Proteomics as an Indicator for the Research of Cell Apoptosisin Toxicology

  

    
TECHNOLOGIES OF PROTEOMIC RESEARCH IN TOXICOLOGY 
  
  

    
Methods 
    
Principles
    
Application
    
Advantages
    
Disadvantages
  
  

    
Proteins Separation 
  
  

    
Two-dimensional gel electrophoresis (2-DE)
    
Molecular Weight (MW); Isoelectric    point (pl)
    
Far-ranging cell samples
    
Visualization Distinguish    phosphorylation; Relative low cost
    
Difficult to detect low abundance    proteins; Difficult to detect    hydrophobic and strongly acid basic proteins; Time comusing
  
  

    
Differences gel electrophoresis (2-DIGE)
    
Molecular Weight (MW); Isoelectric    point (pl)
      
Cydye Fluorescent tags
    
Small amount of samples than 2-DE;    Micro cutting samples
    
The heightened accuracy; Eliminate the    differences between the glue
    
Can only detect denatured proteins
  
  

    
Liquid Chromatography (LC)
    
Chromatography
    
A single protein or simple sample    separation; Membrane protein; Low abundance proteins;
    
High throughput; Automation analysis;    Fast speed and high sensitivity
    
Hardly detect complex mixture of    proteins; More cost
  
  

    
Isobaric tags for relative and absolute quantitation (iTARQ)
    
Isotope-coded covalent tags
    
Membrane proteins; Nucleoprotein;    Extracellular protein; Low abundance proteins; Strong basic proteins
    
High flux; Proteome dynamic monitoring    of multiple time points; Quantitative sensitivity; Low sample amount
    
No visual spot detection; more cost
  
  

    
Proteins identification 
  
  

    
Matrix-Assisted Laser Desorption/Ionization
      
Time of Flight Mass Spectrometry (MALDI-TOF-MS)
    
Gasification and ionization of    peptides; Soft ionization
      
Utilize m/z value
    
Single sample point
    
High Sensitivity;
      
High accuracy;
      
High resolution
    
Dependence of high proteins purity
  
  

    
Surface-Enhanced Laser Desorption/Ionization
      
Time of Flight Mass Spectrometry (SELDI-TOF-MS)
    
Chip technology;
      
Gasification and ionization of    peptides; Soft ionization
    
Complex samples; Serum; Urine; Clinical    Specimens
    
Protein purification unnecessary;    Informative; High Sensitivity; Relation with protein-protein interaction
    
Bad repetitiveness
  
  

    
Electro spray Ionization
      
Mass Spectrometry
    
Coulomb force between the positive charge;    Different m/z value
    
Unstable proteins
    
More soft ionization than MALDI;    Integrated Ion; High sensitivity; High degree of automation
    
Expensive; High apparatus requirement
  

Table 1:  Technologies of proteomic research in toxicology.

Regulation of apoptosis

Apoptosis, also known as programmed cell death, is the energy-dependent process of cell death [10], accompany by cellular or extracellular protein expression. Apoptosis plays important roles in physiology and pathology, and can be triggered by numerous stimuli [11]. Proteomics-- the simultaneous analysis and quantification of protein abundance, is used to analyze the characteristic of biochemical events, resulting in changes in cellular morphology and death [12]. It is applied in exogenous stimulate toxicology, verifying the apoptosis pathway for diagnosis biomarker and drug development, integrating miRNA, mRNA and proteomic profiling approaches toxicology to identify the biological pathways both in vivo and in vitro systems, and revealing the regulation of apoptosis.

As a naturally occurring mycotoxin and food contaminant, Ochratoxin A (OTA) has various toxicological effects, especially nephrotoxicity, as an inducer focusing on proteomic approaches. An iTRAQ-based mitoproteomics approach is used to explore possible toxicity mechanisms of OTA and potential protective regulations of N-acetyl-L-cysteine (NAC) using the mitochondria of Human Embryonic Kidney 293 (HEK 293) cells. The significantly different proteins are mainly involved in the perturbation of the mitochondrial electron transport chain, inhibition of protein synthesis, and induction of stress response and cell death. NAC could almost completely reverse the adverse effects of OTA at the protein level to protect cell death. Using the data of proteomics, a hypothetical model of OTA-induced mitochondria damage is proposed to provide a framework for the toxicity mechanism [13]. To further reveal the mechanism of cytotoxicity, an iTRAQ-based quantitative proteomics approach is used in identifying differentially expressed proteins between Lon Protease 1 knock-down cells and control. Defense against oxidative stress in the mitochondria, regulating protein synthesis, modification and repair, maintaining thebalance of carbohydrate metabolism, and assisting in mtDNA maintenance are realized the protective function processes through the results. Meanwhile the method of combination of RNAi and iTRAQ-based quantitative proteomics paves the way to gain a better understanding of the toxicity mechanisms [14]. OTA-induced early hepatotoxicity also been revealed by combining multi-omics methods [15]. The comprehensive understanding of toxicological effects is also conducive to the further exploration of detoxification mechanism.

