Volume: 20, Issue: 4

Original Research

Phosphoproteomics Reveals the AMPK Substrate Network in Response to DNA Damage and Histone Acetylation

Yuejing Jiang, Xiaoji Cong, Shangwen Jiang, Ying Dong, Lei Zhao, Yi Zang, Minjia Tan, Jia Li

AMP-activated protein kinase (AMPK) is a conserved energy sensor that plays roles in diverse biological processes via phosphorylating various substrates. Emerging studies have demonstrated the regulatory roles of AMPK in DNA repair, but the underlying mechanisms remain to be fully understood. Herein, using mass spectrometry-based proteomic technologies, we systematically investigate the regulatory network of AMPK in DNA damage response (DDR). Our system-wide phosphoproteome study uncovers a variety of newly-identified potential substrates involved in diverse biological processes, whereas our system-wide histone modification analysis reveals a link between AMPK and histone acetylation. Together with these findings, we discover that AMPK promotes apoptosis by phosphorylating apoptosis-stimulating of p53 protein 2 (ASPP2) in an irradiation (IR)-dependent manner and regulates histone acetylation by phosphorylating histone deacetylase 9 (HDAC9) in an IR-independent manner. Besides, we reveal that disrupting the histone acetylation by the bromodomain BRD4 inhibitor JQ-1 enhances the sensitivity of AMPK-deficient cells to IR. Therefore, our study has provided a resource to investigate the interplay between phosphorylation and histone acetylation underlying the regulatory network of AMPK, which could be beneficial to understand the exact role of AMPK in DDR.
研究问题： 1. 在DNA损伤修复中AMPK的调控功能有哪些？主要通过磷酸化调控哪些底物来完成？ 2. AMPK在组蛋白翻译后修饰中的功能有哪些？通过什么底物完成调控？ 3. AMPK对组蛋白翻译后修饰的调控在提高肿瘤细胞电离辐射敏感性方面的应用。 研究方法： 在本研究中，我们结合基因敲除技术、先进的基于生物质谱的定量蛋白质磷酸化组学技术和组蛋白修饰分析技术，建立了对AMPK功能缺乏条件下DNA损伤的生物模型中蛋白磷酸化修饰和组蛋白修饰交互调控行为进行研究的平台，揭示了一系列DNA损伤中受AMPK调控的底物蛋白，并反映了在DNA损伤中AMPK可以通过磷酸化底物蛋白间接调控组蛋白的乙酰化。 主要结果1： 获得AMPK参与DNA损伤反应的磷酸化蛋白组数据。 主要结果2： 发现AMPK在DNA损伤反应中通过磷酸化p53结合蛋白2（ASPP2）来促进细胞凋亡。 主要结果3： 发现AMPK通过磷酸化组蛋白去乙酰化酶9（HDAC9）来调节组蛋白的乙酰化水平。 主要结果4： 发现AMPK缺陷细胞在DNA损伤中对BRD4抑制剂JQ-1敏感性增强。 数据链接：https://www.iprox.org/page/SSV024.html;url=1579003580788LG0U

Page 597-613

Original Research

A Global Multiregional Proteomic Map of the Human Cerebral Cortex

Zhengguang Guo, Chen Shao, Yang Zhang, Wenying Qiu, Wenting Li, Weimin Zhu, Qian Yang, Yin Huang, Lili Pan, Yuepan Dong, Haidan Sun, Xiaoping Xiao, Wei Sun, Chao Ma, Liwei Zhang

The Brodmann area (BA)-based map is one of the most widely used cortical maps for studies of human brain functions and in clinical practice; however, the molecular architecture of BAs remains unknown. The present study provided a global multiregional proteomic map of the human cerebral cortex by analyzing 29 BAs. These 29 BAs were grouped into 6 clusters based on similarities in proteomic patterns: the motor and sensory cluster, vision cluster, auditory and Broca’s area cluster, Wernicke’s area cluster, cingulate cortex cluster, and heterogeneous function cluster. We identified 474 cluster-specific and 134 BA-specific signature proteins whose functions are closely associated with specialized functions and disease vulnerability of the corresponding cluster or BA. The findings of the present study could provide explanations for the functional connections between the anterior cingulate cortex and sensorimotor cortex and for anxiety-related function in the sensorimotor cortex. The brain transcriptome and proteome comparison indicates that they both could reflect the function of cerebral cortex, but show different characteristics. These proteomic data are publicly available at the Human Brain Proteome Atlas (www.brain-omics.com). Our results may enhance our understanding of the molecular basis of brain functions and provide an important resource to support human brain research.

