Study On Signal Transduction Process In A549 Cells Responding To Stimulation Of Exogenous Metal Ions By Proteomic Investigation

Cells by activating a series of biological processes, including metabolism, transcription, growth and differentiation, respond to various exogenous stimuli, such as metal pressure, stimulation of growth factors and hormones, etc. The excess or deficiency of essential metal elements or the existence of non-essential metal elements will interfere with the nomal biological function of responding to exogenous stimuli. Trace non-essential metals, such as cadmium, mercury and lead, are still toxic. And essential metals, such as zinc and copper, are an indispensable composition of proteins, especially enzymes and transcription factors. But the excess essential metals are also toxic. Cell evolved, therefore, to form the fine biological regulation systems to balance metals level in cells by controlling the influx, screen, storage, transport, and efflux of metals. All organisms are able to adjust the expression patterns of proteome or genome to reduce or avoid metal damage. They respond adaptively to exogenous stimulus or pressure of heavy metals by the homeostasis system of cells. These expression patterns of genome or proteome induced by metal treatment or exposure not only have tissue-dependent specificity, cells-dependent specificity and concentration-dependent specificity, but also have time-dependent specificity. The exploration of differential expressions and changes in time-dependent manner in genes, proteins and metal elements help to observe kinetic response process of cells to exogenous stumili. This is very important to understand the mechanism of homeoshtasis, defense and metal toxicity in cells.In this work, A549 cell line was used as cell model. We described time-dependent response of proteome in cells to exposure of exogenous essential metal ion zinc and non-essential metal ion cadmium by proteomics method of high-throughput and high resolution two dimension electrophoresis-mass spectrometry (2DE-MS). Moreover, we investigated time-dependent change in the expression of proteins of zinc homeostasis and cadmium toxicity in cells after treatment of exogenous heavy metal ions for different periods using the Western blot, RT-PCR and Bradford. Meanwhile, we investigated the time-dependent change in contents of heavy metal elements or free ions by combining inductively coupled plasma-mass spectrometry (ICP-MS) with confocal technology. And we preliminarily explored the process of cells kineticly responding to the exogenous metal ions in time-dependent manner after exposure for different periods. The work of this paper mainly includes the following five parts:(1) By matrix-assisted laser desorption ionization tandem time of flight (MALDI-TOF/TOF) mass spectrometry, we determined the composition of bacitracin zinc and obtained almost all sequence ions of bacitracin zinc A, a side chain-cyclized dodecapeptide antibiotic, by single tandem mass (MS/MS) spectrum under optimal instrumental conditions. The amino acid sequence of bacitracin zinc A was analyzed and validated by de novo sequencing. In addition, the structure characteristics of this typical metal polypeptide antibiotic were further elucidated using infrared spectrometry (IR), ultra violet spectrometry (UV) and inductively coupled plasma-atomic emission spectrometry (ICP-AES).(2) We investigated the differential expressions and transcriptions of key proteins in the signal path of zinc homeostasis in A549 cells after exogenous zinc exposure using Western blot and RT-PCR technology. Their features of time-dependent expression or transcription were observed, including metal transcription factor (MTF-1), metallothionein (MT-1) and zinc transporters (ZIP-1 and ZnT-1). The expressions of these proteins conservatively changed after exogenous zinc exposure for different periods (0-24 h), and their expressions were mainly induced by zinc exposure. Interestingly, the expressions of these proteins showed biggest abundance ratios at exogenous zinc exposure for 9 h. Therefore, we speculated the signal process of zinc homeostasis regulated by these key proteins.(3) We profiled the differential expression of whole proteome in A549 cells responding to exogenous zinc ion stress after different periods by 2DE technology coupled with silver staining and Coomassie brilliant blue (CBB) staining. The differentially expressed proteins spots were categorized and analyzed. The differential expression rules were found as four expressed patterns. The expression of these proteins was always higher at 9 h than 24 h after cadmium exposure, which means that long-time exposure would mainly further reduce expression of these proteins. Therefore, we put forward that the time-dependent characteristic of differential expression of proteins in cells has an important influence on investigation of proteomics and metal homeostasis mechanism. Additionally, the differentially expressed protein spots were analyzed and identified by MALDI TOF/TOF and Mascot database. These hit proteins were classified according to biological processes and molecular functions, which help to further comprehensively understand the signal transduction process of zinc homeostasis after exogenous zinc stimulus.(4) The time-dependent change rules of total protein concentrations in whole cells, cytoplasm and nucleus after exogenous zinc exposure were observed by Bradford method and ICP-MS technology. Moreover, the time-dependent changes in the content and distribution of free Zn(Ⅱ) in cells after exposure were presented using confocal microscopy imaging techniques. In addition, the time-dependent characteristic and the signal transduction process induced by adaptive response of cells to exogenous zinc exposure were further explored.(5) Similar to investigation of Zn(II), we first obtained differentially expressed protein profiling of whole proteins in A549 cells exposed to Cd(II), and their four expression patterns and time-dependent expression rules were investigated. Interestingly, we found that the significantly different expression patterns and responsive rules of proteome in A549 cells after cadmium exposure were different from those of zinc exposure:The expression of cadmium-resposive proteins conservatively changed after exogenous cadmium exposure for different periods (0-24 h), and the expression is mainly induced by cadmium exposure. Interestingly, the expressions of these proteins showed a biggest abundance ratio at 24 h exposure; The expression of cadmium-reponsive proteins were always higher at 24 h than 4.5 h. Exposure to exogenous cadmium for longer time would mainly increase the expression of these proteins. The time-dependent change in expression of key cadmium toxicity proteins, in concentrations of total protein and metal ions such as Cd, Zn, Cu, Co and Mn, were further studied using Western blot, Bradford and ICP-MS. Based on these finding, we preliminary discussed the signal process of cadmium toxicity.Our results showed that the responses of proteomes and cells to different metal ions were different, but they always showed a time-dependent feature. The discovery of this rule means that specific time-dependent response and expression caused by adaptive stress of cells play an important role in studying proteome and signal mechanism. Our findings would provide a new perspective for studying cellular metal homeostasis and toxicity mechanisms.