Perfluorooctanoic acid (PFOA), one of the most commonly used perfluorinated compounds in commercial products, has been associated with a number of adverse health effects [16]. However, the molecular mechanisms involved in PFOA toxicity are still not well characterized. By investigating the alterations in protein profilein human nontumor hepatic cells, the proteomic study proposed that the inhibition of some proteins, including GRP78, HSP27, CTSD and hnRNPC may be involved in the activation of p53, which consequently triggers the apoptotic process [17]. This has shined a new light on the molecular mechanisms responsible for PFOA-mediated toxicity in human liver cells.

Drug-induced liver injury (DILI) is a significant clinical problem, commonly classified into intrinsic (dose-dependent and predictable) and idiosyncratic (non-dose-dependent and unpredictable) hepato toxicity [18]. A novel high-through put proteomic approach based on 2D-nano-LC-MS/MS was applied to simultaneously evaluate the alterations of global protein expression involved in the response of levo-tetrahydropalmatine (l-THP) treatment in normal human liver cells. Further analysis by Western-blot confirmed that mTOR and MEK2 proteins were expressed at lower levels compared with control, and provided detailed evidence to support that l-THP is capable of inducing apoptosis in mammalian liver cells [10]. This is helpful to evaluate drugs in therapeutic doses.

To identify the biological pathways, proteomics lead researchers to havea deeper comprehension of the actual active players of cellular function comparing to genomics. Because mRNA is a disposable message and only a limited amount of it gets translated into proteins [20-22]. To find the regulation of apoptosis in megakaryocytes, a combined proteomic, transcriptomic and bioinformatic approach is used to elucidate the molecular mechanisms, underlying Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) signaling via its receptor VPAC1. Comparison of genomic and proteomic data, provide novel insights into toxic mechanisms and is necessary for human health risk assessment [23]. After two-dimensional difference gel electrophoresis and tandem MS, 2384 differentially expressed genes/proteins are detected. The majority of the 120 proteins modulate by PACAP belong to the class of "cell cycle and apoptosis" proteins, and ingenuity pathway analysis further identify apoptosis as the highest scored network with NF-κB as a key-player [24]. The viewpoint NF-κBas well as p38, ERK 1/2 and JNK 1/2MAPKplays critical roles in immune response also detect by 2-DE [25], to reveal the function of protein and signals.

The molecular mechanism of the apoptosis effect of SNX-2112, a novel inhibitor of Hsp90 currently used as an anti-tumor drug, induces apoptosis inmultiple tumor cell lines is poorly understood. Combination of transcriptomics and proteomics approaches further reveal that caspase signals originate from mitochondria dysfunction, mediate by Akt signaling pathway inactivity [26]. The results from combination of transcriptomics and proteomics demonstrate the therapeutic potential of SNX-2112 against human chronic myeloid leukemia [27]. There are many examples of proteomics research playing important roles in the field of cell apoptosis.

Biomarkers finding and disease research

The cell apoptosis and dysfunctional are often occurrence and development with disease. Biomarkers are indicators of a specific biological state because of the proteomic methodology has rapidly evolved in the past ten years [28]. Proteomics and metabolomics, in combination with conventional endpoints and transcriptomics, have helped in generating mechanistic hypotheses and identifying or qualifying biomarkers. Toxicologists and pathologists are working to detect biomarkers and their applications as reliable predictors of pathological conditions, such as neurodegenerative diseases, types of cancer, chronic obstructive pulmonary disease, urinary tract obstruction, is important for clinical disease management.

A surplus of death signals is associated with neurodegenerative diseases [29] because the death signals maynot only lead to complete cell death but may also render cells dysfunctional [1]. The specific diagnostic and prognostic biomarkers are helpful in managing neurodegenerative diseases. To identify relevant biomarkers and therapeutic targets for these disorders, the cerebrospinal fluid (CSF) proteome are focused on the mechanisms underlying neuronal death in neurodegenerative diseases. Compared with the secretome of apoptotic and surviving cerebellar granule neurons (CGNs), a totalof 1375 proteins are identified in CGN-conditioned media by using two different heavy isotopelabels follow by liquid chromatography couple with Fourier transform tandem mass spectrometry. Among these proteins, 47 are differentially expressed in the supernatants of apoptotic and surviving neurons. It should be considered when using targeted quantitative proteomics approaches to characterize or validate CSF biomarkers of neurodegenerative disorders [30], which is a noteworthy advance in specific diagnostic.