Page 614-632

Original Research

Identification of Age-associated Proteins and Functional Alterations in Human Retinal Pigment Epithelium

Xiuxiu Jin, Jingyang Liu, Weiping Wang, Jiangfeng Li, Guangming Liu, Ruiqi Qiu, Mingzhu Yang, Meng Liu, Lin Yang, Xiaofeng Du, Bo Lei

Retinal pigment epithelium (RPE) has essential functions, such as nourishing and supporting the neural retina, and is of vital importance in the pathogenesis of age-related retinal degeneration. However, the exact molecular changes of RPE during aging remain poorly understood. Here, we isolated human primary RPE (hRPE) cells from 18 eye donors distributed over a wide age range (10–67 years old). A quantitative proteomic analysis was performed to analyze changes in their intracellular and secreted proteins. Age-group related subtypes and age-associated proteins were revealed and potential age-associated mechanisms were validated in ARPE-19 and hRPE cells. The results of proteomic data analysis and verifications suggest that RNF123- and RNF149-related protein ubiquitination plays an important role in protecting hRPE cells from oxidative damage during aging. In older hRPE cells, apoptotic signaling-related pathways were up-regulated, and endoplasmic reticulum organization was down-regulated both in the intracellular and secreted proteomes. Our work paints a detailed molecular picture of hRPE cells during the aging process and provides new insights into the molecular characteristics of RPE during aging and under other related clinical retinal conditions.

Page 633-647

Original Research

Profiling the Bisecting N-acetylglucosamine Modification in Amniotic Membrane via Mass Spectrometry

Qiushi Chen, Yuanliang Zhang, Keren Zhang, Jie Liu, Huozhen Pan, Xinran Wang, Siqi Li, Dandan Hu, Zhilong Lin, Yun Zhao, Guixue Hou, Feng Guan, Hong Li, Siqi Liu, Yan Ren

Bisecting N-acetylglucosamine (GlcNAc), a GlcNAc linked to the core β-mannose residue via a β1,4 linkage, is a special type of N-glycosylation that has been reported to be involved in various biological processes, such as cell adhesion and fetal development. This N-glycan structure is abundant in human trophoblasts, which is postulated to be resistant to natural killer cell-mediated cytotoxicity, enabling a mother to nourish a fetus without rejection. In this study, we hypothesized that the human amniotic membrane, which serves as the last barrier for the fetus, may also express bisected-type glycans. To test this hypothesis, glycomic analysis of the human amniotic membrane was performed, and bisected N-glycans were detected. Furthermore, our proteomic data, which have been previously employed to explore human missing proteins, were analyzed and the presence of bisecting GlcNAc-modified peptides was confirmed. A total of 41 glycoproteins with 43 glycopeptides were found to possess a bisecting GlcNAc, and 25 of these glycoproteins were reported to exhibit this type of modification for the first time. These results provide insights into the potential roles of bisecting GlcNAc modification in the human amniotic membrane, and can be beneficial to functional studies on glycoproteins with bisecting GlcNAc modifications and functional studies on immune suppression in human placenta.
研究问题： 平分型N-乙酰葡萄糖胺（GlcNAc）是一种特殊的N-糖基化修饰。据报道它参与了多种生物学过程，例如细胞粘附和胎儿发育。这种N-糖结构大量存在于人类滋养层细胞中；据推测它对自然杀伤细胞介导的细胞毒性具有抗性，从而使母体能够在不产生排斥的情况下滋养胎儿。在这项研究中，我们假设作为胎儿最后屏障的羊膜也可能表达这种平分型N糖从而保护胎儿。为了验证这一假设，我们对人羊膜进行了糖组学分析，并检测到了平分型N糖。此外，我们对蛋白质组学数据进行了分析，并确认存在含有平分型N糖的糖肽，这些糖肽来源于41个糖蛋白，其中25个糖蛋白是首次被报道有这种修饰。这些结果将有助于含有平分型GlcNAc糖蛋白的功能研究和人类胎盘免疫抑制的功能研究。 研究方法： 本研究开展了人羊膜的糖组学实验并分析了产生的糖组学数据，还深度分析了相关的蛋白质组学数据。 主要结果1： 通过多级质谱技术确定了人羊膜上的N糖含有平分型GlcNAc。 主要结果2： 这些含有平分型GlcNAc的N糖来源于41个糖蛋白，这其中有25个属于是第一次被报道含有这种修饰。 主要结果3： 在我们以往产出的人膀胱和肾脏的蛋白质组学数据中没有发现有同样的修饰的肽段。 数据连接：https://db.cngb.org/search/project/CNP0001701/

Page 648-656

Original Research

High VHL Expression Reverses Warburg Phenotype and Enhances Immunogenicity in Kidney Tumor Cells

Songbiao Zhu, Wenxi Ding, Yuling Chen, Weixuan Wang, Renhua Xu, Chongdong Liu, Xiaohui Liu, Haiteng Deng

