Display, engineering, and applications of antigen-specific T cell receptors

The use of T cell receptors (TCRs) as potential therapeutic agents provides an opportunity to target a greatly expanded array of antigens, compared to those now targeted with monoclonal antibodies. With the advent of new display technologies and TCR formats for in vitro engineering, it should be possible to generate high-affinity TCRs against virtually any peptide antigen that is shown to bind to a major histocompatibility complex (MHC) molecule (e.g. peptides derived from viral antigens or from self proteins that are associated with the transformed phenotype). What remains, however, are challenges associated with effective targeting of very low numbers of cell surface antigens (pepMHC), fewer than the case for conventional monoclonal antibody-based therapies. This hurdle might be overcome with the attachment of more effective payloads for soluble TCR approaches, or by using TCR gene transfer into T cells that can then be adoptively transferred into patients. There is considerable work to be done on the physiological aspects of either approach, including pharmacokinetic studies in the case of soluble TCRs, and T cell trafficking, persistence, and autoreactivity studies in the case of adoptively transferred T cells. As with the field of monoclonal antibodies, it will take time to explore these issues, but the potential benefits of TCR-based therapies make these challenges worth the effort.     

Mutation in MRPS34 Compromises Protein Synthesis and Causes Mitochondrial Dysfunction

The evolutionary divergence of mitochondrial ribosomes from their bacterial and cytoplasmic ancestors has resulted in reduced RNA content and the acquisition of mitochondria-specific proteins. The mitochondrial ribosomal protein of the small subunit 34 (MRPS34) is a mitochondria-specific ribosomal protein found only in chordates, whose function we investigated in mice carrying a homozygous mutation in the nuclear gene encoding this protein. The Mrps34 mutation causes a significant decrease of this protein, which we show is required for the stability of the 12S rRNA, the small ribosomal subunit and actively translating ribosomes. The synthesis of all 13 mitochondrially-encoded polypeptides is compromised in the mutant mice, resulting in reduced levels of mitochondrial proteins and complexes, which leads to decreased oxygen consumption and respiratory complex activity. The Mrps34 mutation causes tissue-specific molecular changes that result in heterogeneous pathology involving alterations in fractional shortening of the heart and pronounced liver dysfunction that is exacerbated with age. The defects in mitochondrial protein synthesis in the mutant mice are caused by destabilization of the small ribosomal subunit that affects the stability of the mitochondrial ribosome with age.     

Defining Sequence Space and Reaction Products within the Cyanuric Acid Hydrolase (AtzD)/Barbiturase Protein Family

Cyanuric acid hydrolases (AtzD) and barbiturases are homologous, found almost exclusively in bacteria, and comprise a rare protein family with no discernible linkage to other protein families or an X-ray structural class. There has been confusion in the literature and in genome projects regarding the reaction products, the assignment of individual sequences as either cyanuric acid hydrolases or barbiturases, and spurious connection of this family to another protein family. The present study has addressed those issues. First, the published enzyme reaction products of cyanuric acid hydrolase are incorrectly identified as biuret and carbon dioxide. The current study employed (13)C nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry to show that cyanuric acid hydrolase releases carboxybiuret, which spontaneously decarboxylates to biuret. This is significant because it revealed that homologous cyanuric acid hydrolases and barbiturases catalyze completely analogous reactions. Second, enzymes that had been annotated incorrectly in genome projects have been reassigned here by bioinformatics, gene cloning, and protein characterization studies. Third, the AtzD/barbiturase family has previously been suggested to consist of members of the amidohydrolase superfamily, a large class of metallohydrolases. Bioinformatics and the lack of bound metals both argue against a connection to the amidohydrolase superfamily. Lastly, steady-state kinetic measurements and observations of protein stability suggested that the AtzD/barbiturase family might be an undistinguished protein family that has undergone some resurgence with the recent introduction of industrial s-triazine compounds such as atrazine and melamine into the environment.     

