Subunit‐Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin‐binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site‐specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N‐terminal glycine is unveiled after protease‐mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin‐specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit‐specific labeling of ubiquitin chains when studying how DUBs process these chains.     

N末端改造提高GH11家族木聚糖酶热稳定性的研究进展

木聚糖酶具有重要的工业应用价值,在食品、饲料、造纸等领域有着良好的发展前景。不同应用领域对木聚糖酶的酶学性质具有不同要求,耐热性是木聚糖酶在实际生产过程中最有应用价值的性质之一;因此,许多研究者致力于木聚糖酶耐热性机制的研究。研究发现GH11家族木聚糖酶的热稳定性与其N末端区域密切相关,可以通过N末端改造的策略有效提升GH11家族木聚糖酶的热稳定性。本文主要综述了影响GH11家族木聚糖酶热稳定性的因素、N末端改造提升木聚糖酶热稳定性的技术和方法,并对耐热木聚糖酶的应用前景进行展望,以期为GH11家族木聚糖酶的耐热性研究提供一定的参考。     

HCV核心抗原N-端片段生物素化表达质粒的构建及表达

目的构建HCV核心(C)抗原N-端1～130aa片段的生物素化表达质粒,并在大肠杆菌中进行表达。方法PCR扩增HCV C抗原N-端1～130aa片段的编码基因,拼接上生物素-蛋白连接酶底物肽序列(BSP),构建重组原核表达质粒pGEX4T-1-CN130BSP,转化大肠杆菌Rosetta,IPTG诱导表达。表达产物经SDS-PAGE后,电转至硝酸纤维素膜上,以抗HCVC抗原的单克隆抗体和辣根过氧化物酶标记的亲和素对表达产物进行分析。结果PCR扩增出约466bp的目的基因片段;质粒pGEX4T-1-CN130BSP经PCR及双酶切鉴定,与预期结果一致,测序鉴定基因无突变;SDS-PAGE显示表达产物相对分子质量约为42000,22℃诱导16h可溶性表达产物含量较高;表达的重组蛋白可被抗HCV C抗原的单克隆抗体所识别,并能特异性结合辣根过氧化物酶标记的亲和素。结论已成功构建了HCV C抗原N-端1～130aa片段生物素化表达质粒,并表达出带生物素标签的GST融合蛋白,为进一步研制HCV双抗原夹心检测试剂奠定了基础。     

Conformational Stabilization of Gp41-Mimetic Miniproteins Opens Up New Ways of Inhibiting HIV-1 Fusion

Inhibition of the HIV-1 fusion process constitutes a promising strategy to neutralize the virus at an early stage before it enters the cell. In this process, the envelope glycoprotein (Env) plays a central role by promoting membrane fusion. We previously identified a vulnerability at the flexible C-terminal end of the gp41 C-terminal heptad repeat (CHR) region to inhibition by a single-chain miniprotein (named covNHR-N) that mimics the first half of the gp41 N-terminal heptad repeat (NHR). The miniprotein exhibited low stability, moderate binding to its complementary CHR region, both as an isolated peptide and in native trimeric Envs, and low inhibitory activity against a panel of pseudoviruses. The addition of a disulfide bond stabilizing the miniprotein increased its inhibitory activity, without altering the binding affinity. Here, to further study the effect of conformational stability on binding and inhibitory potency, we additionally stabilized these miniproteins by engineering a second disulfide bond stapling their N-terminal end, The new disulfide-bond strongly stabilizes the protein, increases binding affinity for the CHR target and strongly improves inhibitory activity against several HIV-1 strains. Moreover, high inhibitory activity could be achieved without targeting the preserved hydrophobic pocket motif of gp41. These results may have implications in the discovery of new strategies to inhibit HIV targeting the gp41 CHR region.     

Isolation and partial characterisation of two low molecular weight durum wheat (Triticum durum) glutenins

Two low molecular weight durum wheat (Triticum durum Desf) glutenins, DSG-1 and DSG-2 (durum-wheat sulphur-rich glutenin fractions) were isolated from two cultivars, Mondur of good technological quality and Kidur of poor technological quality. The glutenin fraction, composed mostly of DSG protein, was extracted using a low concentration of NA-tetradecanoate and then fractionted by by using molecular sieve chromatography (Bio-Gel P 30). The amino acid compositions and the N-terminal sequences of the pure DSG proteins were determined, and their hydrophobic characteristics, calculated on the basis of these data, showed that DSG-2 is more hydrophobic than DSG-1. The amino acid compositions of DSG-1 and DSG-2 were different. The N-terminal amino acids of DSG-1 and DSG-2 were also different but were identical for each of the two cultivars. In the case of DSG-1, in addition to the main chain a minor chain was found in which the first three amino acids of the main chain were missing. The minor chain represented about 30% int he DSG-1 of mondur and almost 50% in Kidur.     

