N-豆蔻酰化是一种蛋白质修饰方式，也是常见的蛋白质脂化类型之一。N-豆蔻酰化通过改变蛋白构象、稳定性及细胞定位，影响细胞信号转导、代谢途径重编程以及参与各种生理及病理过程。研究表明N-豆蔻酰化转移酶（NMT）在各类恶性肿瘤中异常表达参与肿瘤发生、发展。因此，靶向NMT抑制剂具有良好抗癌效果，为癌症复发及转移治疗提供了潜在靶点。文章主要介绍了蛋白质N -豆蔻酰化系统并综述其在肿瘤发生、发展中的作用及机制，总结近5年靶向蛋白质N-豆蔻酰化治疗癌症的最新研究进展。

利用聚合酶链式反应从苦参植物叶片中扩增出热应激蛋白基因hsp编码区序列,并成功构建了pET22b-hsp原核表达载体,将其转入大肠杆菌BL21(DE3)表达菌株,在不同温度、时间和不同浓度的IPTG诱导下实现了HSP的体外表达,获得了hsp编码区序列,长度为498 bp,编码166个氨基酸,分子质量约17.8 ku的小分子热应激蛋白(HSP17.8).对此蛋白的物理化学性质、同源进化树、二级结构以及三维结构等生物信息学分析表明:HSP17.8为稳定的亲水性蛋白,其具有多个N-豆蔻酰化位点、磷酸化位点和N-糖基化位点;与羽扇豆应激蛋白HSP的同源性最高,亲缘关系最近;HSP17.8蛋白的结构特点是α螺旋与β折叠依次出现,形成了β1-α1-β2-α2-β3-α3-β4-α4-β5的折叠方式,且同一方向的β折叠被α螺旋包裹在空间结构的内部,构成了立体结构的框架.首次从苦参中成功克隆热应激蛋白基因hsp17.8编码区序列,并在体外得到了其最佳的诱导表达条件,分析了HSP17.8的理化特性、进化关系及结构特点,为该蛋白结构、功能研究奠定了实验基础.

目的 体外获得人巨细胞病毒(human cytomegalovirus, HCMV)临床病毒株 UL148 RNA及探讨其基因功能.方法 将感染HCMV的新生儿尿液接种人胚肺细胞,分离HCMV临床病毒株,并经多重PCR鉴定.扩增UL148基因,经双酶切后克隆入pGEM-T-Easy质粒,构建重组质粒,并置于T7启动子下游,经重组质粒、PCR产物、双酶切产物电泳鉴定及序列测定.克隆成功的质粒以32P标记,进行体外转录RNA.根据UL148序列特征应用生物信息学分析其翻译后修饰位点.结果 成功在体外获得HCMV临床病毒株,经电泳及序列测定证实重组质粒构建成功,在T7 RNA聚合酶作用下,体外获得UL148 RNA.翻译后修饰位点显示UL148基因存在1个与细胞黏附相关的特征序列,1个豆荚外源凝集素β-链标记位点、2个N-豆蔻酰化位点,1个酪氨酸激酶Ⅱ磷酸化位点,7个蛋白激酶C磷酸化位点,1个cAMP/cGMP-依赖性蛋白激酶磷酸化位点和2个N-糖基化位点,1个跨膜区域.结论 UL148基因编码产物可能为一个病毒黏附分子,参与宿主细胞的信号转导作用.

