Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases

We report a method for site-specifically incorporating l-lysine derivatives into proteins in mammalian cells, based on the expression of the pyrrolysyl-tRNA synthetase (PylRS)-tRNA^Pyl pair from Methanosarcina mazei. Different types of external promoters were tested for the expression of tRNA^Pyl in Chinese hamster ovary cells. When tRNA^Pyl was expressed from a gene cluster under the control of the U6 promoter, the wild-type PylRS-tRNA^Pyl pair facilitated the most efficient incorporation of a pyrrolysine analog, N^ε-tert-butyloxycarbonyl-l-lysine (Boc-lysine), into proteins at the amber position. This PylRS-tRNA^Pyl system yielded the Boc-lysine-containing protein in an amount accounting for 1% of the total protein in human embryonic kidney (HEK) 293 cells. We also created a PylRS variant specific to N^ε-benzyloxycarbonyl-l-lysine, to incorporate this long, bulky, non-natural lysine derivative into proteins in HEK293. The recently reported variant specific to N^ε-acetyllysine was also expressed, resulting in the genetic encoding of this naturally-occurring lysine modification in mammalian cells.     

A new detection method for arginine-specific ADP-ribosylation of protein -- a combinational use of anti-ADP-ribosylarginine antibody and ADP-ribosylarginine hydrolase

Arginine-specific ADP-ribosylation is one of the posttranslational modifications of proteins by transferring one ADP-ribose moiety of NAD to arginine residues of target proteins. This modification, catalyzed by ADP-ribosyltransferase (Art), is reversed by ADP-ribosylarginine hydrolase (AAH).

In this study, we describe a new method combining an anti-ADP-ribosylarginine antibody (ADP-R-Arg Ab) and AAH for detection of the target protein of ADP-ribosylation. We have raised ADP-R-Arg Ab with ADP-ribosylated histone and examined the reactivity of the antibody with proteins treated by Art and/or AAH, as well as in situ ADP-ribosylation system with mouse T cells. Our results indicate that the detection of ADP-ribosylated protein with ADP-R-Arg Ab and AAH is a useful tool to explore the target proteins of ADP-ribosylation. We applied the method to search endogenously ADP-ribosylated protein in the rat, and detected possible target proteins in the skeletal muscle, which has high Art activity.     

Possible role of membrane proteins in mercury resistance of Enterobacter aerogenes

Mercury resistance shown by a strain of Enterobacter aerogenes was found to be determined by a plasmid. The resistance appeared to be not due to enzymatic volatilization of mercury, but due to the alteration in cellular permeability to mercury.Comparison of the outer membrane proteins was made between the resistant cells and the sensitive counterparts obtained by the treatment with mitomycin C, showing that two proteins with molecular weight of 46,000 and 44,000 had disappeared from the outer membrane along with the plasmid by the curing. These results suggest that the two membrane proteins mediating the cellular permeability to mercury compound may be responsible for the mercury resistance of the strain.     

Antimicrobial Activity of 3-Phenylimidazolidine-2,4-diones and Related Compounds

The structure-activity relationships of 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were investigated by the agar dilution method using Sclerotinia sclerotiorum as a test microbe. Several compounds were tested for antimicrobial spectrum in vitro with other pathogenic microbes and for foliage protective activity in green house tests with rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. It was found that the antimicrobial activity was enhanced when the 1-position of imidazolidine ring was substituted by an alkyl group but was reduced when the 5-position was substituted by alkyl groups. Generally, 3-(3′,5′-dichlorophenyl)imidazolidine-2,4-dione derivatives were active against Scierotiniaceae, Corticiaceae, Dematiaceae, Polystigmataceae or Pleosporaceae. In green house tests, some of these compounds showed high protective activity against rice sheath blight, rice brown spot, damping-off of cucumber and kidney bean stem rot. Results of the green house tests on the above mentioned diseases correlate well with those of in vitro tests except in the case of kidney bean stem rot.     

Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells

Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-l-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein-protein interactions in cells.     