Clear cell renal cell carcinoma (ccRCC) is a frequently occurring renal cancer. The Von Hippel-Lindau disease tumor suppressor VHL, a known tumor suppressor gene, is frequently mutated in about 50% of patients with ccRCC. However, it is unclear whether VHL influences the progression of ccRCC tumors expressing wild-type VHL. In the present study, we found that higher expression of VHL was correlated with the better disease-free survival (DFS) in ccRCC patients using The Cancer Genome Atlas (TCGA) datasets. We revealed that VHL overexpression in ccRCC cells inhibited epithelial-mesenchymal transition (EMT), sterol regulatory element-binding protein 1 (SREBP1)-regulated triglyceride synthesis, and cell proliferation. Proteomic analysis provided us a global view that VHL regulated four biological processes, including metabolism, immune regulation, apoptosis, and cell movement. Importantly, we found that VHL overexpression led to up-regulated expression of proteins associated with antigen processing and interferon-responsive proteins, thus rendering ccRCC cells more sensitive to interferon treatment. We defined an interferon-responsive signature (IRS) composed of ten interferon-responsive proteins, whose mRNA expression levels were positively correlated with DFS in ccRCC patients. Taken together, our results propose that the subset of ccRCC patients with high VHL expression benefit from immunotherapy.
肾透明细胞癌（ccRCC）是肾癌中发病率最高的亚型。抑癌基因VHL在50%的ccRCC中发生突变，抑制HIF1的降解，促进肾细胞癌的发生发展。但是目前尚不清楚野生型VHL的表达水平与ccRCC肿瘤进展存在着什么样的关系。在本项研究中，通过分析TCGA数据，我们发现高表达VHL基因肾癌患者的无病生存期显著长于低表达VHL基因的患者。通过构建VHL过表达的ccRCC细胞系，我们揭示VHL过表达降低细胞的迁移速度，抑制SREBP1介导的甘油三酯合成，并且降低细胞的生长速度。定量蛋白质组学分析系统地揭示了VHL蛋白调控了ccRCC中四类生物学过程，代谢、免疫、细胞凋亡和细胞运动。更重要的是我们发现VHL过表达上调抗原处理和呈递相关蛋白，以及干扰素响应蛋白的表达，增加ccRCC细胞的免疫原性和对干扰素的敏感性。我们通过蛋白质组学数据分析定义了一个包含10个差异蛋白的干扰素响应基因群（Interferon Responsive Signature，IRS），其表达水平与ccRCC患者VHL基因表达正相关，可以用来预测患者的无病生存期。综上所述，我们的研究结果揭示了VHL高表达在ccRCC中的生物学功能，加深了我们对ccRCC免疫治疗的认识。

Page 657-669

Original Research

New Metabolic Alterations and A Predictive Marker Pipecolic Acid in Sera for Esophageal Squamous Cell Carcinoma

Lei Liu, Jia Wu, Minxin Shi, Fengying Wang, Haimin Lu, Jibing Liu, Weiqin Chen, Guanzhen Yu, Dan Liu, Jing Yang, Qin Luo, Yan Ni, Xing Jin, Xiaoxia Jin, Wen-Lian Chen

Esophageal squamous cell carcinoma (ESCC) is a major histological subtype of esophageal cancer with a poor prognosis. Although several serum metabolomic investigations have been reported, ESCC tumor-associated metabolic alterations and predictive biomarkers in sera have not been defined. Here, we enrolled 34 treatment-naive patients with ESCC and collected their pre- and post-esophagectomy sera together with the sera from 34 healthy volunteers for a metabolomic survey. Our comprehensive analysis identified ESCC tumor-associated metabolic alterations as represented by a panel of 12 serum metabolites. Notably, postoperative abrosia and parenteral nutrition substantially perturbed the serum metabolome. Furthermore, we performed an examination using sera from carcinogen-induced mice at the dysplasia and ESCC stages and identified three ESCC tumor-associated metabolites conserved between mice and humans. Notably, among these metabolites, the level of pipecolic acid was observed to be progressively increased in mouse sera from dysplasia to cancerization, and it could be used to accurately discriminate between mice at the dysplasia stage and healthy control mice. Furthermore, this metabolite is essential for ESCC cells to restrain oxidative stress-induced DNA damage and cell proliferation arrest. Together, this study revealed a panel of 12 ESCC tumor-associated serum metabolites with potential for monitoring therapeutic efficacy and disease relapse, presented evidence for refining parenteral nutrition composition, and highlighted serum pipecolic acid as an attractive biomarker for predicting ESCC tumorigenesis.
研究问题 食管癌是一类在全球发病率与致死率分别排名第九和第六的恶性肿瘤，而食管鳞癌（Esophageal squamous cell carcinoma，ESCC）是其最常见的组织学分型，约占中国所有食管癌患者病例的90%。ESCC患者在早期阶段缺乏有效的筛查手段，因此大多数患者初诊时即处于进展期，这是导致患者预后不良的一个重要因素。已有的研究报道在肿瘤患者的外周血中存在异常表达的代谢产物，这些代谢物可用于肿瘤诊断、以及疗效和预后的评估。因此，开展ESCC患者血清代谢谱的深入研究，有望鉴定和发现可用于预测ESCC发生的新型生物标志物。 研究方法 为了阐明ESCC的血清代谢谱变化特征，我们招募了34名初治的ESCC患者，这些患者均接受食管切除术，我们收集了他们术前和术后的空腹血血清标本。另外，我们招募了34名健康志愿者，并采集其空腹血血清标本。此外，我们构建了致癌物诱导的ESCC小鼠模型，并分别采集了对照小鼠、以及处于食管异型增生期（Epithelial squamous dysplasia）和食管ESCC癌变期的血清标本。我们对这些血清标本进行了代谢组学检测和分析，全面解析了与ESCC相关的血清代谢谱变化，并发现了血清中可预测ESCC发生的新型代谢物生物标志物。 主要结果 1. 鉴定了与ESCC直接相关的血清代谢谱变化，该变化主要由一组12个血清代谢物来表征；另外发现术后的禁食和肠外营养干预可扰动患者的血清代谢谱。 2. 确定了小鼠和人类之间变化趋势一致的三个ESCC相关血清代谢物：哌啶酸（Pipecolic acid）、1-油酰甘油（1-Oleoylglycerol）和亚磷酸（Phosphoric acid）。其中，哌啶酸在模型小鼠血清中的水平从异型增生期到癌变期逐渐递增。并且，该血清代谢物可准确区分异型增生期小鼠和健康对照组小鼠。 3. 功能研究发现，哌啶酸可帮助ECSS细胞对抗氧化应激诱导的DNA损伤和细胞周期阻滞、并维持细胞在不利条件下的存活。 综上，这项研究发现了一组12个血清代谢物标志物，该标志物组有望用于ESCC治疗疗效的监测和疾病复发的预测。同时，本研究为进一步优化ESCC患者的肠外营养液组分配比提供了坚实的科学依据。此外，本研究发现血清中的哌啶酸是预测ESCC发生的新型潜在生物标志物。 数据链接 https://www.biosino.org/node/project/detail/OEP001955

