We performed molecular phylogenetic analyses of glutamine synthetase (GS) genes in order to investigate their evolutionary history. The analyses were done on 30 DNA sequences of the GS gene which included both prokaryotes and eukaryotes. Two types of GS genes are known at present: the GSI gene found so far only in prokaryotes and the GSII gene found in both prokaryotes and eukaryotes. Our study has shown that the two types of GS gene were produced by a gene duplication which preceded, perhaps by >1000 million years, the divergence of eukaryotes and prokaryotes. The results are consistent with the facts that (t0 GS is a key enzyme of nitrogen metabolism found in all extant life forms and (fi) the oldest biological fossils date back 3800 million years. Thus, we suggest that GS genes are one of the oldest existing and functioning genes in the history of gene evolution and that GSI genes should also exist in eukaryotes. Furthermore, our study may stimulate investigation on the evolution of "preprokaryotes," by which we mean the organisms that existed during the era between the origin of life and the divergence of prokaryotes and eukaryotes.Glutamine synthetase (GS) is a key enzyme in nitrogen metabolism; it has dual functions in two essential biochemical reactions, ammonia assimilation and glutamine biosynthesis (1, 2). It is also one of the few amide synthetases found in organisms. Prokaryotes and eukaryotes were once thought to synthesize different GSs: GSI for the former and GSII for the latter. It is now known, however, that GSII is also present in bacteria belonging to Rhizobiaceae (3-6), Frankiaceae (7), and Streptomycetaceae (8, 9). GSI, by contrast, has not been found in any eukaryote.Glutamine produced by GS is essential for protein synthesis, and its amide nitrogen is donated to synthesize many essential metabolites. It is thus natural to consider GS as present in, and probably indispensable to, all organisms. In view of the central roles played by GS, it is reasonable to believe that the GS gene is extremely old. From the sequence alignment of GSI from Salmonella typhimurium and GSII from alfalfa (10), we could observe that the differences in amino acids between them was 0.75 per site. This value is quite large compared with those for other proteins, suggesting also that the GSI and GSII genes share a very old comnmon ancestor.The aforementioned discovery of the GSII gene in plant symbiotic bacteria led to the suggestion that the gene had originated from host plants through lateral gene transfer (3). This was later questioned by the further findings of the GSII gene in plant nonsymbiotic actinomycetes (8, 9). Shatters and Kahn (6) have suggested that the common ancestor of the GSII genes in Rhizobiaceae and in the host plant must be older than the plant itself, and have argued against the gene transfer.In this paper we have traced the evolutionary history of the GS genes, using our own nucleotide sequence data and others' data from prokaryotic and eukaryotic species in order to estimate the age of...

Increased tryptophan (Trp) catabolism in the tumor microenvironment (TME) can mediate immune suppression by upregulation of interferon (IFN)-γ-inducible indoleamine 2,3-dioxygenase (IDO1) and/or ectopic expression of the predominantly liver-restricted enzyme tryptophan 2,3-dioxygenase (TDO). Whether these effects are due to Trp depletion in the TME or mediated by the accumulation of the IDO1 and/or TDO (hereafter referred to as IDO1/TDO) product kynurenine (Kyn) remains controversial. Here we show that administration of a pharmacologically optimized enzyme (PEGylated kynureninase; hereafter referred to as PEG-KYNase) that degrades Kyn into immunologically inert, nontoxic and readily cleared metabolites inhibits tumor growth. Enzyme treatment was associated with a marked increase in the tumor infiltration and proliferation of polyfunctional CD8 lymphocytes. We show that PEG-KYNase administration had substantial therapeutic effects when combined with approved checkpoint inhibitors or with a cancer vaccine for the treatment of large B16-F10 melanoma, 4T1 breast carcinoma or CT26 colon carcinoma tumors. PEG-KYNase mediated prolonged depletion of Kyn in the TME and reversed the modulatory effects of IDO1/TDO upregulation in the TME.

Proteome analysis plays a key role in the elucidation of the functions and applications for numerous proteins. For proteome analyses, various microplate-and microarray-based techniques have been developed by a number of researchers. Their intent was to immobilize proteins on the surface of a solid substrate in a site-directed manner while retaining structure and native biological function. In this review, we focus on recent advances in immobilization methodology for proteins/enzymes on a surface, including those using the affinity peptides screened by random peptide library systems. We also discuss applications of the affinity peptide-mediated immobilization method in fields related to proteome analysis, particularly our recent work concerning immunoassay and protein-protein interaction analysis.

No abstract

Dodecapeptides that exhibit a high affinity specific to a polystyrene surface (PS-tags) were screened using an Escherichia coli random peptide display library system, and the compounds were used as a peptide tag for the site-specific immobilization of proteins. The various PS-tags obtained after 10 rounds of biopanning selection were mainly composed of basic and aliphatic amino acid residues, most of which were arranged in close proximity to one another. Mutant-type glutathione S-transferases (GSTs) fused with the selected PS-tags, PS19 (RAFIASRRIKRP) and PS23 (AGLRLKKAAIHR) at their C-terminus, GST-PS19 and GST-PS23, when adsorbed on the PS latex beads had a higher affinity than the wild-type GST, and the specific remaining activity of the immobilized mutant-type GSTs was approximately 10 times higher than that of the wild-type GST. The signal intensity detected for GST-PS19 and GST-PS23 adsorbed on hydrophilic and hydrophobic PS surfaces using an anti-peptide antibody specific for the N-terminus peptide of GST was much higher than that for the wild-type GST. These findings indicate that the mutant-type GSTs fused with the selected peptide tags, PS19 and PS23, could be site-specifically immobilized on the surface of polystyrene with their N-terminal regions directed toward the solution. Thus, the selected peptide tags would be useful for protein immobilization in the construction of enzyme-linked immunosorbent assay (ELISA) systems and protein-based biochips.

Streptomyces hygroscopicus, which produces the glutamine synthetase inhibitor phosphinothricin, possesses at least two genes (glnA and glnB) encoding distinct glutamine synthetase isoforms (GSI and GSII) Glutamine synthetase (GS), a pivotal enzyme for nitrogen metabolism, is found in at least three distinct forms. In studies reported to date, one of these forms, GSI, has been primarily associated with procaryotes and another, GSII, has been associated with eucaryotes. A third type of GS has been recently found in the anaerobe Bacteriodes fragilis (22). These three types of enzymes are distinct in their primary as well as tertiary structures. GSI is composed of 12 subunits (443 to 474 amino acids each); GSII has 8 subunits (332 to 378 amino acids each) (see Table 2 for references); and the Bacteriodes fragilis enzyme has 6 subunits (729 amino acids) (22). Sequence alignments show that the GSI and GSII families are only 15% identical (40) and that GSI has an extended C terminus (see Fig. 6 for references), which includes an adenylylation site important for the posttranslational control of activity (43). Thermolability of GSII has been widely used to differentiate it from GSI (13,16,17).The strict association of one of these two enzyme families with procaryotes and the other with eucaryotes was first called into question by studies of nitrogen metabolism in nodulating bacteria such as Rhizobium (13,18,33), Agrobacterium (17), Bradyrhizobium (7), and Frankia (16)

No abstract

No abstract