We have identified a series of novel insulin receptor partial agonists (IRPAs) with a potential to mitigate the risk of hypoglycemia associated with the use of insulin as an antidiabetic treatment. These molecules were designed as dimers of native insulin connected via chemical linkers of variable lengths with optional capping groups at the N-terminals of insulin chains. Depending on the structure, the maximal activation level (%Max) varied in the range of ∼20-70% of native insulin, and EC50 values remained in sub-nM range. Studies in minipig and dog demonstrated that IRPAs had sufficient efficacy to normalize plasma glucose levels in diabetes, while providing reduction of hypoglycemia risk. IRPAs had a prolonged duration of action, potentially making them suitable for once-daily dosing. Two lead compounds with %Max values of 30 and 40% relative to native insulin were selected for follow up studies in the clinic.

Recent advances in the field of cancer biology have accelerated the discovery and development of novel biopharmaceuticals. At the forefront of these drug development efforts are high-throughput screening, compressed timelines, and limited sample quantities, all characteristic of the discovery space. To meet program targets, large numbers of protein variants must be produced, screened, and characterized, presenting a daunting analytical challenge. Additionally, the higher-order structure is paramount for protein function and must be monitored as a critical quality attribute. Matrix-assisted laser desorption/ionization mass spectrometry has been utilized as an ultra-fast, automatable, sample-sparing analytical tool for biomolecules. Our group has published applications integrating hydrogen-deuterium exchange mass spectrometry with matrix-assisted laser desorption/ionization mass spectrometry for the rapid conformational characterization of small proteins, the current work expands this application to monoclonal and bi-specific antibodies. This study demonstrates the ability of the methodology, matrix-assisted laser desorption/ionization hydrogen-deuterium exchange mass spectrometry, to detect conformational differences between bi-specific antibodies from different expression hosts. These conformational differences were validated by orthogonal techniques including circular dichroism, nuclear magnetic resonance, and size-exclusion chromatography hydrogen-deuterium exchange mass spectrometry. This work demonstrates the utility of applying the developed methodology as a rapid conformational screening tool to triage samples for further analytical characterization.

Myc, a transcription factor with oncogenic activity, is upregulated by amplification, translocation, and mutation of the cellular pathways that regulate its stability. Inhibition of the Myc oncogene by various modalities has had limited success. One Myc inhibitor, Omomyc, has limited cellular and in vivo activity. Here, we report a mini‐protein, referred to as Mad, which is derived from the cellular Myc antagonist Mxd1. Mad localizes to the nucleus in cells and is 10‐fold more potent than Omomyc in inhibiting Myc‐driven cell proliferation. Similar to Mxd1, Mad also interacts with Max, the binding partner of Myc, and with the nucleolar upstream binding factor. Mad binds to E‐Box DNA in the promoters of Myc target genes and represses Myc‐mediated transcription to a greater extent than Omomyc. Overall, Mad appears to be more potent than Omomyc both in vitro and in cells.

This chapter discusses four unique aspects of archaeal aa-tRNA formation that led to a much deeper understanding of this process not only in the Archaea, but in all domains of life. The topics are: (i) processing of half-tRNA genes to mature tRNA in Nanoarchaeum equitans, (ii) RNA-dependent cysteine synthesis in methanogens, (iii) pyrrolysyl-tRNA formation in the Methanosarcinaceae, and (iv) glutaminyl- tRNA synthesis in archaea. The bulge-helix-bulge (BHB) motifs postulated to form at the intron-exon junctions of archaeal tRNAs show divergence from the canonical structure. Once mature tRNA has been generated each tRNA species needs to be acylated (charged) with the correct amino acid. This is primarily achieved by the direct attachment of an amino acid to the corresponding tRNA by an aminoacyl-tRNA synthetase. However, since many organisms lack the complete set of 20 aminoacyl-tRNA synthetases (aaRSs), many biochemical, genetic, and genomic studies revealed the existence of an essential indirect two-step pathway that also provides correctly charged aa-tRNA. The aminoacyl-tRNA synthetases are an ancient family of enzymes that esterify an amino acid to the 3' end of the cognate tRNA species. The current understanding of the Methanothermobacter thermautotrophicus Glu-tRNAGln amidotransferase GatDE is discussed. Discovery of the tRNA-dependent cysteine biosynthetic route in M. jannaschii may have implications that reach far beyond the only problem of the formation of Cys-tRNACys in three methanogenic archaea. The Methanosarcinaceae are an exception among the methanogens, as they are able to use compounds like methanol, methylated thiols, and methylamines as energy sources.

Significance Translation requires aminoacyl-tRNAs that are mainly formed by acylating tRNAs with the corresponding amino acids. Methanogenic archaea synthesize Cys-tRNA in an unusual indirect fashion. They attach a precursor amino acid, phosphoserine, to tRNA Cys , which is then converted to cysteine. This study shows that the indirect Cys-tRNA formation is carried out in a multienzyme complex assembled by a translation factor. Complex formation markedly promotes reaction efficiency. Because the indirect Cys-tRNA formation is the ancestral pathway of Cys biosynthesis in archaea, this complex may represent a remnant of a primordial machinery for Cys coding.