Page 670-687

Original Research

Global Profiling of 2-hydroxyisobutyrylome in Common Wheat

Ning Zhang, Lingran Zhang, Linjie Li, Junyou Geng, Lei Zhao, Yan Ren, Zhongdong Dong, Feng Chen

As a novel post-translational modification (PTM), lysine 2-hydroxyisobutyrylation (Khib) is considered to regulate gene transcriptional activities in eukaryotic cells; however, the functions of Khib-modified proteins in plants remain unknown. Here, we report that Khib is an evolutionarily-conserved PTM in wheat and its progenitors. A total of 3348 Khib sites on 1074 proteins are identified in common wheat (Triticum aestivum L.) by using affinity purification and mass spectroscopy of 2-hydroxyisobutyrylome. Bioinformatic data indicate that Khib-modified proteins participate in a wide variety of biological and metabolic pathways. Immunoprecipitation confirms that Khib-modified proteins are present endogenously. A comparison of Khib and other main PTMs shows that Khib-modified proteins are simultaneously modified by multiple PTMs. Using mutagenesis experiments and co-immunoprecipitation assays, we demonstrate that Khib on K206 of phosphoglycerate kinase (PGK) is a key regulatory modification for its enzymatic activity, and mutation on K206 affects the interactions of PGK with its substrates. Furthermore, Khib modification of low-molecular-weight proteins is a response to the deacetylase inhibitors nicotinamide and trichostatin. This study provides evidence to promote our current understanding of Khib in wheat plants, including the cooperation between Khib and its metabolic regulation.

Page 688-701

Original Research

Expanding the Coverage of Metabolic Landscape in Cultivated Rice with Integrated Computational Approaches

Xuetong Li, Hongxia Zhou, Ning Xiao, Xueting Wu, Yuanhong Shan, Longxian Chen, Cuiting Wang, Zixuan Wang, Jirong Huang, Aihong Li, Xuan Li

