both the proliferation and migration of cervical cancer cells.
All the deubiquitinase (DUB) sequences were classified into USPs and non-USPs. Feature vectors, including 188D, n-gram, and 400D dimensions, were extracted from these sequences and subjected to binary classification via the Weka software. Next, thirty human USPs were also analyzed to identify conserved motifs and ascertained evolutionary relationships. Experimentally, more than 90 unique DUB-encoding plasmids were transfected into HeLa cell lines to assess alterations in KLF6 protein levels and to isolate a specific DUB involved in
KLF6 regulation. Subsequent experiments utilized both wild-type (WT) USP26 overexpression and shRNA-mediated USP26 knockdown to examine changes in KLF6 protein levels. The half-life experiment was performed to assess the influence of USP26 on KLF6 protein stability. Immunoprecipitation was applied to confirm the USP26-KLF6 interaction, and ubiquitination assays to explore the role of USP26 in
KLF6 deubiquitination. Additional cellular assays were conducted to evaluate the effects of USP26 on HeLa cell proliferation and migration.
1. Among the extracted feature vectors of 188D, 400D, and n-gram, all 12 classifiers demonstrated excellent performance. The RandomForest classifier demonstrated superior performance in this assessment. Phylogenetic analysis of 30 human USPs revealed the presence of nine unique motifs, comprising zinc finger and ubiquitin-specific protease domains. 2. Through a systematic screening of the deubiquitinase library, USP26 was identified as the sole DUB associated with KLF6. 3. USP26 positively regulated the protein level of
KLF6, as evidenced by the decrease in
KLF6 protein expression upon shUSP26 knockdown in both 293T and Hela cell lines. Additionally, half-life experiments demonstrated that USP26 prolonged the stability of KLF6. 4. Immunoprecipitation experiments revealed a strong interaction between USP26 and
KLF6. Notably, the functional interaction domain was mapped to amino acids 285-913 of USP26, as opposed to the 1-295 region. 5. WT USP26 was found to attenuate the ubiquitination levels of
KLF6. However, the mutant USP26 abrogated its deubiquitination activity. 6. Functional biological assays demonstrated that overexpression of USP26 inhibited both proliferation and migration of HeLa cells. Conversely, knockdown of USP26 was shown to promote these oncogenic properties.
1. At the protein sequence level, members of the USP family can be effectively differentiated from non-USP proteins. Furthermore, specific functional motifs have been identified within the sequences of human USPs. 2. The deubiquitinating enzyme USP26 has been shown to target KLF6 for deubiquitination, thereby modulating its stability. Importantly, USP26 plays a pivotal role in the modulation of proliferation and migration in cervical cancer cells.