Assays for transglutaminases in cell death.
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.
Melino
G
G
IDI-IRCCS Biochemistry Lab, University of Rome Tor Vergata, Italy.
Candi
E
E
Steinert
P M
PM
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D015415:Biomarkers; D016923:Cell Death / Q000502:physiology; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods; D016384:Consensus Sequence; D017931:DNA Primers; D004789:Enzyme Activation; D006168:Guinea Pigs; D022781:Hepatocytes / Q000201:enzymology / Q000648:ultrastructure; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D015603:Keratinocytes / Q000201:enzymology*; D007700:Kinetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012867:Skin / Q000166:cytology / Q000201:enzymology; D018411:Spodoptera; D014162:Transfection / Q000379:methods; D011503:Transglutaminases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
D015415:Biomarkers; D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D011503:Transglutaminases