Proteomic tools for the characterization of cell death mechanisms in drug discovery

This review focuses on the use of proteomic tools for the characterization of cell death mechanisms that have contributed to drug discovery efforts. Resistance to cell death plays a major role in the development of many diseases, including numerous types of malignancies. Using a multitude of proteomic approaches, including protein–protein interaction studies, phosphorylation site mapping, ubiquitination site identification, and differential quantitative approaches, various cellular death pathways such as apoptosis and necroptosis have been investigated. These studies have aided in the development of therapeutic strategies or allowed dissection of clinical results to evaluate the success of clinical trials in addition to contributing to our understanding of these biological pathways. Here, we address the new wave of discoveries enabled by advancements in mass spectrometric technology and bioinformatic infrastructure that will hopefully lead to clinically efficacious strategies to overcome resistance to apoptosis and therefore offer improved treatment options for patients.     

Proteomic analysis of early urinary biomarkers of renal changes in type 2 diabetic patients

Diabetic nephropathy (DN) is a complication associated with diabetes, leading to end-stage renal disease (ESRD). Despite significant progress in understanding DN, the cellular mechanisms leading to the renal damage are incompletely defined. In this study, with the aim to identify urine biomarkers for the early renal alterations in type 2 diabetes mellitus (T2D), we performed urinary proteomic analysis of 10 normoalbuminuric patients with T2D, 12 patients with type 2 DN (T2DN), and 12 healthy subjects. Proteins were separated by 2-DE and identified with ESI-Q-TOF MS/MS. Comparing the patients proteomic profiles with those of normal subjects, we identified 11 gradually differently changed proteins. The decreased proteins were the prostatic acid phosphatase precursor, the ribonuclease and the kallikrein-3. Eight proteins were progressively increased in both patients groups: transthyretin precursor, Ig κ chain C region, Ig κ chain V-II region Cum, Ig κ-chain V-III region SIE, carbonic anhydrase 1, plasma retinol-binding protein, β-2-microglobulin precursor, β-2-glycoprotein 1. The proteomic analysis allowed us to identify several increased urinary proteins, not only in T2DN but also in T2D patients in which the microalbuminuria was in the normal range. These patterns of urinary proteins might represent a potential tool for a better understanding of diabetic renal damage.     

Proteomic Research of Molecular Defects in Globozoospermia

Globozoospermia is a form of teratozoospermia characterized by round‐headed spermatozoa and lack of acrosome. These spermatozoa cannot penetrate the zona pellucida of the oocyte, resulting in unsuccessful fertilization and infertility. When intracytoplasmic sperm injection is performed, the fertilization rate tends to be low. Until now, the causes of this disorder remain to be elucidated; however, mutations of some genes segregating on an autosomal recessive mode have been associated with this infertile condition. DPY19L2 (dpy‐19‐like 2 [Caenorhabditis elegans]) codes for a transmembrane protein expressed predominantly in spermatids, with specific localization limited to the internal nuclear membrane. Genetic defects in the DPY19L2 gene have been demonstrated the most frequent genetic cause of globozoospermia; however, intracellular molecular pathways related to its encoded protein are largely unknown. In this issue of Proteomics Clinical Applications, Guo and co‐workers investigate the proteome of gloobozoospermic spermatozoa. The authors identified 491 proteins that are differentially expressed in globozoospermia (370 are upregulated and 121 are downregulated in DPY19L2‐deficient globozoospermic sperm). Notably, the molecular defects identified by the authors are closely related to biological processes involved in acrosome formation, chromatin composition, sperm‐egg binding, and fertilization.     

Anticancer effects of aloe-emodin on HepG2 cells: Cellular and proteomic studies

Aloe-emodin (AE) is one of the main bioactive anthraquinones of Rheum palmatum, a widely used herbal medicine. Several recent studies suggested that AE possesses potent anticancer properties, although the mechanisms are yet to be fully elucidated. The present study aimed to identify the molecular targets of AE in a human hepatocellular carcinoma cell line, HepG2. We first found that AE was more cytotoxic and effective in inducing apoptosis and cell cycle arrest than its analog emodin (EM). Proteomic study using 2-D DIGE revealed that AE affected multiple proteins associated with oxidative stress, cell cycle arrest, antimetastasis, and hepatitis C virus replication. For example, peroxiredoxins (PRDX) and DJ-1, both of which are redox-sensitive proteins, were among those markedly up-regulated, suggesting the presence of oxidative stress in AE-treated cells. Further biochemical studies demonstrated that AE enhanced the intracellular level of reactive oxygen species and oxidation of PRDX-2, -4, and DJ-1. In addition, AE inhibited DNA synthesis via up-regulation of the CDK4 inhibitor p16 and inhibition of Rb phosphorylation. Furthermore, AE was able to decrease cell migration via up-regulation of the metastasis inhibitor, nm23. Taken together, AE induced anticancer effects in HepG2 cells via multiple pathways by affecting different protein targets.     

