Protein Microarray Characterization of the S-Nitrosoproteome

Nitric oxide (NO) mediates a substantial part of its physiologic functions via S-nitrosylation, however the cellular substrates for NO-mediated S-nitrosylation are largely unknown. Here we describe the S-nitrosoproteome using a high-density protein microarray chip containing 16,368 unique human proteins. We identified 834 potentially S-nitrosylated human proteins. Using a unique and highly specific labeling and affinity capture of S-nitrosylated proteins, 138 cysteine residues on 131 peptides in 95 proteins were determined, defining critical sites of NO''s actions. Of these cysteine residues 113 are novel sites of S-nitrosylation. A consensus sequence motif from these 834 proteins for S-nitrosylation was identified, suggesting that the residues flanking the S-nitrosylated cysteine are likely to be the critical determinant of whether the cysteine is S-nitrosylated. We identify eight ubiquitin E3 ligases, RNF10, RNF11, RNF41, RNF141, RNF181, RNF208, WWP2, and UBE3A, whose activities are modulated by S-nitrosylation, providing a unique regulatory mechanism of the ubiquitin proteasome system. These results define a new and extensive set of proteins that are susceptible to NO regulation via S-nitrosylation. Similar approaches could be used to identify other post-translational modification proteomes.It is known that NO regulates the majority of its physiologic function through S-nitrosylation (1). Protein-assisted or small molecule, S-nitrosoglutathione (GSNO)¹ trans-nitrosylation, oxidative S-nitrosation, and metalloprotein-catalyzed S-nitrosylation are the prominent cellular mechanisms that are utilized to S-nitrosylate proteins (2). A number of proteins are known to be S-nitrosylated and this post-translational modification can either activate or inactivate a protein''s biologic activity (1, 3). A number of attempts at probing tissue-specific S-nitrosoproteomes have been made, but the results of these are limited to proteins that are S-nitrosylated to a great degree and which are present at high concentrations (2, 4–6). Recently, to investigate determinants of S-nitrosylation, yeast and human target protein microarrays have been studied. However, these assay were limited because of the small number of proteins present on the chip (7). In addition, many proteins that are known to be S-nitrosylated have been studied through a targeted and biased approach (8). To overcome these shortcomings, we report the use of a 16,368 human protein microarray chip to better define the human S-nitrosoproteome.Ubiquitin is a 76-amino-acid long polypeptide that can be covalently added to lysine residues on targeted proteins either as single monomers or in chains. Ubiquitination of proteins can dramatically alter their function or localization depending on the number of ubiquitin attached and the nature of their linkages. The most well characterized ubiquitin-mediated process is targeting of the protein for degradation by the 26S proteasome, which occurs via poly-ubiquitination linked together through lysine 48 on the ubiquitin monomers. Ubiquitination occurs in a three-step enzymatic process in which the third enzyme, the ubiquitin protein ligase (E3) determines protein target specificity (9). NO S-nitrosylates the RING finger E3 ligases, parkin and XIAP, modifying their function (10, 11). In the case of parkin, S-nitrosylation transiently activates its E3 ligase activity, but ultimately inhibits its activity (12). In contrast, XIAP''s E3 ligase activity is unaffected by S-nitrosylation, but its anti-apoptotic function is compromised (11). Using the 16,368 human protein microarray, we identify a number of NO-regulated E3 ligases, the majority of which are activated by NO-dependent S-nitrosylation.     

Circumventing the heterogeneity and instability of human serum albumin-interferon-alpha2b fusion protein by altering its orientation

Albuferon is a novel long-acting interferon resulted from the direct genetic fusion of human albumin and interferon-alpha2b (HSA-IFN-alpha2b). Albuferon, co-developed by Human Genome Sciences Inc. and Novartis, is currently in late stage development for the treatment of hepatitis C. It was unexpected that HSA-IFN-alpha2b secreted from Pichia pastoris migrated as doublets on non-reducing SDS-PAGE and was prone to form covalent aggregates in aqueous solution. The heterogeneity and instability of HSA-IFN-alpha2b lowered its recovery rate to about 10% and necessitated lyophilized formulation. Site-directed mutagenesis revealed that the heterogeneity and instability of HSA-IFN-alpha2b was caused by the incomplete disulfide bridge formation between Cys1 and Cys98 of IFN-alpha2b. To alleviate the structural perturbation of IFN-alpha2b by HSA, IFN-alpha2b-HSA fusion protein, in which IFN-alpha2b was located at the N-terminus, was created. IFN-alpha2b-HSA was shown to be homogeneous and stable at 37 degrees C for at least 10 days. The improved homogeneity and stability of IFN-alpha2b-HSA increased the recovery rate by 2.5-fold and made the development of stable solution formulation possible. In vitro antiviral assays showed that both fusion proteins retained the activity of IFN-alpha2b, and the EC(50) of HSA-IFN-alpha2b, and IFN-alpha2b-HSA was calculated to be 120+/-12.5, and 160+/-1 1.3ng/ml, respectively. The increased recovery rate and the possibility of solution formulation of IFN-alpha2b-HSA may compensate for its slightly decreased in vitro activity, and makes it to be a promising therapeutic agent that deserves further evaluation.     

光诱导角膜交联及检测技术的研究进展

由于圆锥角膜疾病导致越来越多的人患有近视,常见的矫正方法有佩戴近视眼镜、隐形眼镜等.随着科技的进步,利用光对近视等眼科疾病进行屈光矫正已经成为当前临床中常用的方法.使用光诱导角膜胶原蛋白发生交联,从而达到治疗圆锥角膜疾病、提高患者视力水平的目的,这是一种新型的光治疗眼睛疾病的方法.同时这种方法由于无侵入性、对操作者能力依赖性小等优势成为新的研究热点.本文阐述光诱导角膜交联的基本原理,并介绍其发展历程,分析现有的各种交联方法和角膜检测技术的原理,并对现有交联方法和检测方法的优缺点进行讨论.最后,本文对光诱导角膜交联和检测技术的最新进展进行系统的论述,并对未来的发展趋势进行展望.     