Genome-scale metabolomics analysis is increasingly used for pathway and function discovery in the post-genomics era. The great potential offered by developed mass spectrometry (MS)-based technologies has been hindered, since only a small portion of detected metabolites were identifiable so far. To address the critical issue of low identification coverage in metabolomics, we adopted a deep metabolomics analysis strategy by integrating advanced algorithms and expanded reference databases. The experimental reference spectra and in silico reference spectra were adopted to facilitate the structural annotation. To further characterize the structure of metabolites, two approaches were incorporated into our strategy, i.e., structural motif search combined with neutral loss scanning and metabolite association network. Untargeted metabolomics analysis was performed on 150 rice cultivars using ultra-performance liquid chromatography coupled with quadrupole-Orbitrap MS. Consequently, a total of 1939 out of 4491 metabolite features in the MS/MS spectral tag (MS2T) library were annotated, representing an extension of annotation coverage by an order of magnitude in rice. The differential accumulation patterns of flavonoids between indica and japonica cultivars were revealed, especially O-sulfated flavonoids. A series of closely-related flavonolignans were characterized, adding further evidence for the crucial role of tricin-oligolignols in lignification. Our study provides an important protocol for exploring phytochemical diversity in other plant species.
植物在生长发育和抵御外界胁迫的过程中产生了上百万种代谢物，包括生物碱、萜类、黄酮等，这些代谢物复杂多变的结构赋予其多样的生物功能及药理活性，是具有极大研究价值的宝库。代谢物是上游基因和蛋白调控活动的终端产物，代谢物的直接积累造就了产量、抗逆性等在农业生产上起着决定性作用的表型性状。代谢物是连接基因与表型之间的重要桥梁。近年来，代谢组学方法越来越多地应用于生物功能、通路解析等基础研究，也开始在改良作物营养品质、产量、抗性等育种实践中发挥关键作用，成为作物遗传改良的重要技术基础。现代质谱技术的迅速发展为代谢组学提供了高分辨率、高灵敏度的分析手段，产生的代谢组学数据，数量庞大且结构多变，给鉴定分析工作带来了极大的挑战。由于标准参考谱图的缺乏，仅一小部分检测到的代谢物能得到结构鉴定，严重制约了下游功能及途径的解析，成为代谢组学研究的瓶颈。在方法学上，如何提高代谢组检测与定性的覆盖度和准确性，成为代谢组学研究的关键科学问题。在生物学上，代谢物结构注释水平的提升，有助于下游大量次生代谢途径及生物功能的解析，有助于农作物遗传改良育种的应用研究。 针对植物代谢组的结构注释这一难题，我们整合了算法工具以及大规模的参考谱图库，发展了深度代谢组学分析策略。我们以1）实验标准谱图和2）通过机器学习获得的大量代谢物结构预测而来的理论谱图，作为参考，有效地提升了结构注释的覆盖度和成功率。为了进一步注释参考谱图库中未收录的代谢物结构，我们发展了基于谱图特征结构搜索和中性丢失扫描的方法，用以注释未知谱图中碎片峰所对应的结构骨架、以及中性丢失所对应的修饰基团。同时我们整合了基于代谢物相关性网络的方法，用以进一步注释代谢物的结构和潜在的相互作用关系。我们利用高性能UPLC-Q-Orbitrap质谱平台对150个水稻核心种质进行了非靶向代谢组学实验分析，构建了包含4491个代谢物信号的二级谱图标签库，其中1939个得到了结构注释。通过我们的研究，水稻代谢组的结构注释覆盖度提升了一个数量级上，扩展了对水稻（和其它农作代谢物多样性的认知。得益于水稻代谢物结构的高水平注释，我们揭示了多种修饰类别（碳糖基化、氧糖基化、氧硫酸化）的黄酮在粳稻、籼稻亚种中的差异积累模式，有助于次生代谢相关修饰酶类的遗传定位和功能研究。通过代谢物相关性网络的构建，我们注释了一系列在木质素合成过程中发挥关键作用的黄酮木质素，进一步支持了单子叶不同于双子叶植物的木质素合成通路。 这些研究成果为对植物/农作物的代谢多样性探索和功能研究提供了一个关键的技术方法。

Page 702-714

Original Research

Global Landscape of Native Protein Complexes in Synechocystis sp. PCC 6803

Chen Xu, Bing Wang, Lin Yang, Lucas Zhongming Hu, Lanxing Yi, Yaxuan Wang, Shenglan Chen, Andrew Emili, Cuihong Wan

Synechocystis sp. PCC 6803 (hereafter: Synechocystis) is a model organism for studying photosynthesis, energy metabolism, and environmental stress. Although known as the first fully sequenced phototrophic organism, Synechocystis still has almost half of its proteome without functional annotations. In this study, by using co-fractionation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), we define 291 multi-protein complexes, encompassing 24,092 protein–protein interactions (PPIs) among 2062 distinct gene products. This information not only reveals the roles of photosynthesis in metabolism, cell motility, DNA repair, cell division, and other physiological processes, but also shows how protein functions vary from bacteria to higher plants due to changes in interaction partners. It also allows us to uncover the functions of hypothetical proteins, such as Sll0445, Sll0446, and Sll0447 involved in photosynthesis and cell motility, and Sll1334 involved in regulation of fatty acid biogenesis. Here we present the most extensive PPI data for Synechocystis so far, which provide critical insights into fundamental molecular mechanisms in cyanobacteria.
集胞藻PCC6803是研究光合作用、能量代谢和环境胁迫的模式生物。虽然是第一个完成基因组测序的光能营养型生物，但它仍然有大约一半的蛋白质没有功能注释。为了更深入了解集胞藻6803的蛋白质功能，我们利用共馏分和液相色谱-质谱串联技术（LC/MS/MS）鉴定了其291个蛋白复合物，包含2062个蛋白质和24,092个蛋白质-蛋白质相互作用（PPI）。这些数据不仅揭示了光系统在新陈代谢、细胞运动、DNA修复、细胞分裂和其他生理过程中的作用，而且还展示了从细菌到高等植物由于蛋白质相互作用的变化而导致蛋白质功能的变化。通过蛋白质相互作用，我们还能发现蛋白质的功能，例如，Sll0445、Sll0446和Sll0447参与光合作用和细胞运动，以及Sll1334在调节脂肪酸生物合成中的作用。在本研究中，我们给出了迄今为止最丰富的集胞藻的蛋白质相互作用的数据，该结果将为研究蓝藻的基本分子机制提供了重要的指导和数据支持。

