tRNA Processing and Subcellular Trafficking Proteins Multitask in Pathways for Other RNAs

This article focuses upon gene products that are involved in tRNA biology, with particular emphasis upon post-transcriptional RNA processing and nuclear-cytoplasmic subcellular trafficking. Rather than analyzing these proteins solely from a tRNA perspective, we explore the many overlapping functions of the processing enzymes and proteins involved in subcellular traffic. Remarkably, there are numerous examples of conserved gene products and RNP complexes involved in tRNA biology that multitask in a similar fashion in the production and/or subcellular trafficking of other RNAs, including small structured RNAs such as snRNA, snoRNA, 5S RNA, telomerase RNA, and SRP RNA as well as larger unstructured RNAs such as mRNAs and RNA-protein complexes such as ribosomes. Here, we provide examples of steps in tRNA biology that are shared with other RNAs including those catalyzed by enzymes functioning in 5′ end-processing, pseudoU nucleoside modification, and intron splicing as well as steps regulated by proteins functioning in subcellular trafficking. Such multitasking highlights the clever mechanisms cells employ for maximizing their genomes

Assay and Purification of Omega-Amidase/Nit2, a Ubiquitously Expressed Putative Tumor Suppressor, That Catalyzes the Deamidation of the Alpha-Keto Acid Analogues of Glutamine and Asparagine

omega-Amidase (omega-amidodicarboxylate amidohydrolase, EC 3.5.1.3) isolated from rat liver cytosol is a versatile enzyme that catalyzes a large number of amidase, transamidase, and ester hydrolysis reactions. omega-Amidase activity toward alpha-ketoglutaramate and alpha-ketosuccinamate (the alpha-keto acid analogues of glutamine and asparagine, respectively) is present in mammalian tissues, tumors, plants, bacteria, and fungi. Despite its versatility, widespread occurrence, and high specific activity, the enzyme has been little studied, possibly because the assay procedure previously required a substrate (alpha-ketoglutaramate) that is not commercially available. Here we report a simplified method for preparing alpha-ketoglutaramate and an assay procedure that measures alpha-ketoglutarate formation from alpha-ketoglutaramate in a 96-well plate format. We also describe a 96-well plate assay procedure that measures omega-amidase-catalyzed hydroxaminolysis of commercially available succinamic acid. The product, succinyl hydroxamate, yields a stable brown color in the presence of acidic ferric chloride that can be quantitated spectrophotometrically with negligible background interference. The two assay procedures (hydrolysis of alpha-ketoglutaramate and hydroxaminolysis of succinamate) were employed in purifying omega-amidase approximately 3600-fold from rat liver cytosol. The ratio of alpha-ketoglutaramate hydrolysis to succinamate hydroxaminolysis remained constant during the purification. omega-Amidase has recently been shown to be identical to Nit2, a putative tumor suppressor protein. It is anticipated that these new assay procedures will help to characterize the function of omega-amidase/Nit2 in tumor suppression, will provide the basis of high-throughput procedures to search for potent inhibitors and enhancers of omega-amidase, and will assist in identifying biological interactions between nitrogen metabolism and tumor biology

Commercially available angiotensin II At₂ receptor antibodies are nonspecific.

Commercially available angiotensin II At₂ receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available At₂ receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and At₂ receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or At₂ receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of At₂ receptor binding or gene expression. We conclude that none of these commercially available At₂ receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study At₂ receptor expression

Identification of the Putative Tumor Suppressor Nit2 as Omega-Amidase, an Enzyme Metabolically Linked to Glutamine and Asparagine Transamination

The present report identifies the enzymatic substrates of a member of the mammalian nitrilase-like (Nit) family. Nit2, which is widely distributed in nature, has been suggested to be a tumor suppressor protein. The protein was assumed to be an amidase based on sequence homology to other amidases and on the presence of a putative amidase-like active site. This assumption was recently confirmed by the publication of the crystal structure of mouse Nit2. However, the in vivo substrates were not previously identified. Here we report that rat liver Nit2 is omega-amidodicarboxylate amidohydrolase (E.C. 3.5.1.3; abbreviated omega-amidase), a ubiquitously expressed enzyme that catalyzes a variety of amidase, transamidase, esterase and transesterification reactions. The in vivo amidase substrates are alpha-ketoglutaramate and alpha-ketosuccinamate, generated by transamination of glutamine and asparagine, respectively. Glutamine transaminases serve to salvage a number of alpha-keto acids generated through non-specific transamination reactions (particularly those of the essential amino acids). Asparagine transamination appears to be useful in mitochondrial metabolism and in photorespiration. Glutamine transaminases play a particularly important role in transaminating alpha-keto-gamma-methiolbutyrate, a key component of the methionine salvage pathway. Some evidence suggests that excess alpha-ketoglutaramate may be neurotoxic. Moreover, alpha-ketosuccinamate is unstable and is readily converted to a number of hetero-aromatic compounds that may be toxic. Thus, an important role of omega-amidase is to remove potentially toxic intermediates by converting alpha-ketoglutaramate and alpha-ketosuccinamate to biologically useful alpha-ketoglutarate and oxaloacetate, respectively. Despite its importance in nitrogen and sulfur metabolism, the biochemical significance of omega-amidase has been largely overlooked. Our report may provide clues regarding the nature of the biological amidase substrate(s) of Nit1 (another member of the Nit family), which is a well-established tumor suppressor protein), and emphasizes a) the crucial role of Nit2 in nitrogen and sulfur metabolism, and b) the possible link of Nit2 to cancer biology

AT₂ receptor protein expression in adrenal, spleen, kidney and brainstem of wild-type and AT₂ knockout mice.

<p>Angiotensin II AT₂ receptor protein expression was studied by western blotting. Protein extracts were separated by SDS-PAGE electrophoresis and exposed to three different anti-AT₂ receptor antibodies as indicated by catalog number at the top of the picture. The scale on the left indicates the size in kDa according to the positions of the protein ladder. The reported size of the AT₂ receptor is about 50-55 kDa. Note that each antibody tested generated different immunoreactivity patterns, that these patterns do not correlate with the presence or absence of the target protein, and that in all cases there is no difference between band intensity obtained from tissues from AT₂ knockout and wild-type mice. The figure represents a typical experiment repeated two times in individual samples.</p