A set of survival signals is predominantly associated with cancer. Systemic mining of cancer exoproteome/secretome has emerged as a pivotal strategy for delineation of molecular pathways with mechanistic importance in cancer development, as well as the discovery of diagnostic/prognostic biomarkers [31]. Cancer cells have the ability to evade apoptotic signals and promote survival beyond their normal life span. This is a hallmark of tumorigenesis, and proteomics analysis is often used in the area of cancer therapy and biomarker screening [32]. Pediatric as trocytomas, a leading cause of death associated with cancer, are the most common primary central nervous system tumors found in children. It progresses rapidly and invades surrounding tissues. The check sample analyze by 2D SDS-PAGE, mass spectrometry (MALDI-TOF), RT2 miRNA PCR Array System, shows that vimentin, calreticulin and 14-3-3 epsilon protein are hub proteins in these neoplasm's. The novel proteins and micro RNAs with expression changes on pediatric astrocytoma could serve as biomarkers of tumor progression [33]. This article indicated that the large-scale analyses allow a fairly accurate prediction of different cellular processes altered in tumors. Gastric cancer is the fourth most common cancer and the second leading cause of cancer-related deaths worldwide [34]. To examine the background of lymph node metastasis (LNM) in gastric cancer, the proteomic approach is used. Members protein of the beta-3integrin (ITGB3) pathway are significantly enriched, which has not been observed before. This report suggests a novel role for the integrin pathway in the development of LNM in gastric cancer [35]. It has indicated possible benefits for proteomic studies.

Nasopharyngeal carcinoma (NPC) is a leading lethal malignancy, especially prevalent in the Cantonese region of southern China [36]. To identify novel biomarkers for the early detection of NPC patients, 2D-DIGE combine with MALDI-TOF-MS analysis is performed to identify differentially expressed proteins in the carcinogenesis and progression of NPC, using LCM-purified normal nasopharyngeal epithelial tissues and various stages of NPC biopsies [37]. The results determine that the differentially expressed proteins ENO1 and CYPA are two potential novel biomarkers for the early detection of NPC, and detect both of them improving the diagnose accuracy significantly. The combining-biomarker methods enhance the stability and efficiency of disease diagnosis.

In developing worlds, long-term exposure to pollution irritants causes an inflammatory response in the lungs, resulting in narrowing of the small airways and breaking down of lung tissue [38]. Chronic obstructive pulmonary disease (COPD) develops as a significant and chronic inflammatory response to inhaled irritants. Chronic bacterial infections may also add to this inflammatory state [39-40]. Because of that, COPD has becoming one of the leading causes of morbidity and mortality around the world. However, the exact mechanisms of COPD and its progression are still poorly understood. There is an increasing need to be able to perform proteomic studies on patients with COPD, to describe the association with disease specificity, severity, progress and prognosis as well as monitor the efficacy of therapies. Proteomic results in 60 patients diagnosed with broncho-alveolar lavage revealed inflammatory signaling, free radical scavenging and oxidative stress response, glycolysis and gluconeogenesis pathways correlated with the COPD. The most relevant signaling link was through the NF-κB pathway [41]. Induced sputumis analyzed by cysteine-specific two-dimensional difference gel electrophoresis (2D-DIGE) coupled with mass spectrometry to identify proteins involved in COPD pathogenesis [42]. Another proteomic analysis highlights waysto identify novel biomarkers for diagnosis, therapy, and prognosis in COPD. In the results, through the detection of lung tissue, broncho-alveolar lavage, sputum and serum, surfactant protein-A (SP-A) protein may represent a helpful biomarker in the early detection of COPD and other relate disorders. Sputum polymeric immunoglobulin receptor (PIGR) -- a glycosylated secretory component, is significantly elevated in the bronchial and alveolar epithelium to moderate COPD [43-44].

Urinary tract obstruction (UTO) is a commonly noted disorder on prenatal ultrasound that has the potential to lead to permanent loss of renal function [45]. An in vitro model has been developed which involves mechanical stretch of proximaltubule cells grown on flexible plates, which mimics the physiological conditions during UTO. This study employs a one dimensional SDS-PAGE fractionation procedure, following by in-gel digest and LC-MS/MS analysis in a semi-quantitative experiment, using spectral counting to relatively quantify changes in protein expression following the established model of UTO. Quantitative analysis shows 135 increased and 182 decreased expression proteins, providing a more complete characterization of changes in protein abundance as a result of stretch than previous studies, supporting a number of previously undescribed proteins in proximal tubule cells that may play a role in UTO [12]. To believe proteomics will be a bigger stage in disease prevention and control.

The integration of proteome research evidence into clinical practice is going to require extensive studies on analytical and clinical validity of biomarkers in large cohorts of patients, establishing the necessary laboratory infrastructure, education of healthcare and genetic professionals, establishing counseling services for patients and establishing external quality assurance testing [46-47]. It is important to the development of personalized medicine, adapting the evaluation of predictive and prognostic biomarkers, and the application of cancer-type specificdrugs that targets individuals for vanquishing diseases.