Page 715-727

Original Research

Comparative Secretome Analysis of Magnaporthe oryzae Identified Proteins Involved in Virulence and Cell Wall Integrity

Ning Liu, Linlu Qi, Manna Huang, Deng Chen, Changfa Yin, Yiying Zhang, Xingbin Wang, Guixin Yuan, Rui-Jin Wang, Jun Yang, You-Liang Peng, Xunli Lu

Plant fungal pathogens secrete numerous proteins into the apoplast at the plant–fungus contact sites to facilitate colonization. However, only a few secretory proteins were functionally characterized in Magnaporthe oryzae, the fungal pathogen causing rice blast disease worldwide. Asparagine-linked glycosylation 3 (Alg3) is an α-1,3-mannosyltransferase functioning in the N-glycan synthesis of N-glycosylated secretory proteins. Fungal pathogenicity and cell wall integrity are impaired in Δalg3 mutants, but the secreted proteins affected in Δalg3 mutants are largely unknown. In this study, we compared the secretomes of the wild-type strain and the Δalg3 mutant and identified 51 proteins that require Alg3 for proper secretion. These proteins were predicted to be involved in metabolic processes, interspecies interactions, cell wall organization, and response to chemicals. Nine proteins were selected for further validation. We found that these proteins were localized at the apoplastic region surrounding the fungal infection hyphae. Moreover, the N-glycosylation of these proteins was significantly changed in the Δalg3 mutant, leading to the decreased protein secretion and abnormal protein localization. Furthermore, we tested the biological functions of two genes, INV1 (encoding invertase 1, a secreted invertase) and AMCase (encoding acid mammalian chinitase, a secreted chitinase). The fungal virulence was significantly reduced, and the cell wall integrity was altered in the Δinv1 and Δamcase mutant strains. Moreover, the N-glycosylation was essential for the function and secretion of AMCase. Taken together, our study provides new insight into the role of N-glycosylated secretory proteins in fungal virulence and cell wall integrity.
在致病真菌与植物互作的过程中，病原菌会分泌大量的外泌蛋白来帮助侵染。稻瘟菌引起的稻瘟病严重影响水稻产量；但是在稻瘟菌中，仅报道了少量的分泌蛋白的功能。α-1, 3-甘露糖转移酶Alg3影响了分泌蛋白N-糖链的合成。但是在稻瘟菌中，Alg3的缺失影响了哪些蛋白的分泌，导致突变体∆alg3的致病力和细胞壁完整性受到影响，目前还不清楚。本研究中，作者通过比较稻瘟菌野生型和突变体∆alg3的分泌蛋白质组，鉴定了51个依赖Alg3来正确分泌的蛋白。它们主要参与代谢过程，种间互作，细胞壁组织等过程。作者验证了其中的9个蛋白，发现它们均定位于侵染菌丝外周的质外体空间；并且在突变体∆alg3中，这9个蛋白的N-糖基化水平都受到了破坏，导致了它们不正常的定位和分泌。进一步，作者对其中的两个基因进行了功能研究，INV1和AMCase分别编码可分泌的 invertase 和 chitinase；在突变体∆inv1和 ∆amcase中，稻瘟菌的致病力和细胞壁完整性都受到了严重影响，并且作者证明了AMCase的N-糖基化修饰对于其分泌和功能是必需的。本研究通过比较分泌蛋白组学的方法，鉴定到了新的影响稻瘟菌致病力和细胞壁完整性的N-糖基化分泌蛋白。

Page 728-746

Original Research

Structural and Functional Analyses of Hub MicroRNAs in An Integrated Gene Regulatory Network of Arabidopsis

Zhaoxu Gao, Jun Li, Li Li, Yanzhi Yang, Jian Li, Chunxiang Fu, Danmeng Zhu, Hang He, Huaqing Cai, Lei Li