Proteomic characterization of differentially expressed proteins in breast cancer: Expression of hnRNP H1, RKIP and GRP78 is strongly associated with HER-2/neu status

The human epidermal growth factor receptor type 2 (HER‐2/neu) oncoprotein is overexpressed in about 30% of breast cancers and associates with metastatic phenotypes of breast tumours. Dissecting the HER‐2/neu‐modulated molecules in cancer will be helpful in elucidating the underlying molecular mechanisms of HER‐2/neu‐driven tumourigenesis. We investigated the differential proteome profiles between microdissected HER‐2/neu‐positive and ‐negative tumours and unambiguously identified 21 proteins with diverse biological functions by peptide sequencing and NCBInr database interrogation. Six proteins were up‐regulated whereas 15 were down‐regulated in the HER‐2/neu‐positive tumours. Differential expressions of heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), 78 kDa glucose‐regulated protein (GRP78/Bip) and Raf‐1 kinase inhibitor protein (RKIP), which have not been previously reported as being linked to HER‐2/neu signalling, were further verified. Immunohistochemical staining on tissue microarray sections demonstrated a positive correlation of hnRNP H1 (p = 0.008) and negative correlations of GRP78 and RKIP (p = 0.018 and 0.013, respectively) with HER‐2/neu. Heregulin α1 enhanced hnRNP H1, but reduced GRP78 and RKIP expression in BT474 cells in a dose‐dependent manner, providing evidence of crosstalk between HER‐2/neu signalling and these modulators. Our studies have identified novel modulators that are likely to be intricately involved in HER‐2/neu‐driven tumour proliferation, invasion and metastasis.     

Proteomic characterization of thiazolidinedione regulation of obese adipose secretome in Zucker obese rats

Signaling molecules released by adipose tissue have been implicated in inflammation, adipocyte dysfunction and systemic insulin resistance. In this study, we used 2-D LC-MS/MS and quantitative proteomics approaches to characterize the obese adipose secretory proteins that are responsive to the thiazolidinediones class of PPAR-γ agonizts. We first showed the differential secretion profiling between obese and lean adipose tissue; 87 proteins were detected from the conditioned medium of adipose tissue of Zucker obese rats compared with 31 from lean rats. A total of 57 proteins comprising immune factors, inflammatory molecules, collagens, proteases, and extracellular matrix proteins were detected from obese, but not lean adipose tissue. More importantly, a quantitative proteomics approach using (18) O proteolytic labeling allowed quantification of the difference in the secretion levels of 77 proteins, and thiazolidinediones treatment suppressed the secretion of most of the obese adipose tissue secretome, thus resembling a lean tissue. We have demonstrated an application of identifying the obese adipose secretome and characterizing the regulation of adipose secretion in obesity and insulin resistance. Our data provide the first evidence of changes in adipose secretion in obesity at a global level and show that such changes are correlated with systemic insulin resistance.     

Real time in vitro measurement of oxygen uptake rates for HEPG2 liver cells encapsulated in alginate matrices

Quantitative non-invasive measurement of critical physiological parameters is necessary to assess the functionality and applicability of tissue engineered matrices. Advancements in fiber optic sensors have made it possible for measuring parameters such as oxygen, glucose, and aminoacids necessary for viable tissue growth. In this study, we have devised an experimental protocol to measure in real time, the oxygen uptake rate (OUR) values for a selected liver cell line (HEPG2) when grown (a) on cover glass slides, and (b) encapsulated within alginate based hydrogel matrices. For both cases, the oxygen uptake rates of HEPG2 cells at selected time points varied in close co-relation with cell proliferation and metabolic activity during the 7-day culture period. This investigation concludes that OUR can be used as an indicative parameter to assess the metabolic activity of cells encapsulated within a matrix. The study also presents a fiber optic sensing technology as a non-invasive diagnostic tool to monitor cell behavior and activity.     

