Tissue transglutaminase (TGase) is involved in the regulation of several biological events including cellular differentiation and apoptosis. The expression and activation of TGase are up-regulated in response to retinoic acid (RA), leading to the protection of several cell lines against N-(4-hydroxyphenyl)retinamide (HPR)-induced apoptosis. The anti-apoptotic mechanisms of TGase are poorly understood at this time. We examined the interaction of TGase with the retinoblastoma (Rb) protein, a substrate of TGase that is also implicated in cell survival functions. In cells undergoing HPR-induced apoptosis, Rb is degraded. This degradation is blocked when cells are pretreated with RA, an important regulator of TGase. In vitro studies revealed that TGase protects Rb from caspase-induced degradation in a transamidation-dependent manner. Experiments performed with fibroblasts from Rb-/- mice further demonstrated that the presence of Rb was required for TGase to exhibit antiapoptotic activity in response to RA treatment. Microinjection of Rb-/- cells with a transamidation-defective TGase mutant and Rb afforded no protection from HPR-induced apoptosis. Taken together, these findings suggest that the ability of TGase to modify Rb via transamidation underlies the ability of TGase to provide protection against apoptotic insults and to ensure that cells remain viable during differentiation.

Retinoic acid (RA) and its various synthetic analogs affect mammalian cell growth, differentiation, and apoptosis.Whereas treatment of the human leukemia cell line HL60 with RA results in cellular differentiation, addition of the synthetic retinoid, N-(4-hydroxyphenyl) retinamide (HPR), induces HL60 cells to undergo apoptosis.Moreover, pretreatment of HL60 cells as well as other cell lines (i.e.NIH3T3 cells) with RA blocks HPRinduced cell death.In attempting to discover the underlying biochemical activities that might account for these cellular effects, we found that monodansylcadaverine (MDC), which binds to the enzyme (transamidase) active site of tissue transglutaminase (TGase), eliminated RA protection against cell death and in fact caused RA to become an apoptotic factor, suggesting that the ability of RA to protect against apoptosis is linked to the expression of active TGase.Furthermore, it was determined that expression of exogenous TGase in cells exhibited enhanced GTP binding and transamidation activities and mimicked the survival advantage imparted by RA.We tested whether the ability of this dual function enzyme to limit HPR-mediated apoptosis was a result of the ability of TGase to bind GTP and/or catalyze transamidation and found that GTP binding was sufficient for the protective effect.Moreover, excessive transamidation activity did not appear to be detrimental to cell viability.These findings, taken together with observations that the TGase is frequently up-regulated by environmental stresses, suggest that TGase may function to ensure cell survival under conditions of differentiation and cell stress.

The cDNA for the chloroplast-located homolog of bacterial RecA protein, designated recA-AT, was placed in a plasmid appropriate for in vitro transcription and translation, Translation with S-35-labeled Met permitted demonstration of uptake of the protein product into isolated pea chloroplasts, and processing to a mature size, Preliminary evidence for the first amino acid was estimated from results using both S-35-Met and H-3-Leu for in vitro transcription and translation, followed by uptake into chloroplasts and processing, The labeled protein was subject to sequential amino acid hydrolyses, and radioactivity was measured in each round, Induction of gene transcription in leaves infiltrated with the DNA-damaging agent, methyl methanesulfonate was shown by Northern blot analysis, Further constructs were made for over-expression of the gene in E. coli; and one out of many tried permitted production of some soluble protein. Extracts from transformed bacteria were shown to have RecA activity using the "POM" assay [Bertrand et al, (1993) Nucl. Acids Res, 21: 3653] for DNA strand transfer. The protein was purified to close to homogeneity using methods developed for E. coli RecA isolation.

The Rho GDP-dissociation inhibitors (GDIs) negatively regulate Rho-family GTPases 1 , 2 . The inhibitory activity of GDI derives both from an ability to bind the carboxy-terminal isoprene of Rho family members and extract them from membranes 3 , 4 , and from inhibition of GTPase cycling between the GTP- and GDP-bound states 4 , 5 . Here we demonstrate that these binding and inhibitory functions of rhoGDI can be attributed to two structurally distinct regions of the protein. A carboxy-terminal folded domain of relative molecular mass 16,000 ( M r 16K) binds strongly to the Rho-family member Cdc42, yet has little effect on the rate of nucleotide dissociation from the GTPase. The solution structure of this domain shows a β-sandwich motif with a narrow hydrophobic cleft that binds isoprenes, and an exposed surface that interacts with the protein portion of Cdc42. The amino-terminal region of rhoGDI is unstructured in the absence of target and contributes little to binding, but is necessary to inhibit nucleotide dissociation from Cdc42. These results lead to a model of rhoGDI function in which the carboxy-terminal binding domain targets the amino-terminal inhibitory region to GTPases, resulting in membrane extraction and inhibition of nucleotide cycling.