Prediction methods for the progression of type 1 diabetes (T1D), based on current genetic and autoantibody testing, lack sensitivity and specificity. In this R21 proposal we describe proteomics based methods of study aimed at the identification and detection of specific markers of beta cell destruction and active anti islet immunity. The overall strategy of this proposal is to continue our efforts in mapping tissue specific, tissue restricted, and disease induced gene products of human islets. From previous work we have at our disposal a map of human islet tissue restricted transcripts. In collaboration with experts in mass spectroscopy of proteins (Dr. Caputos' group), we now propose to take advantage of this unique information to enhance studies of soluble human islet proteins with specific applications in mind. We propose that the study of the proteome of purified human islets and those cultured in interferon alpha ex vivo, will lead to the identification of tissue restricted and/or disease associated proteins, some of which will represent potentially useful serum markers of nascent type 1 diabetes and/or its progression. We hypothesize that some of these markers will represent by-products of inflammatory processes that precede and/or drive beta cell destruction, where as other markers, representing proteins either specific to or highly restricted to islet tissue, will provide evidence of beta cell damage or correlate with beta cell mass. We propose that due to beta cell destruction, some of these markers may be present in serum in forms that reflect incomplete post-translational modification. Drawing from the model of release of cardiac muscle specific proteins following myocardial ischemia, in Specific Aim One, we propose to study serum proteins in the rat BB model of human T1D with the goal of determining whether islet specific proteins are released into circulation following the destruction of beta cells. Using informatics tools and traditional protein sequencing methods we will identify serum proteins and peptides that correlate with beta cell destruction and diabetes.
In Specific aim two, we will study the proteome of purified human islets, with the goals of identifying the most prevalent islet tissue specific proteins, with a particular focus on identifying their levels of post translational modification. In parallel, we will develop a SELDI-TOF based immunoassay chip, composed of antibodies recognizing beta cell specific, neuroendocrine tissue specific and/or islet inflammation-associated protein markers. The choice of markers is based on our knowledge of abundant islet transcripts and information we obtain from our islet proteome characterization. Having established the performance characteristics of the Islet chip, we will return to the rat model and retrospectively search for these markers in serum and determine their association with beta cell damage and diabetes. We have drawn together an interdisciplinary research team with the common goal of developing of markers of the destruction of beta cells of the endocrine pancreas.
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| Caputo, Emilia; Miceli, Roberta; Motti, Maria Letizia et al. (2014) AurkA inhibitors enhance the effects of B-RAF and MEK inhibitors in melanoma treatment. J Transl Med 12:216 |
