Proteomic Identification Of A New Diagnostic Marker In Breast Cancer And Multi-enzyme Complexes In Maize Endosperm

In the post genome era, proteomics technologies were widely applied to various fields. In this study, proteomic technologies were used in two parts of the research: voladation of S100A11 protein as a new diagnostic protein mark in breast cancer; isolation, identification and functional analysis of multi-enzyme complexes in maize endosperm. The main technologies involved in this reasearch: two dimensional electrophoresis (2-DE), matrix-assisted laser desorption/ionization time of flight mass spectrometry, two dimensional affinity electrophoresis (2-DAE), liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/M S), western blot, immunohistochemistry, zymogram analysis and yeast two-hybrid assay, etc.Recent immunohistochemistry analyses revealed that calcium-binding protein S100A11 was overexpressed in most human tumor tissues, suggesting that it may be a putative tumor suppressor. However, there is a controversial viewpoint among authors regarding the expression pattern and functions of S100A11 in breast tumors. To evaluate whether S100A11 could be considered as a novel diagnostic marker in breast carcinoma, the method of differential proteomics, western blotting, immunohisto- chemistry were used to detect the expression pattern and subcellular localization of S100A11. Statistical analyses also suggest that pecific up-regulated of S100A11 did not correlate with other prognostic factors. Our data support that S100A11 is a novel diagnostic marker, and its ubiquitous expression increased in different types of breast carcinoma. Analysis of S100A11 expression in breast cancer may be an effective tool to help with detecting early-stage breast cancer.Starch is a major storage product of seeds and/or storage organs in higher plants. It serves as a main source of food and other industrial materials. The phloem provides a means for moving sucrose from the leaf source tissue to the endosperm, and then, sucrose is converted into ADP-glucose (ADP-Glc), an essential precursor and substrate for starch biosynthesis. ADP-glucose pyrophosphorylase (AGPase) is a committed enzyme responsible for the production of ADP-Glc. A group of enzymes including granule-bound starch synthases (GBSS), GBSSI and GBSSII, are responsible for producing amylose. A second group of starch synthases (SS), designated as SSI, SSII, SSIII, and SSIV, are exclusively involved in amylopectin biosynthesis in combination with starch branching enzymes (SBEs). Meanwhile, a range of plastidial enzymes with starch-degrading capabilities, such as debranching enzymes (DBE) and starch phosphorylase (SP), has been shown to play important roles in the process of starch degradation and turnover. Although individual enzymatic steps in starch metabolic pathways have been adequately revealed over the last decades, detailed mechanisms of starch synthesis remain unknown. It is believed that starch metabolism is under the control of a complex metabolic network. Indeed, regulatory mechanisms may operate through functional interactions among multiple starch metabolizing enzymes. To date, only few protein complexes have been discovered, and little is known about their dynamic changes during maize endosperm development. In this work, a new proteomic method, two dimensional affinity electrophoresis (2-DAE) coupled with LC-ESI-MS/MS, was esbilished for exploring some starch-binding proteins/complexes from developing maize endosperms. Crude proteins extracted from maize endosperm at four representative developing stages, i.e.7-9days after pollination (DAP), 16-18 DAP, 26-28 DAP, and 35-37 DAP, were used to examine the dynamic formation of starch-binding proteins/complexes by 2-DAE in the presence of different affinity substrates, i.e. sucrose,β-limit dextrin, glycogen and amylopectin, respectively. Protein spots with altered shifting mobilities were subsequently collected for identification using liquid chromatography-electrospray ionization tandem mass spectrometry, followed by protein verification by zymogram analysis and protein interaction validated by yeast two-hybrid assay. In total, two individual enzymes and eight protein complexes were 吉林大学博士学位论文identified, seven of which were novel to maize and exhibited differential substrate-binding activities. It was observed that sucrose synthase isoform SUS-SH1, starch branching enzyme IIb, pyruvate orthophosphate dikinase 1, and ADP-glucose pyrophosphorylase were presented in one complex at 16-37 DAP, implying its possible function in diverting carbon from sucrose into amino acids. In late developmental stages at 35-37 DAP, five new protein complexes containing enzymes involved in starch biosynthesis, lignin biosynthesis, amino acid biosynthesis, terpene biosynthesis, and glycolysis were also found. It suggest that at late endosperm development starch biosynthesis enzymes could associated with enzymes involved in other metabolic pathways for more advanced carbon partitioning. This paper provides the first evidence that two dimensional affinity electrophoresis (2-DAE) coupled with LC-ESI-MS/MS identification is a powerful approach for the identification of protein complexes associated with various substrates, i.e. sucrose, limit dextrin, and amylopectin. This is also the first report of seven new multi-enzyme complexes. Our findings substantiate the long standing notion that starch metabolism requires the formation of functional protein complexes, and further support that starch metabolism is cross-talking with various other metabolic pathways by forming different protein complexes. Current findings provided an important insight into understanding of starch metabolic mechanism in maize.