Synthetic tRNA for selenoprotein production is described by D. Söll et al. in their Communication on page 1441 ff. The tRNA is a substrate for three E. coli proteins: seryl-tRNA synthetase (SerRS), selenocysteine synthase (SelA) generating Sec-tRNAUTu, and EF-Tu for Sec-tRNAUTu transport to the ribosome, which allows site-specific Sec insertion into proteins. This system has general utility in protein engineering, molecular biology, and disease research.

Enjoying UTu in concert: A synthetic tRNA (tRNAUTu) was used as a substrate for three E. coli proteins: seryl-tRNA synthetase (SerRS) forming Ser-tRNAUTu, selenocysteine (Sec) synthase (SelA) generating Sec-tRNAUTu, and EF-Tu for Sec-tRNAUTu transport to the ribosome (see scheme). tRNAUTu can be used by the ribosome, thus allowing site-specific Sec insertion into proteins, including formate dehydrogenase H, selenoglutaredoxin, and glutathione peroxidase. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Synthetische tRNA für die Selenoproteinproduktion beschreiben D. Söll et al. in der Zuschrift auf S. 1481 ff. Die tRNA ist ein Substrat für drei E.-coli-Proteine: seryl-tRNA-Synthetase (SerRS), Selenocystein-Synthase (SelA), die Sec-tRNAUTu liefert, und EF-Tu für den Transport von Sec-tRNAUTu zum Ribosom, was die ortsspezifische Insertion von Sec in Proteine ermöglicht. Das System ist für das Protein-Engineering, die Molekularbiologie und die medizinische Forschung nützlich.

Class I and II aminoacyl‐tRNA synthetases (AARSs) attach amino acids to the 2′‐ and 3′‐OH of the tRNA terminal adenosine, respectively. One exception is phenylalanyl‐tRNA synthetase (PheRS), which belongs to Class II but attaches phenylalanine to the 2′‐OH. Here we show that two Class II AARSs, O‐phosphoseryl‐ (SepRS) and pyrrolysyl‐tRNA (PylRS) synthetases, aminoacylate the 2′‐ and 3′‐OH, respectively. Structure‐based‐phylogenetic analysis reveals that SepRS is more closely related to PheRS than PylRS, suggesting that the idiosyncratic feature of 2′‐OH acylation evolved after the split between PheRS and PylRS. Our work completes the understanding of tRNA aminoacylation positions for the 22 natural AARSs.

5‐Methylaminomethyl‐2‐selenouridine (mnm5Se2U) is found in the first position of the anticodon in certain tRNAs from bacteria, archaea and eukaryotes. This selenonucleoside is formed in Escherichia coli from the corresponding thionucleoside mnm5S2U by the monomeric enzyme YbbB. This nucleoside is present in the tRNA of Methanococcales, yet the corresponding 2‐selenouridine synthase is unknown in archaea and eukaryotes. Here we report that a bipartite ybbB ortholog is present in all members of the Methanococcales. Gene deletions in Methanococcus maripaludis and in vitro activity assays confirm that the two proteins act in trans to form in tRNA a selenonucleoside, presumably mnm5Se2U. Phylogenetic data suggest a primal origin of seleno‐modified tRNAs.

O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep(218), Sep(222)). This system has general utility in protein engineering, molecular biology, and disease research.

In Archaea selenocysteine (Sec) is synthesized in three steps. First seryl-tRNA synthetase acylates tRNA(Sec) with serine to generate Ser-tRNA(Sec). Then phosphoseryl-tRNA(Sec) kinase (PSTK) forms Sep-tRNA(Sec) , which is converted to Sec-tRNA(Sec) by Sep-tRNA:Sec-tRNA synthase (SepSecS) in the presence of selenophosphate produced by selenophosphate synthetase (SelD). A complete in vivo analysis of the archaeal Sec biosynthesis pathway is still unavailable, and the existence of a redundant pathway or of a rescue mechanism based on the conversion of Sep-tRNA(Sec) to Cys-tRNA(Sec) during selenium starvation, cannot be excluded. Here we present a mutational analysis of Sec biosynthesis in Methanococcus maripaludis strain Mm900. Sec formation is abolished upon individually deleting the genes encoding SelD, PSTK or SepSecS; the resulting mutant strains could no longer grow on formate while growth with H(2) + CO(2) remained unaffected. However, deletion of the PSTK and SepSecS genes was not possible unless the selenium-free [NiFe]-hydrogenases Frc and Vhc were expressed. This required the prior deletion of either the gene encoding SelD or that of HrsM, a LysR-type regulator suppressing transcription of the frc and vhc operons in the presence of selenium. These results show that M. maripaludis Mm900 is facultatively selenium-dependent with a single pathway of Sec-tRNA(Sec) formation.