Hydroxylation of the Acetyltransferase NAA10 Trp38 Is Not an Enzyme-Switch in Human Cells

NAA10 is a major N-terminal acetyltransferase (NAT) that catalyzes the cotranslational N-terminal (Nt-) acetylation of 40% of the human proteome. Several reports of lysine acetyltransferase (KAT) activity by NAA10 exist, but others have not been able to find any NAA10-derived KAT activity, the latter of which is supported by structural studies. The KAT activity of NAA10 towards hypoxia-inducible factor 1α (HIF-1α) was recently found to depend on the hydroxylation at Trp38 of NAA10 by factor inhibiting HIF-1α (FIH). In contrast, we could not detect hydroxylation of Trp38 of NAA10 in several human cell lines and found no evidence that NAA10 interacts with or is regulated by FIH. Our data suggest that NAA10 Trp38 hydroxylation is not a switch in human cells and that it alters its catalytic activity from a NAT to a KAT.     

Thaumatin-like protein in kiwifruit

A 21-kDa protein extracted from woody tissue underlying the pedicel of kiwifruit (Actinidia deliciosa var deliciosa [A Chev] Liang and Ferguson) was purified to homogeneity by cation exchange, gel filtration, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) on a 15% gel and electro-transfer onto a polyvinylidene difluoride (PVDF) membrane, followed by excision of the band of interest. Amino terminal sequence analysis of this band revealed a high degree of homology with basic endochitinase (EC 3.2.1.14) in orange and thaumatin-like proteins in tobacco, barley, maize and tomato. This is the first record of a thaumatin-like protein in a subtropical berryfruit. Potential implications of this finding and the need for future research are discussed. © 1999 Society of Chemical Industry     

Cyanidin suppresses neoplastic cell transformation by directly targeting phosphatidylinositol 3-kinase

Cyanidin, an anthocyanidin found in fruits and vegetables, has been reported to possess anti-cancer effects. However, there is no study on the chemopreventive effect of cyanidin against neoplastic cell transformation and its molecular mechanisms. In the present study, we compare the anti-carcinogenic effects of cyanidin and cyanidin-3-glucoside (C3G) and investigate their underlying mechanisms. The inhibitory effect of cyanidin on EGF-induced cell transformation was higher than those of C3G in JB6 P+ mouse epidermal (JB6 P+) cells. Both cyanidin and C3G showed dose-dependent radical scavenging activities. It is indicated that the divergent inhibitory effects of cyanidin and C3G are not due to their antioxidant activities. We found that cyanidin, but not C3G, inhibited the EGF-induced Akt/p70S6K phosphorylation. Moreover, cyanidin directly suppressed the activity of PI3K by binding to PI3K directly in an ATP-competitive manner, which indicates that PI3K is one of the molecular targets of cyanidin.     

Rhizopus sp.052凝乳酶的部分酶学性质

Rhizopus sp.052在pH1~7范围内,4℃条件下稳定。凝乳酶的最适作用温度为40℃,55℃保持20min完全失去活性,与小牛凝乳酶性质相似,而热失活的温度低于小牛凝乳酶。Rhizopussp.052凝乳酶对热不稳定的特性有利于干酪副产品乳清的加工处理。Rhizopussp.052凝乳酶对酪蛋白的水解能力比蛋白酶低,比小牛凝乳酶高,可以用于干酪的生产。对N-末段15个氨基酸序列GTGSVPVTDYQNDVE进行检索,结果表明,Rhizopus sp.052凝乳酶与其他菌株相比不完全相同,尚未见有研究者对其进行研究和报道。     

NAA10 p.(D10G) and NAA10 p.(L11R) Variants Hamper Formation of the NatA N-Terminal Acetyltransferase Complex

The majority of the human proteome is subjected to N-terminal (Nt) acetylation catalysed by N-terminal acetyltransferases (NATs). The NatA complex is composed of two core subunits—the catalytic subunit NAA10 and the ribosomal anchor NAA15. Furthermore, NAA10 may also have catalytic and non-catalytic roles independent of NatA. Several inherited and de novo NAA10 variants have been associated with genetic disease in humans. In this study, we present a functional analysis of two de novo NAA10 variants, c.29A>G p.(D10G) and c.32T>G p.(L11R), previously identified in a male and a female, respectively. Both of these neighbouring amino acids are highly conserved in NAA10. Immunoprecipitation experiments revealed that both variants hamper complex formation with NAA15 and are thus likely to impair NatA-mediated Nt-acetylation in vivo. Despite their common impact on NatA formation, in vitro Nt-acetylation assays showed that the variants had opposing impacts on NAA10 catalytic activity. While NAA10 c.29A>G p.(D10G) exhibits normal intrinsic NatA activity and reduced monomeric NAA10 NAT activity, NAA10 c.32T>G p.(L11R) displays reduced NatA activity and normal NAA10 NAT activity. This study expands the scope of research into the functional consequences of NAA10 variants and underlines the importance of understanding the diverse cellular roles of NAA10 in disease mechanisms.