Assays for transglutaminases in cell death
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Published on: May 6, 2011
G Melino1, E Candi, P M Steinert
1IDI-IRCCS Biochemistry Lab, University of Rome Tor Vergata, Italy.
Abstract:
Several in vivo and in vitro experimental model systems demonstrate a direct relationship between the expression and activity of tissue transglutaminase [tTG; also called transglutaminase type 2 (TGase 2)] and programmed cell death or apoptosis. This is based on mRNA and protein studies, sense and antisense transfection, identification of N epsilon-(gamma-glutamyl)-lysine cross-links in extracted apoptotic bodies, and in blue mouse experiments. In the epidermis, apoptosis occurs under particular conditions in the proliferative basal layer with the involvement of the tTG enzyme. However, in epidermal keratinocytes other TGases (TGase 1, TGase 3, and perhaps TGase X) are normally activated in a terminal differentiation program, called cornification, that leads to cell death. These cells perform their functions after death, providing an elastic physical and permeability barrier to the skin. In fact, TGase 1 mutations cause the skin disease lamellar ichthyosis. Because all TGases share strong similarities in structure and function, being involved in mechanisms of cell death, this chapter describes the current assays for TGases at the mRNA, protein, and enzymatic levels. We also describe procedures to produce, purify, and characterize recombinant TGases, to identify mutation in disease, to isolate cross-linked bodies, and to analyze the N epsilon-(gamma-glutamyl)-lysine isopeptide cross-links. Finally, we discuss general rules for the interpretation and comparison of these events in cell death.
Insights
Tissue transglutaminase (tTG) is linked to programmed cell death. Other transglutaminases (TGases) are crucial for skin barrier formation during terminal differentiation and cell death.
Area of Science:
- Biochemistry
- Cell Biology
- Dermatology
Background:
- Tissue transglutaminase (tTG), also known as transglutaminase type 2 (TGase 2), plays a role in programmed cell death (apoptosis).
- Epidermal keratinocytes utilize other TGases (TGase 1, 3, X) for terminal differentiation, a process leading to cell death that forms the skin barrier.
- Mutations in TGase 1 are associated with the skin disease lamellar ichthyosis.
Purpose of the Study:
- To explore the relationship between tissue transglutaminase (tTG) expression/activity and programmed cell death.
- To detail methodologies for assessing TGase function in cell death and disease.
- To provide guidelines for interpreting TGase involvement in cell death mechanisms.
Main Methods:
- In vivo and in vitro experimental models.
- mRNA and protein expression studies, transfection techniques.
- Analysis of N epsilon-(gamma-glutamyl)-lysine cross-links in apoptotic bodies.
- Production, purification, and characterization of recombinant TGases.
- Mutation identification and isolation of cross-linked bodies.
Main Results:
- Demonstrated a direct correlation between tTG expression/activity and apoptosis across various model systems.
- Confirmed tTG involvement in epidermal apoptosis under specific conditions.
- Highlighted the distinct roles of other TGases in keratinocyte terminal differentiation and skin barrier function.
Conclusions:
- TGases are fundamentally involved in cell death mechanisms, both apoptosis and terminal differentiation.
- Understanding TGase function is critical for studying cell death and related diseases like lamellar ichthyosis.
- Standardized assays and interpretation frameworks are necessary for comparative analysis of TGase activity in cell death.
