H.C, Prashanth: Identification and characterization of novel keratin associated proteins using a genetic interaction screening system. - Bonn, 2009. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5N-17718
@phdthesis{handle:20.500.11811/4081,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5N-17718,
author = {{Prashanth H.C}},
title = {Identification and characterization of novel keratin associated proteins using a genetic interaction screening system},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2009,
month = jul,

note = {Keratins are an integral and important constituent of the epithelial cytoskeleton and protect epithelial cells against stress. In addition to their well-established function as cytoskeletal scaffolds in epithelia, recent findings have revealed that beyond maintenance of cytoarchitecture, keratins play important role in cell proliferation, growth, survival and organelle transport along with its associated proteins. To understand these novel keratin functions and to address the molecular mechanisms, the identification of keratin-associated proteins was a major prerequisite. The aim of the present study was to find novel keratin associated proteins that might provide missing links. To this end, screening for the KAPs was carried out by a genetic approach ‘Sos recruitment’ technique based on temperature sensitive selection of positive interacting candidates in yeast to isolate K5 and K14 interacting proteins. As the first step, cDNA library was constructed with 500,000 cDNA fragments (size ranging from 500 bp to 4 kb) isolated from human skin sample. Yeast-two-hybrid screen was performed using head, rod and tail domains of epidermal keratins K5 and K14 as baits against cDNA library.
Screening in yeast yielded more than 2400 interacting candidates, out of which 200 selected cDNA fragments were sequenced.
It was observed that proteins affiliated with diverse functions seem to interact with K5 and K14. To name a few, small GTPase family members proteins (Rab34, EP164), membrane binding proteins (LYPD3), WW domain binding proteins (WBP1, TAPT1 etc), transcription factors (AP2β, STAT6), translation elongation factor (EEF1A1), 14-3-3theta a member of 14-3-3 family proteins along with many hypothetical and un-annotated proteins like p86DM were identified as interacting partners.
K14 head domain interacting candidates ‘Rab34’ a small GTPase of the ras superfamily reported to be involved in the regulation of lysosome morphogenesis (Jordens et al., 2001) and in formation of macropinosomes (Wang and Hong, 2005), regulator protein ‘14-3-3 theta’, transcription factor ‘AP2-β’ a member AP-2 family of helix-span-helix transcription factors which determine the cell-type-restricted proliferation and the suppression of terminal differentiation of epithelia and additional tissues, and the anannotated protein ‘p86DM’ were selected for further analysis.
A direct interaction of full length K14 with AP-2β, and Rab34 was confirmed in cytoplasm of mammary epithelial MCF-7 cells by bimolecular fluorescence complementation (BiFC) technique.
These results strongly suggest:
1. Ras-related protein Rab-34 belonging to small GTPase Rab family directly interacts with K14. Understanding the significance of this interaction and further in-depth studies might definitely help in knowing process of import and regulation of melanosome by keratinocytes.
2. Direct interaction between K14 with AP-2β strongly suggests that AP-2β is sequestered in the cytoplasm by interaction with K14 and can be hypothesized that sequestration by distinct keratins represents a novel mechanism to regulate the activity of AP-2 transcription factors.
3. Preliminary set of results from the functional studies of p86DM displayed encouraging results and thus can be considered as a promising candidate and requires thorough investigation to reveal the importance of its direct interaction with actin.
It is worthwhile to note that, for the first time a detailed screen has been undertaken to identify keratin-associated proteins and less than 10% of the candidates obtained from Y2H screen have been sequenced so far. Further analysis of the already identified proteins and identifying the preserved un-sequenced samples will be beneficial towards understanding the molecular mechanism of keratin regulated functions.},

url = {http://hdl.handle.net/20.500.11811/4081}
}

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