Journal ArticleDOI
A new method of high-speed cellular protein separation and insight into subcellular compartmentalization of proteins
TLDR
This work identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged T GM-2 peptide fragments and proposed how trafficking of such proteins between cellular compartments can occur to regulate cell function.Abstract:
Transglutaminase (TGM)-2 is a ubiquitous protein with important cellular functions such as regulation of cytoskeleton, cell adhesion, apoptosis, energy metabolism, and stress signaling. We identified several proteins that may interact with TGM-2 through a discovery-based proteomics method via pull down of flag-tagged TGM-2 peptide fragments. The distribution of these potential binding partners of TGM-2 was studied in subcellular fractions separated by density using novel high-speed centricollation technology. Centricollation is a compressed air-driven, low-temperature stepwise ultracentrifugation procedure where low extraction volumes can be processed in a relatively short time in non-denaturing separation conditions with high recovery yield. The fractions were characterized by immunoblots against known organelle markers. The changes in the concentrations of the binding partners were studied in cells expressing short hairpin RNA against TGM-2 (shTG). Desmin, mitochondrial intramembrane cleaving protease (PARL), protein tyrosine kinase (NTRK3), and serine protease (PRSS3) were found to be less concentrated in the 8.5%, 10%, 15%, and 20% sucrose fractions (SFs) from the lysate of shTG cells. The Golgi-associated protein (GOLGA2) was predominantly localized in 15% SF fraction, and in shTG, this shifted to predominantly in the 8.5% SF and showed larger aggregations in the cytosol of cells on immunofluorescent staining compared to control. Based on the relative concentrations of these proteins, we propose how trafficking of such proteins between cellular compartments can occur to regulate cell function. Centricollation is useful for elucidating biological function at the molecular level, especially when combined with traditional cell biology techniques.read more
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Bioanalysis of Eukaryotic Organelles
Chad P. Satori,Michelle M. Henderson,Elyse A. Krautkramer,Vratislav Kostal,Mark M. Distefano,Edgar A. Arriaga +5 more
TL;DR: The role that organelle analysis has played in understanding biology is highlighted as it enables a more specific description of the molecular, biochemical, and physiological processes associated with diseases, embryonic development, tissue differentiation, organism aging, disease treatments, and organism response to pathogens.
Journal ArticleDOI
Mechanistic role of transglutaminase-2 in focal adhesions.
Evelyn Png,Aihua Hou,Louis Tong +2 more
TL;DR: Taken together with previous findings, TG-2 binds paxillin non-covalently, and JNK can phosphorylate pxillin, these processes critically regulate corneal epithelial adhesion and migration.
Journal ArticleDOI
Transglutaminase-2 is critical for corneal epithelial barrier function via positive regulation of Claudin-1.
TL;DR: In this paper , the role of transglutaminase (TG)-2 in corneal barrier function and its potential regulation of epithelial junctional proteins and transcription factors was investigated.
References
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Journal ArticleDOI
Transglutaminases: crosslinking enzymes with pleiotropic functions
Laszlo Lorand,Robert M. Graham +1 more
TL;DR: Having knowledge of transglutaminases is essential for understanding the aetiologies of diverse hereditary diseases of the blood and skin, and various autoimmune, inflammatory and degenerative conditions.
Journal Article
An SV40-immortalized human corneal epithelial cell line and its characterization.
Kaoru Araki-Sasaki,Y Ohashi,T Sasabe,Kozaburo Hayashi,Hiroshi Watanabe,Yasuo Tano,Hiroshi Handa +6 more
TL;DR: The authors have established an SV40-immortalized human corneal epithelial cell line with properties similar to normal cornea-specific, 64-kD cytokeratin in addition to five major insoluble proteins.
Journal ArticleDOI
Tissue transglutaminase in normal and abnormal wound healing: Review article
TL;DR: The view of the function of tissue transglutaminase in wound repair under normal and pathological situations is described and its potential as a strategic therapeutic target in the development of new treatments to improve wound healing and prevent scarring is highlighted.
Journal ArticleDOI
Distinct Nuclear Localization and Activity of Tissue Transglutaminase
TL;DR: In this paper, the localization and activity of tissue transglutaminase in the nucleus of SH-SY5Y cells was examined, showing that 93% of the transglUTaminase activity was localized to the cytosol.
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Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications
Russell Collighan,Martin Griffin +1 more
TL;DR: The functions of the enzyme tissue transglutaminase (TG2) in the extracellular matrix (ECM) both as a matrix stabiliser through its protein cross-linking activity and as an important cell adhesion protein involved in cell survival are summarized.