Genetic-code evolution for protein synthesis with non-natural amino acids

The genetic encoding of synthetic or “non-natural” amino acids promises to diversify the functions and structures of proteins. We applied rapid codon-reassignment for creating Escherichia coli strains unable to terminate translation at the UAG “stop” triplet, but efficiently decoding it as various tyrosine and lysine derivatives. This complete change in the UAG meaning enabled protein synthesis with these non-natural molecules at multiple defined sites, in addition to the 20 canonical amino acids. UAG was also redefined in the E. coli BL21 strain, suitable for the large-scale production of recombinant proteins, and its cell extract served the cell-free synthesis of an epigenetic protein, histone H4, fully acetylated at four specific lysine sites.     

Quantitative analysis of glycosaminoglycans,chondroitin/dermatan sulfate,hyaluronic acid,heparan sulfate,and keratan sulfate by liquid chromatography–electrospray ionization–tandem mass spectrometry

We developed a method using liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS) with a selected reaction monitoring (SRM) mode for simultaneous quantitative analysis of glycosaminoglycans (GAGs). Using one-shot analysis with our MS/MS method, we demonstrated the simultaneous quantification of a total of 23 variously sulfated disaccharides of four GAG classes (8 chondroitin/dermatan sulfates, 1 hyaluronic acid, 12 heparan sulfates, and 2 keratan sulfates) with a sensitivity of less than 0.5 pmol within 20 min. We showed the differences in the composition of GAG classes and the sulfation patterns between porcine articular cartilage and yellow ligament. In addition to the internal disaccharides described above, some saccharides derived from the nonreducing terminal were detected simultaneously. The simultaneous quantification of both internal and nonreducing terminal saccharides could be useful to estimate the chain length of GAGs. This method would help to establish comprehensive “GAGomic” analysis of biological tissues.     

A set of external reference controls/probes that enable quality assurance between different microarray platforms

RNA external standards, although important to ensure equivalence across many microarray platforms, have yet to be fully implemented in the research community. In this article, a set of unique RNA external standards (or RNA standards) and probe pairs that were added to total RNA in the samples before amplification and labeling are described. Concentration–response curves of RNA external standards were used across multiple commercial DNA microarray platforms and/or quantitative real-time polymerase chain reaction (RT–PCR) and next-generation sequencing to identify problematic assays and potential sources of variation in the analytical process. A variety of standards can be added in a range of concentrations spanning high and low abundances, thereby enabling the evaluation of assay performance across the expected range of concentrations found in a clinical sample. Using this approach, we show that we are able to confirm the dynamic range and the limit of detection for each DNA microarray platform, RT–PCR protocol, and next-generation sequencer. In addition, the combination of a series of standards and their probes was investigated on each platform, demonstrating that multiplatform calibration and validation is possible.     

Wide-range protein photo-crosslinking achieved by a genetically encoded N(ε)-(benzyloxycarbonyl)lysine derivative with a diazirinyl moiety

A derivative of N(ε)-benzyloxycarbonyl-L-lysine with a photo-reactive diazirinyl group, N(ε)-[((4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzyl)oxy)carbonyl]-L-lysine, was site-specifically incorporated into target proteins in mammalian cells. The incorporated photo-crosslinker is able to react not only with residues as distant as about 15 ? but also with those in closer proximity, thus enabling "wide-range" photo-crosslinking of proteins.     

Efficient decoding of the UAG triplet as a full-fledged sense codon enhances the growth of a prfA-deficient strain of Escherichia coli

We previously reassigned the amber UAG stop triplet as a sense codon in Escherichia coli by expressing a UAG-decoding tRNA and knocking out the prfA gene, encoding release factor 1. UAG triplets were left at the ends of about 300 genes in the genome. In the present study, we showed that the detrimental effect of UAG reassignment could be alleviated by increasing the efficiency of UAG translation instead of reducing the number of UAGs in the genome. We isolated an amber suppressor tRNA(Gln) variant displaying enhanced suppression activity, and we introduced it into the prfA knockout strain, RFzero-q, in place of the original suppressor tRNA(Gln). The resulting strain, RFzero-q3, translated UAG to glutamine almost as efficiently as the glutamine codons, and it proliferated faster than the parent RFzero-q strain. We identified two major factors in this growth enhancement. First, the sucB gene, which is involved in energy regeneration and has two successive UAG triplets at the end, was expressed at a higher level in RFzero-q3 than RFzero-q. Second, the ribosome stalling that occurred at UAG in RFzero-q was resolved in RFzero-q3. The results revealed the importance of "backup" stop triplets, UAA or UGA downstream of UAG, to avoid the deleterious impact of UAG reassignment on the proteome.