MicroRNAs (miRNAs) are trans-acting small regulatory RNAs that work coordinately with transcription factors (TFs) to shape the repertoire of cellular mRNAs available for translation. Despite our growing knowledge of individual plant miRNAs, their global roles in gene regulatory networks remain mostly unassessed. Based on interactions obtained from public databases and curated from the literature, we reconstructed an integrated miRNA network in Arabidopsis that includes 66 core TFs, 318 miRNAs, and 1712 downstream genes. We found that miRNAs occupy distinct niches and enrich miRNA-containing feed-forward loops (FFLs), particularly those with miRNAs as intermediate nodes. Further analyses revealed that miRNA-containing FFLs coordinate TFs located in different hierarchical layers and that intertwined miRNA-containing FFLs are associated with party and date miRNA hubs. Using the date hub MIR858A as an example, we performed detailed molecular and genetic analyses of three interconnected miRNA-containing FFLs. These analyses revealed individual functions of the selected miRNA-containing FFLs and elucidated how the date hub miRNA fulfills multiple regulatory roles. Collectively, our findings highlight the prevalence and importance of miRNA-containing FFLs, and provide new insights into the design principles and control logics of miRNA regulatory networks governing gene expression programs in plants.
研究问题： 植物中由microRNA（miRNA）、转录因子及靶基因构成的基因调控网络是什么样的？其中miRNA对网络结构和网络基序（network motif）构成有怎样的影响？网络中miRNA与转录因子的相互关系如何？ 研究方法： 在本研究中，从拟南芥中已知miRNA出发，通过系统预测这些miRNA的靶基因，并对公共数据库和文献中已有的调控关系数据进行重新分析和整理，构建了一个包括miRNA、核心转录因子、以及下游靶基因的调控网络；通过对miRNA节点与转录因子、靶基因节点在网络中的位置信息进行比较，搜索网络中富含的网络基序，并将含miRNA的前馈环路（feed-forward loop）在网络中的层级位置与结构和miRNA所处的层级有关进行关联，对网络结构进行解析；以靶向多个MYB类转录因子的枢纽miR858为例进行了详细的分子和遗传研究。 主要结果1： 构建了拟南芥中一个包括66个核心转录因子、318个miRNA和1712个下游靶基因的调控网络。 主要结果2： 发现含miRNA的前馈环路，特别是miRNA处于中间节点的环路在网络中显著富集。 主要结果3： 鉴定了枢纽miRNA（hub miRNA），根据是影响多个生物过程还是只影响单一生物过程，分为约会型枢纽miRNA和集会型枢纽miRNA。 主要结果4： 对三个由miR858衔接的前馈环路进行了详细的分子和遗传研究，阐明了约会型枢纽miRNA发挥多种调控作用的网络基础。

Page 747-764

Original Research

Defining A Global Map of Functional Group-based 3D Ligand-binding Motifs

Liu Yang, Wei He, Yuehui Yun, Yongxiang Gao, Zhongliang Zhu, Maikun Teng, Zhi Liang, Liwen Niu

Uncovering conserved 3D protein–ligand binding patterns on the basis of functional groups (FGs) shared by a variety of small molecules can greatly expand our knowledge of protein–ligand interactions. Despite that conserved binding patterns for a few commonly used FGs have been reported in the literature, large-scale identification and evaluation of FG-based 3D binding motifs are still lacking. Here, we propose a computational method, Automatic FG-based Three-dimensional Motif Extractor (AFTME), for automatic mapping of 3D motifs to different FGs of a specific ligand. Applying our method to 233 naturally-occurring ligands, we define 481 FG-binding motifs that are highly conserved across different ligand-binding pockets. Systematic analysis further reveals four main classes of binding motifs corresponding to distinct sets of FGs. Combinations of FG-binding motifs facilitate the binding of proteins to a wide spectrum of ligands with various binding affinities. Finally, we show that our FG–motif map can be used to nominate FGs that potentially bind to specific drug targets, thus providing useful insights and guidance for rational design of small-molecule drugs.
研究问题： 配体小分子官能团在蛋白质中保守性的结合模块的计算不仅可以帮助我们深入理解蛋白质小分子识别机制，也可以在药物分子的设计以及优化中提供必要的理论指导，提高药物研发效率。 研究方案： 我们提出了一种新的可以用于自动化为配体小分子官能团提取其在所结合蛋白质中保守性的结合模块的算法AFTME（Automatic Functional Group-based Three-dimensional Motif Extractor，自动化官能团3D结合模块提取器），接着利用AFTME构建大规模官能团与其保守性结合模块的匹配信息库，并对信息库结合模块进行系统性分析，最后利用信息库对药物分子进行预测。 主要结果1： AFTME算法可以自动化识别并提取小分子官能团保守性结合模块。以ATP（Adenosine-5'-Triphosphate，腺苷三磷酸）小分子为例，算法成功识别并提取到了三磷酸结合模块、腺嘌呤结合模块、核糖结合模块。 主要结果2： 官能团结合模块在不同小分子结合口袋中具有可复用性。来自不同种类配体小分子中的相同官能团的结合模块在氨基酸和原子物理化学性质分布上表现一致性，并在统计学上表现出显著相似性。 主要结果3： 系统性分析匹配信息库中全部结合模块，结合模块可被划分为四大类：疏水类、亲水类、混合类和芳香类。每一类结合模块都具有其主导类型的官能团。四类结合模块不同类型的组合方式构成表现出不同强弱程度结合亲和力的配体小分子结合蛋白口袋。 主要结果4： 利用官能团-结合模块匹配信息库可以初步预测药物分子。 算法以及数据库链接： https://github.com/MDhewei/AFTME

Page 765-779

Method

Exploration of Target Spaces in the Human Genome for Protein and Peptide Drugs

Zhongyang Liu, Honglei Li, Zhaoyu Jin, Yang Li, Feifei Guo, Yangzhige He, Xinyue Liu, Yaning Qi, Liying Yuan, Fuchu He, Dong Li