The cluster of differentiation 81 (CD81),a member of the transmembrane 4 superfamily,is primarily found to be expressed in a wide variety of cells including T and B cells of vertebrates as a critical modulator.In the present study,the open reading frame of a CD81 gene homolog (Lja-CD81)was cloned in lamprey,Lampetra japonica,which is 702 bp long and encodes a protein of 233-amino acids.Although Lja-CD81 seems to be close to CD9 molecules in their full-length sequences,Lja-CD81 possesses higher identity to vertebrates' CD81 than to CD9 (including a lamprey CD9) molecules in their large extracellular loops.In addition,it also possesses a myristoylation site (Met-Gly-Val-Glu-Gly-Cys-Leu-Lys) in its N-terminal region which is identical to the N-terminal regions of CD81 molecules.These data suggest that CD9 and CD81 molecules diverged no later than the emergence of jawless vertebrates.The mRNA levels of Lja-CD81 in lymphocytes and supraneural myeloid bodies were up-regulated significantly after stimulation with mixed antigens,and a similar expressional pattern of Lja-CD81 at protein level was also confirmed.Furthermore,Lja-CD81 was found to be co-localized with variable lymphocyte receptor B (VLRB) evenly on the cell membrane of peripheral blood lymphocytes isolated from control group,but they were found to aggregate on one side of the membrane of peripheral blood VLRB+ lymphocytes after stimulation with mixed antigens.All these results indicate that the Lja-CD81 identified in lamprey may play an important role in the immune response of lamprey VLRB+ lymphocytes.

Many geminivirus C4 proteins induce severe developmental abnormalities in plants.We previously demonstrated that Tomato leafcurlYunnan virus (TLCYnV) C4 induces plant developmental abnormalities at least partically by decreasing the accumulation of NbSKη,an ortholog of Arabidopsis BIN2 kinase involved in the brassinosteroid signaling pathway,in the nucleus through directing it to the plasma membrane.However,the molecular mechanism by which the membrane-associated C4 modifies the localization of NbSKη in the host cell remains unclear.Here,we show that TLCYnV C4 is a nucleocytoplasmic shuttle protein,and that C4 shuttling is accompanied by nuclear export of NbSKη.TLCYnV C4 is phosphorylated by NbSKη in the nucleus,which promotes myristoylation of the viral protein.Myristoylation of phosphorylated C4 favors its interaction with exportin-β (XPO I),which in turn facilitates nuclear export of the C4/NbSKη complex.Supporting this model,chemical inhibition of N-myristoyltransferases or exportin-α enhanced nuclear retention of C4,and mutations of the putative phosphorylation or myristoylation sites in C4 resulted in increased nuclear retention of C4 and thus decreased severity of C4-induced developmental abnormalities.The impact of C4 on development is also lessened when a nuclear localization signal or a nuclear export signal is added to its C-terminus,restricting it to a specific cellular niche and therefore impairing nucleocytoplasmic shuttling.Taken together,our results suggest that nucleocytoplasmic shuttling of TLCYnV C4,enabled by phosphorylation by NbSKη,myristoylation,and interaction with exportin-α,is critical for its function as a pathogenicity factor.

Plants utilize intracellular nucleotide-binding leucine-rich repeat domain-containing receptors (NLRs) to recognize pathogen effectors and induce a robust defense response named effector-triggered immunity (ETI).The Arabidopsis NLR protein HOPZ-ACTIVATED RESISTANCE 1 (ZAR1) forms a precomplex with HOPZ-ETI-DEFIClENT 1 (ZED1),a receptor-like cytoplasmic kinase (RLCK) Xll-2 subfamily member,to recognize the Pseudomonas syringae effector HopZ1a.We previously described a dominant mutant of Arabidopsis ZED1,zed1-D,which displays temperature-sensitive autoimmunity in a ZAR1-dependent manner.Here,we report that the RLCKs SUPPRESSOR OF ZED1-D1 (SZE1) and SZE2 associate with the ZAR1-ZED1 complex and are required for the ZED1-D-activated autoimmune response and HopZ1a-triggered immunity.We show that SZE1 but not SZE2 has autophosphorylation activity,and that the N-terminal myristoylation of both SZE1 and SZE2 is critical for their plasma membrane localization and ZED1-D-activated autoimmunity.Furthermore,we demonstrate that SZE1 and SZE2 both interact with ZAR1 to form a functional complex and are required for resistance against P.syringae pv.tomato DC3000 expressing HopZ1a.We also provide evidence that SZE1 and SZE2 interact with HopZ1a and function together with ZED1 to change the intramolecular interactions of ZAR1,leading to its activation.Taken together,our results reveal SZE1 and SZE2 as critical signaling components of HopZ1a-triggered immunity.