Metabolism of Thioamides by Ralstonia pickettii TA

Information on bacterial thioamide metabolism has focused on transformation of the antituberculosis drug ethionamide and related compounds by Mycobacterium tuberculosis. To study this metabolism more generally, a bacterium that grew using thioacetamide as the sole nitrogen source was isolated via enrichment culture. The bacterium was identified as Ralstonia pickettii and designated strain TA. Cells grown on thioacetamide also transformed other thioamide compounds. Transformation of the thioamides tested was dependent on oxygen. During thioamide degradation, sulfur was detected in the medium at the oxidation level of sulfite, further suggesting an oxygenase mechanism. R. pickettii TA did not grow on thiobenzamide as a nitrogen source, but resting cells converted thiobenzamide to benzamide, with thiobenzamide S-oxide and benzonitrile detected as intermediates. Thioacetamide S-oxide was detected as an intermediate during thioacetamide degradation, but the only accumulating metabolite of thioacetamide was identified as 3,5-dimethyl-1,2,4-thiadiazole, a compound shown to derive from spontaneous reaction of thioacetamide and oxygenated thioacetamide species. This dead-end metabolite accounted for only ca. 12% of the metabolized thioacetamide. Neither acetonitrile nor acetamide was detected during thioacetamide degradation, but R. pickettii grew on both compounds as nitrogen and carbon sources. It is proposed that R. pickettii TA degrades thioamides via a mechanism involving consecutive oxygenations of the thioamide sulfur atom.     