ANA HEp-2 cells image classification using number,size, shape and localization of targeted cell regions

The ANA HEp-2 medical test is a powerful tool in autoimmune disease diagnostics. The last step of this test, the interpretation of immunofluorescent images by trained experts, represents a potential source of errors and could theoretically be replaced by automated methods. Here we present a fully automatic method for recognition of types of immunofluorescent images produced by the ANA HEp-2 medical test. The proposed method makes use of the difference in number, size, shape and localization of cell regions that are targeted by the antinuclear antibodies – the humoral components of immune system that bind human antigens as a result of the immune system malfunction. The method extracts morphological properties of stained cell regions using a combination of thresholding-based and thresholding-less approaches and applies a conventional machine-learning algorithm for image classification.     

分布非结构化P2P网络资源定位研究

P2P系统是一个分布式系统,其中的资源如何进行定位是一个重要的问题。通过对分布非结构化P2P系统的搜索机制以及现有的改进方法的研究,给出了一种基于语义路由改进算法,并对此算法进行了模拟仿真。     

P2P-Grid环境中基于流言传播的资源查找方法研究

对两层结构的 P2P-Crid 网格模型进行了改进,在超级节点层上增加了分类节点层,即选择超级节点中性能较高的节点存放分类信息.给出了基于流言传播机制的分布式资源查找算法,该算法利用流言传播机制具有传播流言的兴趣随着重复收到某个流言而减少的特点,避免某个节点处理查找信息的负载过大,实现负载平衡,并与传统的泛洪算法进行了比较.模拟实验证明该算法能够缩短资源请求的响应时间,并且减少网格通信开销.     

P2P网络中激励机制研究

由于P2P网络节点的匿名性和贡献资源的自愿性,绝大多数节点缺乏提供服务的积极性,从而引发了P2P网络中的搭便车问题。在分析搭便车问题的基础上,全面介绍了基于微支付、直接互惠和信誉模型等典型激励机制,指出了这些模型中分别存在的隐藏信息,信息不对称和共谋等问题。根据机制可靠性,扩展性和复杂度等衡量因素对各种激励机制进行了比较分析,讨论了代价与效用量化比较和通用激励框架等激励机制未来研究的趋势。     

PS2P网络中视频点播系统的设计与实现*

结合视频点播应用的具体需求,对BitTorrent协议中的分片选择策略、激励机制进行改进,并融合媒体服务器对资源的下载支持,实现了基于PS2P的视频点播系统HyBT。性能分析表明,HyBT能在保证音/视频内容高速传输的前提下,减少点播缓冲时间,使音/视频内容流畅播放,让用户获得良好的视听体验和服务质量。     

反馈机制在P2P网络资源搜索中的应用研究

资源搜索是P2P网络的关键问题。目前P2P网络资源搜索中对反馈机制的应用研究较少,这样每次搜索对成功历史没有充分利用,搜索效率较低。该文针对此问题提出了基于改进的Rumormongering协议的资源发布算法BDFB(BidirectionFeedback),并将其应用到P2P网络资源搜索的反馈机制研究中,充分利用成功历史,提高资源在网络中的知名度,从而提高P2P网络中资源搜索效率。     

P2P网络激励机制模型研究

由于p2p网络节点的匿名性和贡献资源的自愿性,绝大多数节点不愿共享自己的资源,从而导致大量搭便车现象的出现。该文在分析搭便车现象的基础上,介绍了两种基本的激励机制模型,讨论了博弈理论在激励机制模型中的应用。     

Proteomic analysis of high-molecular-weight protein polymers in a doxorubicin-resistant breast-cancer cell line

We recently reported that increased transglutaminase 2 (TGase 2) expression correlates with increased resistance to the cancer drug doxorubicin in breast-cancer cell lines. Interestingly, high-molecular-weight (HMW) proteins also increased with increased TGase 2 expression in the drug-resistant cell lines. TGase 2 is likely to be responsible for the formation of HMW proteins, because TGase 2 catalyzes cross-linking between proteins. Although the role of the HMW proteins is unclear, we demonstrated that TGase 2 inhibition increases drug sensitivity in breast-cancer cells. Herein we find that TGase 2 inhibition by cystamine dramatically reduces the level of HMW proteins. Identification of the HMW proteins may suggest the mechanism of cancer drug resistance associated with aberrant TGase 2 function. To explore the identities of HMW proteins, we performed in-gel tryptic digestions of unresolved HMW proteins and analyzed the resulting peptides using LC-MALDI-MS/MS. Most of the identified proteins were associated with gene regulation, such as polyadenylate-binding proteins, translation initiation factors, and ribonucleoproteins. This finding suggests that TGase 2 may participate in gene regulation, in addition to its role in cell adhesion.