Enrich cell apoptosis databases

Omics analysis has becoming the hottest trend in technology and has brought forth adramatic impact,but this emerging science facing a massive data, it is crucial to support the development of are elative database. Various types of high-throughput-omics data become rapidly available [48], which are useful for proteomic analysis and data relevance.

For formulate proteins involved in the development of renal disease, integration of the gene-centric and protein-centric general databases with those of human kidney tissue and urine proteomes may open a new window for research in nephrology. Proteins present in the kidney and urine provide basic tools for investigation of kidney function and disease. By comparing such databases between healthy and diseased populations, proteins involved in the development of renal disease or new biomarker candidate proteins may be able to be identified [49].

Many quantitative proteome analyses of apoptosis have been performed to gain insight in proteins involved in the process, but the results in large and complex data sets are difficult to evaluate. To consolidate these data, Magnus develop an Apopto Proteomics database for storing, browsing and analyzing the outcome of large-scale proteome analyses toapoptosis, which is available at https:// apoptoproteomics.uio.no. The proteomics data of 52 publications are integrated and unified with protein annotations from UniProt- KB, the caspase substrate database homepage (CASBAH), and gene ontology [1].

Chlamydia is a virulent pathogen that resides in humans, birds and a wide range of animals [50]. It has been difficult to study because of its obligate intra cellular growth habit and lack of a genetic transformation system. Following the full genome sequencing of seven strains of Chlamydia and a rapid expansion of genomic, transcriptomic and proteomic analysis of these pathogens have been completed. The Chlamydia Interactive Database (CIDB) can be cross-queried by researchers for patterns in the data (https://www3.it.deakin. edu.au:8080/CIDB/) [51]. Combining the data of many research groups into a single database and cross-querying from different perspectives should enhance our understanding of the complex cell biology of these pathogens.

Comparison between induction of apoptosis by the intrinsic and the extrinsic signaling pathway revealed slight differences [1]. Furthermore, proteomics has significantly contributed to the field of apoptosis. Regardless of all the pressing challenges, there is no doubt that proteomic as a holistic measure of organism response to exogenous stressors will continue to make a great impact in biological process.

Challenges in proteomics

Although 2DE and 2D-DIGE technologies are the best available approaches for genuine top-down studies, including the analyses of post translational modifications, de novo sequencing, protein identification independent of genome sequences, establishing direct information on intact proteins, and studies of protein isoforms [52- 54], there are also some challenges associated with it.

Under many physiological and pathological conditions, some important regulatory proteins secreted into the serum by the body undergo quantitative or qualitative changes. Unlike liver or kidney, the serum is easy to achieve in clinical, leading the blood proteome to achieve a comprehensive assessment of multiple-organ as possible [55], this is a reason that serum has been used in numerous diagnostic tests. However, the reared difficulties in making use of serum proteomics techniques to screen tumor serum protein markers.

On the one hand, various impurities should be removed to eliminate interference with the test results; low abundance proteins are the most meaningful proteins and the presence of high abundance proteins affects the detection of low abundance proteins. The same difficulties exist either in cerebrospinal fluid (CSF) proteomics and milk proteomics [57-58]. On the other hand, proteomic analysis relies onsample stability and pretreatment, nevertheless different pretreatment methods or sample collection including sample storage time, storage temperature, frozen period, application of protease inhibitor and others may cause different effects, affecting the objectiveness and veracity of evaluating results [56]. Besides, as shown in table1, each of proteins separation and identification methods has their own advantages and disadvantages in the research of toxicology. 2D and MALDI-TOF-MS are suitable for fundamental research, while LC or SELDI-TOF-MS is often used in high throughput detection. It is clear that the resolution of the instrument is limited. Plenty of proteins that exist exceeding the detection limit of electrophoresis-assisted instrument. The need for developing more sophisticated off-gel proteomics and bioinformatics is increasingly warranted [59]. Meanwhile, it is also necessary to avoid the cross-reaction between proteins or antibodies [60].

In recent years, researchers believe living organisms is regulated by proteins and non-coding RNAs from multiple controls, this gap in proteomics research could be overcome in the short-term with the use of multiple omics profiling technologies to acquire corroborative evidence [15,61]. It is not enough to reveal evidence only in one subject, but the networks combining multi-omics methods are premature for now at the least. It depends on the discovery of more comprehensive rules.

Regardlessof all the pressing challenges, there is no doubt that proteomics as a holistic measure of cell apoptosis will continue to make great strides as an indicator for toxicological research, but its further development should provide more accurate and profound information, connecting seamlessly with other regulating factors.

Acknowledgements

This work was supported by Program for New Century Excellent Talents in University (Grant 2014FG046). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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