Widespread Head-to-Head Hydrocarbon Biosynthesis in Bacteria and Role of OleA

Previous studies identified the oleABCD genes involved in head-to-head olefinic hydrocarbon biosynthesis. The present study more fully defined the OleABCD protein families within the thiolase, α/β-hydrolase, AMP-dependent ligase/synthase, and short-chain dehydrogenase superfamilies, respectively. Only 0.1 to 1% of each superfamily represents likely Ole proteins. Sequence analysis based on structural alignments and gene context was used to identify highly likely ole genes. Selected microorganisms from the phyla Verucomicrobia, Planctomyces, Chloroflexi, Proteobacteria, and Actinobacteria were tested experimentally and shown to produce long-chain olefinic hydrocarbons. However, different species from the same genera sometimes lack the ole genes and fail to produce olefinic hydrocarbons. Overall, only 1.9% of 3,558 genomes analyzed showed clear evidence for containing ole genes. The type of olefins produced by different bacteria differed greatly with respect to the number of carbon-carbon double bonds. The greatest number of organisms surveyed biosynthesized a single long-chain olefin, 3,6,9,12,15,19,22,25,28-hentriacontanonaene, that contains nine double bonds. Xanthomonas campestris produced the greatest number of distinct olefin products, 15 compounds ranging in length from C₂₈ to C₃₁ and containing one to three double bonds. The type of long-chain product formed was shown to be dependent on the oleA gene in experiments with Shewanella oneidensis MR-1 ole gene deletion mutants containing native or heterologous oleA genes expressed in trans. A strain deleted in oleABCD and containing oleA in trans produced only ketones. Based on these observations, it was proposed that OleA catalyzes a nondecarboxylative thiolytic condensation of fatty acyl chains to generate a β-ketoacyl intermediate that can decarboxylate spontaneously to generate ketones.There is currently great interest in elucidating the means by which microbes produce nongaseous hydrocarbons for use as specialty chemicals and fuels (8, 40). While many details remain to be revealed, there appear to be several different pathways by which microbes biosynthesize long-chain hydrocarbons. The most studied of the pathways (16) involves the condensation of isoprene units to generate hydrocarbons with a multiple of five carbon atoms (C₁₀, C₁₅, C₂₀, etc.). A more obscure biosynthetic route is a reported decarbonylation of fatty aldehydes to generate a C_n−1 hydrocarbon chain (19). A third mechanism that has received some attention is what has been denoted head-to-head condensation of fatty acids (2, 3, 4, 8, 64). In this pathway, the hydrocarbons are described to arise from the formation of a carbon-to-carbon bond between the carboxyl carbon of one fatty acid and the α-carbon of another fatty acid (3). This condensation results in a particular type of hydrocarbon with chain lengths of C₂₃ to C₃₃ and containing one or more double bonds. One double bond involves the median carbon in the chain at the point of fatty acid condensation. An example of this overall biosynthetic pathway leading to the formation of specific C₂₉ olefinic hydrocarbon isomers from fatty acid precursors has been demonstrated in vivo (61, 63, 64) and in vitro (4, 8).The condensation, elimination of carbon dioxide, and loss of the other carboxyl group oxygen atoms likely require multiple enzyme-catalyzed reactions. Recent patent applications by L. Friedman et al. describe a role for three or four proteins in this biosynthetic pathway (18 September 2008, WO2008/113041; 4 December 2008, WO2008/147781). Most recently, Beller et al. demonstrated the requirement for three genes from Micrococcus luteus in the biosynthesis of long-chain olefins (8). That study also demonstrated in vitro production of olefins by recombinant proteins in the presence of crude cell extracts from Escherichia coli. In another study, Shewanella oneidensis strain MR-1 was shown to produce a head-to-head hydrocarbon (59). A cluster of four genes, oleABCD, was shown to be involved in olefin biosynthesis by that organism.While genetic and biochemical data have provided evidence for Ole proteins producing long-chain olefins in M. luteus and S. oneidensis, there are many outstanding details of the biosynthesis that remain to be elucidated. Moreover, the extent to which microbial and other species produce head-to-head olefins is unclear. A recent patent application by Friedman and Rude (WO2008/113041) presented tables listing genes homologous to the ole genes described by Beller et al. (8). However, the homologs identified included genes from mouse and tree frog, organisms not known to produce head-to-head hydrocarbons. Additionally, hydrocarbon biosynthetic genes from Arthrobacter sp. FB24 were claimed, and that strain was later shown not to produce hydrocarbons under identical conditions for which other Arthrobacter strains did (22). In that context, the present study closely examined the protein sequence families of Ole proteins and the configurations of putative ole genes within genomes to identify those most likely to be involved in head-to-head hydrocarbon biosynthesis. This was followed by experimental testing for the presence of long-chain head-to-head hydrocarbons in representative bacteria from diverse phyla. This study also found that, of closely related bacteria, some produce head-to-head hydrocarbons and others do not.A previous publication investigated in vitro olefin biosynthesis from myristyl-coenzyme A (CoA) (8). That study showed ketone and olefin biosynthesis in vitro and proposed a mechanism requiring the participation of ancillary proteins not encoded in the oleABCD gene cluster. The mechanism proposed fatty acyl oxidation to generate a β-keto acid that is the substrate for the OleA protein.In fact, different mechanisms have been suggested previously for the biosynthesis of head-to-head olefins (3; Friedman and Rude, WO2008/113041; Friedman and Da Costa, WO2008/147781), and different roles for the OleA protein have been proposed (8; Friedman and Rude, WO2008/113041; Friedman and Da Costa, WO2008/147781). It is not possible to deduce the olefinic biosynthetic pathway or individual reaction types based on protein sequence alignments alone, because this pathway is unique, differing markedly from isoprenoid or decarbonylation hydrocarbon biosynthesis pathways. Moreover, the individual Ole proteins are each homologous to proteins that collectively catalyze diverse reactions. In that context, we (i) initiated a more detailed study of the Ole protein superfamilies, (ii) identified likely olefin (ole) biosynthetic genes out of thousands of homologs, (iii) experimentally tested bacteria from different phyla for long-chain olefins, (iv) developed insights into the role of OleA in head-to-head olefin biosynthesis, and (v) propose an alternative mechanism for head-to-head condensation of fatty acyl groups.