After decades of development, protein and peptide drugs have now grown into a major drug class in the marketplace. Target identification and validation are crucial for the discovery of protein and peptide drugs, and bioinformatics prediction of targets based on the characteristics of known target proteins will help improve the efficiency and success rate of target selection. However, owing to the developmental history in the pharmaceutical industry, previous systematic exploration of the target spaces has mainly focused on traditional small-molecule drugs, while studies related to protein and peptide drugs are lacking. Here, we systematically explore the target spaces in the human genome specifically for protein and peptide drugs. Compared with other proteins, both successful protein and peptide drug targets have many special characteristics, and are also significantly different from those of small-molecule drugs in many aspects. Based on these features, we develop separate effective genome-wide target prediction models for protein and peptide drugs. Finally, a user-friendly web server, Predictor Of Protein and PeptIde drugs’ therapeutic Targets (POPPIT) (http://poppit.ncpsb.org.cn/), is established, which provides not only target prediction specifically for protein and peptide drugs but also abundant annotations for predicted targets.
研究问题： 肽药物靶标和蛋白药物靶标各自有什么特点，它们是否和传统的小分子药物靶标存在不同，不同在哪里？是否可以基于成功肽/蛋白药物靶标各自的特点，构建靶标预测模型以指导新的肽/蛋白药物靶标的选择? 研究方法： 系统梳理现在已成功的蛋白、肽和小分子药物的治疗靶标；从序列、结构、功能等多方面对它们展开研究并同小分子药物靶标进行比较；经过特征筛选，利用朴素贝叶斯构建肽/蛋白药物靶标预测模型。 主要结果： 1）首次系统揭示肽和蛋白药物靶标在多方面的特点，并指出肽、蛋白和小分子药物靶标互相之间的不同； 2）成功构建首个肽/蛋白药物靶标预测模型；3）开发肽/蛋白药物靶标预测和注释在线工具POPPIT (http://poppit.ncpsb.org.cn/)。

Page 780-794

Method

A Yeast BiFC-seq Method for Genome-wide Interactome Mapping

Limin Shang, Yuehui Zhang, Yuchen Liu, Chaozhi Jin, Yanzhi Yuan, Chunyan Tian, Ming Ni, Xiaochen Bo, Li Zhang, Dong Li, Fuchu He, Jian Wang

Genome-wide physical protein–protein interaction (PPI) mapping remains a major challenge for current technologies. Here, we reported a high-efficiency BiFC-seq method, yeast-enhanced green fluorescent protein-based bimolecular fluorescence complementation (yEGFP-BiFC) coupled with next-generation DNA sequencing, for interactome mapping. We first applied yEGFP-BiFC method to systematically investigate an intraviral network of the Ebola virus. Two-thirds (9/14) of known interactions of EBOV were recaptured, and five novel interactions were discovered. Next, we used the BiFC-seq method to map the interactome of the tumor protein p53. We identified 97 interactors of p53, more than three-quarters of which were novel. Furthermore, in a more complex background, we screened potential interactors by pooling two BiFC libraries together and revealed a network of 229 interactions among 205 proteins. These results show that BiFC-seq is a highly sensitive, rapid, and economical method for genome-wide interactome mapping.
当前技术开展全基因组规模的蛋白质相互作用连锁图研究仍然面临诸多挑战。我们开发了一种高效酵母双分子荧光互补技术（bimolecular fluorescence complementation, BiFC），并结合二代测序技术（BiFC-seq），用于蛋白质相互作用组（interactome）研究。我们用此技术全面研究了埃博拉病毒 (EBOV)的病毒蛋白间的互作网络，捕获了9对已报道的相互作用和5对新的相互作用。随后，我们用BiFC-seq技术绘制了肿瘤蛋白p53的互作网络，在得到的97个互作分子中超过四分之三为新发现。在更为复杂的背景条件下，我们进一步用此技术以“库对库”的方式完成了两个BiFC文库间的蛋白相互作用筛选，揭示了一个由205个蛋白和229对相互作用组成的网络。这些结果表明BiFC-seq技术可用于绘制全基因组规模蛋白质连锁图，并具有高灵敏度、快速、经济的特点。

Page 795-807

Corrigendum

Corrigendum to ‘‘An Integrated Systems Biology Approach Identifies the Proteasome as A Critical Host Machinery for ZIKV and DENV Replication” [Genomics Proteomics Bioinformatics 19 (1) (2021) 108–122]

Guang Song, Emily M. Lee, Jianbo Pan, Miao Xu, Hee-Sool Rho, Yichen Cheng, Nadia Whitt, Shu Yang, Jennifer Kouznetsova, Carleen Klumpp-Thomas, Samuel G. Michael, Cedric Moore, Ki-Jun Yoon, Kimberly M. Christian, Anton Simeonov, Wenwei Huang, Menghang Xia, Ruili Huang, Madhu Lal-Nag, Hengli Tang, Wei Zheng, Jiang Qian, Hongjun Song, Guo-li Ming, Heng Zhu

Page 808–809