Various lipids and lipid metabolites are bound to and modify the proteins in eukaryotic cells,which are known as'protein lipidation'.There are four major types of the protein lipidation,i.e.myristoylation,palmitoylation,prenylation,and glycosylphosphatidylinositol anchor.N-myristoylation refers to the attachment of 14-carbon fatty acid myristates to the N-terminal glycine of proteins by N-myristoyltransferases (NMT) and affects their physiology such as plasma targeting,subcellular tracking and localization,thereby influencing the function of proteins.With more novel pathogenic N-myristoylated proteins are identified,the N-myristoylation will attract great attentions in various human diseases including infectious diseases,parasitic diseases,and cancers.In this review,we summarize the current understanding of N-myristoylation in physiological processes and discuss the hitherto implication of crosstalk between N-myristoylation and other protein modification.Furthermore,we mention several wellstudied NMT inhibitors mainly in infectious diseases and cancers and generalize the relation of NMT and cancer progression by browsing the clinic database.This review also aims to highlight the further investigation into the dynamic crosstalk of Nmyristoylation in physiological processes as well as the potential application of protein N-myristoylation in translational medicine.

Three-way junctions are characteristic structures of the tubular endoplasmic reticulum (ER) network. Junctions are formed through atlastin (ATL)-mediated membrane fusion and stabilized by lunapark (Lnp). However, how Lnp is preferentially enriched at three-way junctions remains elusive. Here, we showed that Lnp loses its junction localization when ATLs are deleted. Reintro-duction of ATL1 R77A and ATL3, which have been shown to cluster at the junctions, but not wild-type ATL1, relocates Lnp to the junctions. Mutations in the N-myristoylation site or hydrophobic residues in the coiled coil (CC1) of Lnp N-terminus (NT) cause mis-targeting of Lnp. Conversely, deletion of the lunapark motif in the C-terminal zinc finger domain, which affects the homo-oligomerization of Lnp, does not alter its localization. Purified Lnp-NT attaches to the membrane in a myris-toylation-dependent manner. The mutation of hydrophobic residues in CC1 does not affect membrane association, but compromises ATL interactions. In addition, Lnp-NT inhibits ATL-mediated vesicle fusion in vitro. These results suggest that CC1 in Lnp-NT con-tacts junction-enriched ATLs for proper localization;subsequently, further ATL activity is limited by Lnp after the junction is formed. The proposed mechanism ensures coordinated actions of ATL and Lnp in gener-ating and maintaining three-way junctions.

目的 应用生物信息学方法分析人源和禽源H9N2禽流感病毒血凝素蛋白(hemagglutinin,HA)的主要特性,预测可能的B/T细胞抗原表位. 方法 以H9N2禽流感病毒HA蛋白的氨基酸序列一级结构为基础分析其保守性,采用ProtParam预测H9N2禽流感病毒HA蛋白的理化特性,SOPMA预测其二级结构,MotifScan预测翻译后修饰位点,DNAStar分析其序列的亲水性指数、柔韧性指数、可及性参数以及抗原指数并推测B细胞抗原表位的可能位置,采用SYFPEITHI的T细胞表位预测工具分别预测CTL和Th细胞表位. 结果 人源和禽源H9N2禽流感病毒HA蛋白氨基酸序列分别有90和75个变异位点,其中有38个突变位点与氨基酸置换均相同,有14个突变位点相同但氨基酸置换不同.人源和禽源毒株中还各有38和23个特异性突变位点.HA蛋白存在多个糖基化、酰胺化及磷酸化等翻译后修饰位点.除了蛋白激酶C磷酸化位点外,其他类型的翻译后修饰位点在人源和禽源毒株中均有一些变化.经综合各种单参数,HA蛋白均为亲水性蛋白.人源和禽源H9N2禽流感病毒HA蛋白含有4个B细胞表位,8个CTL细胞表位和6个Th细胞表位,以人源和禽源的B细胞和Th细胞表位差异较大,而CTL细胞表位有部分序列相同. 结论 人源和禽源H9N2禽流感病毒HA蛋白的氨基酸序列有一定差异,其B/T细胞抗原表位的不同,可为H9N2禽流感病毒疫苗的研制提参考.