Accelerated evolution of early angiosperms: Evidence from ranunculalean phylogeny by integrating living and fossil data

The new discovery of angiosperm remains in the Jehol Biota of northeastern China contributes to our understanding of the origin and early evolution of flowering plants. The earliest eudicot genus with reproductive organs, Leefructus, was recently documented from the Lower Cretaceous Yixian Formation at 125.8–123.0 Ma, and was reconsidered to be close to the extant family Ranunculaceae based on gross morphology. However, this hypothesis has not been tested using a cladistic approach. To determine the possible allies of Leefructus within extant eudicots, we constructed a 66 morphological data matrix. Molecular and morphological analyses of extant Ranunculales combined with the fossil suggest that it has an affinity with the Ranunculaceae. The earliest fossil record of the eudicots is 127–125 Ma based on tricolpate pollen grains. Thus, we suggest a hypothesis that the basal eudicots might have experienced an accelerated evolution and diversification during the latest Barremian and earliest Aptian, leading to the stem groups of at least six extant families or lineages, 10–15 Myr earlier than currently documented. Angiosperms have undergone multiple uneven pulses of radiation since their origin. Many key character innovations occurred in different stages that could have triggered those radiations in concert with various biotic and abiotic factors.     

Changes in vegetation and soil properties following 6 years of enclosure in riparian corridors

河岸带是维持生物多样性的重要生态系统之一。然而,由于过度放牧引起的植被消耗和过度开垦等人类活动的干扰,河岸带植被多样性和植被盖度受到严重的破坏,甚至威胁了河道的稳定性。围栏封育在退化草地生态系统修复中被广泛应用,但对退化河岸带植被群落和土壤性质的影响尚不明确。本研究的目的旨在明确围封的实施是否会促进河岸带植被群落的物种组成、物种丰富度和物种多样性恢复,土壤氧分如何随围封年限的增加而变化。辽河干流自2012年起被围栏封育管理,本研究在辽河干流河岸带沿岸设置了20个草本群落长期观测样地,记录了2012–2017年样地中植被高度、盖度和个体数量等参数用于物种丰富度和物种多样性的统计分析。同时,分别测定了2012年和2017年植被群落土壤氧分含量,验证了植被群落和土壤氧分对围封的反馈,研究了2012–2017年辽河干流河岸带的围栏封育对物种多样性和土壤氧分的影响。结果表明,随着围封年限的增加,辽河干流河岸带草本群落植被丰富度和多样性显著增加。物种组成方面,菊科植物的优势度显著增加,禾本科植物优势度显著下降。围封后植被群落的恢复和禁止耕作,加速了土壤中磷和钾的消耗,表现为显著降低,土壤有机质含量对围封的响应表现的相对滞后,并没有显著变化。综上所述,本研究为河岸带植被群落物种多样性、物种组成对围封的响应提供了新的见解。     

Phosphorylation of fanconi anemia (FA) complementation group G protein, FANCG, at serine 7 is important for function of the FA pathway

Fanconi anemia (FA) is an autosomal recessive disease of cancer susceptibility. FA cells exhibit a characteristic hypersensitivity to DNA cross-linking agents. The molecular mechanism for the disease is unknown as few of the FA proteins have functional motifs. Several post-translational modifications of the proteins have been described. We and others have reported that the FANCG protein (Fanconi complementation group G) is phosphorylated. We show that in an in vitro kinase reaction FANCG is radioactively labeled. Mass spectrometry analysis detected a peptide containing phosphorylation of serine 7. Using PCR-mediated site-directed mutagenesis we mutated serine 7 to alanine. Only wild-type FANCG cDNA fully corrected FA-G mutant cells. We also tested the effect of human wild-type FANCG in Chinese hamster ovary cells in which the FANCG homologue is mutant. Human FANCG complemented these cells, whereas human FANCG(S7A) did not. Unexpectedly, FANCG(S7A) bound to and stabilized the endogenous forms of the FANCA and FANCC proteins in the FA-G cells. FANCG(S7A) aberrantly localized to globules in chromatin and did not abrogate the internuclear bridges seen in the FA-G mutant cells. Phosphorylation of serine 7 in FANCG is functionally important in the FA pathway.     

A Practicable Li/Na‐Ion Hybrid Full Battery Assembled by a High‐Voltage Cathode and Commercial Graphite Anode: Superior Energy Storage Performance and Working Mechanism

With the rapidly growing demand for low‐cost and safe energy storage, the advanced battery concepts have triggered strong interests beyond the state‐of‐the‐art Li‐ion batteries (LIBs). Herein, a novel hybrid Li/Na‐ion full battery (HLNIB) composed of the high‐energy and lithium‐free Na₃V₂(PO₄)₂O₂F (NVPOF) cathode and commercial graphite anode mesophase carbon micro beads is for the first time designed. The assembled HLNIBs exhibit two high working voltage at about 4.05 and 3.69 V with a specific capacity of 112.7 mA h g^?1. Its energy density can reach up to 328 W h kg^?1 calculated from the total mass of both cathode and anode materials. Moreover, the HLNIBs show outstanding high‐rate capability, long‐term cycle life, and excellent low‐temperature performance. In addition, the reaction kinetics and Li/Na‐insertion/extraction mechanism into/out NVPOF is preliminarily investigated by the galvanostatic intermittent titration technique and ex situ X‐ray diffraction. This work provides a new and profound direction to develop advanced hybrid batteries.