Browsing by Subject "iTRAQ"

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    Evaluation of the mirn23a Cluster through an iTRAQ-based Quantitative Proteomic Approach
    (ACS Publications, 2016-05-06) Ludwig, Katelyn R.; Dahl, Richard; Hummon, Amanda B.; Department of Microbiology and Immunology, School of Medicine
    MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression that are implicated in a number of disease states. MiRNAs can exist as individual entities, or may be clustered and transcribed as a single polycistron. The mirn23a cluster consists of three miRNAs, miR-23a, miR-24-2, and miR-27a. While these miRNAs are transcribed together, they often exist at varying levels in the cell. Despite the fact that the mirn23a cluster is known to play a role in a number of diseases and developmental processes, few direct targets have been identified. In this study, we examined the effects of miR-23a, miR-24-2, miR-27a, or the mirn23a cluster overexpression on the proteome of 70Z/3 pre-B lymphoblast cells. Quantitative mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ) allowed for global profiling of cell lines after miRNA overexpression. We identified a number of targets of each miRNA that contained predicted miRNA seed sequences and are likely direct targets. In addition, we discovered a cohort of shared miRNA targets and cluster targets, demonstrating the importance of studying miRNA clusters in their entirety.
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    Proteomic profiling of TGFBI-null mouse corneas reveals only minor changes in matrix composition supportive of TGFBI knockdown as therapy against TGFBI-linked corneal dystrophies
    (Wiley, 2017) Poulsen, Ebbe Toftgaard; Runager, Kasper; Nielsen, Nadia Sukusu; Lukassen, Marie V.; Thomsen, Karen; Snider, Paige; Simmons, Olga; Vorum, Henrik; Conway, Simon J.; Enghild, Jan J.; Pediatrics, School of Medicine
    TGFBIp is a constituent of the extracellular matrix in many human tissues including the cornea, where it is one of the most abundant proteins expressed. TGFBIp interacts with Type I, II, IV, VI, and XII collagens as well as several members of the integrin family, suggesting it plays an important role in maintaining structural integrity and possibly corneal transparency as well. Significantly, more than 60 point mutations within the TGFBI gene have been reported to result in aberrant TGFBIp folding and aggregation in the cornea, resulting in severe visual impairment and blindness. Several studies have focused on targeting TGFBIp in the cornea as a therapeutic approach to treat TGFBI-linked corneal dystrophies, but the effect of this approach on corneal homeostasis and matrix integrity remained unknown. In the current study, we evaluated the histological and proteomic profiles of corneas from TGFBI-deficient mice as well as potential redundant functions of the paralogous protein POSTN. The absence of TGFBIp in mouse corneas did not grossly affect the collagen scaffold, and POSTN is unable to compensate for loss of TGFBIp. Proteomic comparison of wild-type and TGFBI−/− mice revealed 11 proteins were differentially regulated, including Type VI and XII collagens. However, as these alterations did not manifest at the macroscopic and behavioral levels, these data support partial or complete TGFBI knockdown as a potential therapy against TGFBI-linked corneal dystrophies. Lastly, in situ hybridization verified TGFBI mRNA in the epithelial cells but not in other cell types, supportive of a therapy directed specifically at this lineage.