子計畫 (II) ：神經纖維瘤之蛋白質表現研究

Neurofibromatosis type I(NF1) is a common autosomal dominant disorder, affecting approximately one in every 3500 individuals, and is one of the most common single-gene disorders influencing neurological function in humans. Mutations in the NF1 gene result in abnormal cell growth and differentiation, with a variety of symptoms, typically including benign neurofibromatosis, hyperpigmentation of melanocytes, and harmatomas of the iris.

The NF1 gene encodes a 250kDa protein called neurofibroma, which has several known biochemical functions, including activation of the Ras GTPase, modulation of adenylyl cyclase, and microtubule association. From the pleotropic systems exhibited by NF1 patients, the defects observed in mice carrying targeted mutations on the NF1 gene, and the cellular abnormalities of NF1-deficient cells, it is clear that neurofibromin function is critical in a wide variety of tissues and cell types. Although the gene was mapped to human chromosone 17 and isolated in 1990, the detection of NF1 mutation is still considered ti be a challenges as the gene is large and contain multiple exons.

Understanding why particular tumor type arises in NF1 pateints is an area of active research. Signaling pathway dysregulated as a consequence of neurofibromin loss will involve the pathophysiologic mechanism which becomes more complex; however, proteomics tools offer a new platform for studies of complex biological functions involving large numbers and network of protein. To evaluate the complicate mechanism of neurofibromatosis type 1, proteomics were performed choice to investigate their proteins correlate with NF1 syndromes.

Preliminary study:In order to understand pathophysiology mechanism of neurofibromatosis type 1 at the molecular level, we establish a 2D master pattern of the proteins expressed innerve tissue of NF1 patient. Expression profiles of protein were identified by MALDI-TOF and database searching under Mascort software ( www.expasy.ch/proteome/mascort ). The results show in following figure. Galectins are a family of soluble b -galactoside-binding animal lectin that modulate cell-to-cell adhesion and play a role in tumor progression, pre-mRNA splicing and apoptosis. MAC2 is also known as galectin-3. The murine Mac2 protein is a galactose- and IgE-binding lectin secreted by inflammatory macrophages. Neurofibromas are benign tumors comprised primarily of Schwann cells and fibroblasts. Mast cell infiltration is a well-known phenomenon; however, their role in tumor pathogenesis has been enigmatic. Aldolase isoenzyme patterns pf representative tumors in the human nerve system which indicate

Aldolase A might involved with NF1.

In the recent study we performed nerve proteomics study for a comparison between a facial specimen of neurofibromatosis and a foot lesion of Macrodactyly. We got a persuasive result between these two specimens

on the diagnosis, treatment and prevention if human disease. One of the driving forces in proteomics is the discovery of biomarkers, proteins that change in concentration or state in associated with a specific biological process or disease. By comprehensively examining the different protein expression profiles (expression level, post-translational modification, interaction,etc.) between normal and diseased or drug-treated samples by 2-DE or protein chips, proteomics may provide information on new biomarkers, disease-associated targets and the process of pathogenesis. This technique has been exclusively employed to investigate cancers and other diseases, but there is currently no report concerning the proteomics study on ischemia-reperfusion injury. In this study, we use proteomics to globally analyze

At this post-genomic era, the pathogenesis and management are still unsolved, and an NF1 might have posttranslational modifications in the gene expression. Systematic and quantitative analysis of gene expression is emerging as a valuable tool to diagnostically distinguish between cell types and to differentiate between states (metabolic, activation, pathological)of a particular cell type. Measuring gene expression at the protein level is potentially more informative than the corresponding measurement at the mRNA level. Proteins, the major catalysts of biological function, contain several dimensions of information that collectively indicate the actual rather than the potential functional state as indicated by mRNA analysis. The proteome, the entire set of proteins present in a cell, or tissue, is equivalent to the total expressed genome. Most of the protein are alternatively spliced, and post-translationally modified. Posttranslational modifications include acetylation, phosphorylation, methylation, glycosylation and ubiquitination, just to mention a few. Besides that it is varying over the time course and pathological insults. But only the knowledge of the proteome will really enable the complete understanding of biological systems. The complexity of cellular protein patterns requires the separation and identification of thousands of proteins. The application of proteomics to the study of various diseases is making it possible to characterize the neurofibromatosis in gene/protein expression in response to different ischemia insult.

Plasma is not only the primary clinical specimen but also represents the largest and deepest version of the human proteome present in any sample: in addition to classical “plasma proteins,” it contains all tissue protein (as leakage markers) plus very numerous distinct immunoglobulin sequences, and it has an extraordinary dynamic range in that more than 10 orders of magnitude in concentration separate albumin and the rarest proteins now measured clinically. The plasma proteome holds the promise of a revolution in disease.

Ideally, a biomarker for the diagnosis, staging, and monitoring, neurofibromatosis should include at least the following characteristics: (1) it should be disease-specific, (2) detection in serum is highly desirable, (3) it should appear early in various phenotypest, (4) reflect the extension of neurofibromatosis, (5) it should be indicative of treatment outcome.

Two-dimensional electrophoresis (2-DE) is a powerful and widely used method for analysis of complex protein mixtures extracted from cells, tissues or other pathophysiological samples. The system is based on two-dimensional separation of proteins through isoelectric focusing (IEF) for the 1 st dimension followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the 2 nd dimension. 2-DE has a high performance ability to separate thousands of proteins. In addition, 2-DE is unique in its ability to detect post- and cotranslational modifications, which cannot be predicted from the genome sequence. Identification and characterization of separated proteins is achieved by mass spectrometry. The most significant breakthrough in proteomics has been the mass spectrometric identification of gel-separated proteins. It is much more sensitive, can deal with protein mixtures and offers much higher throughput. It relies on digestion of gel-separated proteins into peptides by a sequence-specific protease such as trypsin. Mass spectrometry replaces the slower and less sensitive chemical degradation methods as the methods of choice for the identification of proteins separated by 2DE. The combination of partial sequence data with peptide masses and appropriate analytical software is sufficient to allow protein identification in complex mixtures of tens of proteins that emerge from each fraction of the chromatograph. Protein sequence database searching will be performed using software provided by public domain ( www.matrixscience.com ). Since the 2DE has the limitation of pH for peptide fractionation, another methods of fractionation using magnetic beads and SELDI kit will be used for the peptide study based on different physical and chemical characters. Then the differential displayed peptide will be identified and sequenced by mass spectrometry. This can afford a high throughput peptides analysis of the neurofibromatosis gene-expression profiles, and functional proteomics. Hopefully, some of these proteins may provide molecular targets for therapy in the future.

This proposal will be the first investigation of neurofibromatosis protein expression profiles by proteome.

Specific Aims:

To get deeper knowledge of molecular mechanism of neurofibromatosis, we need to know how many protein populations were involved and interacted within condition. Proteome is one of the best technology to comprehensive investigate the pathophysiology.