No abstract

Proteins are made from 19 aa and, curiously, one N-alkylamino acid (''imino acid''), proline (Pro). Pro is thought to be incorporated by the translation apparatus at the same rate as the 19 aa, even though the alkyl group in Pro resides directly on the nitrogen nucleophile involved in peptide bond formation. Here, by combining quench-flow kinetics and charging of tRNAs with cognate and noncognate amino acids, we find that Pro incorporates in translation significantly more slowly than Phe or Ala and that other N-alkylamino acids incorporate much more slowly. Our results show that the slowest step in incorporation of N-alkylamino acids is accommodation/peptidyl transfer after GTP hydrolysis on EF-Tu. The relative incorporation rates correlate with expectations from organic chemistry, suggesting that amino acid sterics and basicities affect translation rates at the peptidyl transfer step. Cognate isoacceptor tRNAs speed Pro incorporation to rates compatible with in vivo, although still 3-6 times slower than Phe incorporation from Phe-tRNA Phe depending on the Pro codon. Results suggest that Pro is the only N-alkylamino acid in the genetic code because it has a privileged cyclic structure that is more reactive than other N-alkylamino acids. Our data on the variation of the rate of incorporation of Pro from native Pro-tRNA Pro isoacceptors at 4 different Pro codons help explain codon bias not accounted for by the ''tRNA abundance'' hypothesis.non-natural amino acids ͉ ribosomes ͉ tRNA ͉ EF-Tu ͉ GTPase

Transglutaminase 2 (TG2) is a multifunctional mammalian protein with transamidase and signaling properties. Using selective TG2 inhibitors and tagged nucleophilic amine substrates, we show that the majority of extracellular TG2 is inactive under normal physiological conditions in cell culture and in vivo. However, abundant TG2 activity was detected around the wound in a standard cultured fibroblast scratch assay. To demonstrate wounding-induced activation of TG2 in vivo, the toll-like receptor 3 ligand, polyinosinic-polycytidylic acid (poly(I:C)), was injected in mice to trigger small intestinal injury. Although no TG2 activity was detected in vehicle-treated mice, acute poly(I:C) injury resulted in rapid TG2 activation in the small intestinal mucosa. Our findings provide a new basis for understanding the role of TG2 in physiology and disease.

No abstract

Human transglutaminase 2 (TG2) is believed to play an important role in the pathogenesis of various human disorders including celiac sprue, certain neurological diseases, as well as some types of cancer. Selective inhibition of TG2 should therefore enable further investigation of its role in physiology and disease, and may lead to effective clinical treatment. Recently we showed that certain 3-halo-4-, 5-dihydroisoxazole containing compounds are selective inhibitors of human TG2 with promising pharmacological activities. Here we present definitive evidence that this class of compounds targets the active site of human TG2. Structure-activity relationship studies have provided insights into the structural prerequisites for selectivity, and have led to the discovery of an inhibitor with ca. fifty-fold higher activity than a prototypical dihydroisoxazole inhibitor with good in vivo activity. A method for preparing enantiomerically enriched analogues was also developed. Our studies show that the 5-(S-) dihydroisoxazole is a markedly better inhibitor of human TG2 than its 5-(R-) stereoisomer.