The essential methanogen enzyme Sep-tRNA:Cys-tRNA synthase (SepCysS) converts O-phosphoseryl-tRNACys (Sep-tRNACys) into Cys-tRNACys in the presence of a sulfur donor. Likewise, Sep-tRNA:Sec-tRNA synthase converts O-phosphoseryl-tRNASec (Sep-tRNASec) to selenocysteinyl-tRNASec (Sec-tRNASec) using a selenium donor. While the Sep moiety of the aminoacyl-tRNA substrates is the same in both reactions, tRNACys and tRNASec differ greatly in sequence and structure. In an Escherichia coli genetic approach that tests for formate dehydrogenase activity in the absence of selenium donor we show that Sep-tRNASec is a substrate for SepCysS. Since Sec and Cys are the only active site amino acids known to sustain FDH activity, we conclude that SepCysS converts Sep-tRNASec to Cys-tRNASec, and that Sep is crucial for SepCysS recognition.

Selenocysteine (Sec) is the 21st genetically encoded amino acid found in organisms from all three domains of life. Sec biosynthesis is unique in that it always proceeds from an aminoacyl‐tRNA precursor. Even though Sec biosynthesis in bacteria was established almost two decades ago, only recently the pathway was elucidated in archaea and eukaryotes. While other aspects of Sec biology have been reviewed previously (Allmang and Krol, Biochimie 2006;88:1561–1571, Hatfield et al., Prog Nucleic Acid Res Mol Biol 2006;81:97–142, Squires and Berry, IUBMB Life 2008;60:232–235), here we review the biochemistry and evolution of Sec biosynthesis and coding and show how the knowledge of an archaeal cysteine biosynthesis pathway helped to uncover the route to Sec formation in archaea and eukaryotes. © 2008 IUBMB IUBMB Life, 61(1): 35–39, 2009

Selenocysteine (Sec) is the 21st genetically encoded amino acid found in organisms from all three domains of life. See biosynthesis is unique in that it always proceeds from an aminoacyl-tRNA precursor. Even though See biosynthesis in bacteria was established almost two decades ago, only recently the pathway was elucidated in archaea and eukaryotes. While other aspects of Sec biology have been reviewed previously (Allmang and Krol, Biochimie 2006;88:1561-1.571, Hatfield et al., Prog Nucleic Acid Res Mol Biol 2006;81:97-142, Squires and Berry, IUBMB Life 2008;60:232-235), here we review the biochemistry and evolution of Sec biosynthesis and coding and show how the knowledge of an archaeal cysteine biosynthesis pathway helped to uncover the route to Sec formation in archaea and eukaryotes. (C) 2008 IUBMB

Aminoacyl-tRNAs (aa-tRNAs) are the essential substrates for translation. Most aa-tRNAs are formed by direct aminoacylation of tRNA catalyzed by aminoacyl-tRNA synthetases. However, a smaller number of aa-tRNAs (Asn-tRNA, Gln-tRNA, Cys-tRNA and Sec-tRNA) are made by synthesizing the amino acid on the tRNA by first attaching a non-cognate amino acid to the tRNA, which is then converted to the cognate one catalyzed by tRNA-dependent modifying enzymes. Asn-tRNA or Gln-tRNA formation in most prokaryotes requires amidation of Asp-tRNA or Glu-tRNA by amidotransferases that couple an amidase or an asparaginase to liberate ammonia with a tRNA-dependent kinase. Both archaeal and eukaryotic Sec-tRNA biosynthesis and Cys-tRNA synthesis in methanogens require O-phosophoseryl-tRNA formation. For tRNA-dependent Cys biosynthesis, O-phosphoseryl-tRNA synthetase directly attaches the amino acid to the tRNA which is then converted to Cys by Sep-tRNA: Cys-tRNA synthase. In Sec-tRNA synthesis, O-phosphoseryl-tRNA kinase phosphorylates Ser-tRNA to form the intermediate which is then modified to Sec-tRNA by Sep-tRNA:Sec-tRNA synthase. Complex formation between enzymes in the same pathway may protect the fidelity of protein synthesis. How these tRNA-dependent amino acid biosynthetic routes are integrated into overall metabolism may explain why they are still retained in so many organisms.

A number of archaeal organisms generate Cys-tRNACys in a two-step pathway, first charging phosphoserine (Sep) onto tRNACys and subsequently converting it to Cys-tRNACys. We have determined, at 3.2-Å resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, α4 synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (αβ)2 phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.

A novel chemolithoautotrophic and hyperthermophilic member of the genus Ignicoccus was isolated from a submarine hydrothermal system at the Kolbeinsey Ridge, to the north of Iceland. The new isolate showed high similarity to the two species described to date, Ignicoccus islandicus and Ignicoccus pacificus, in its physiological properties as well as in its unique cell architecture. However, phylogenetic analysis and investigations on the protein composition of the outer membrane demonstrated that the new isolate was clearly distinct from I. islandicus and I. pacificus. Furthermore, it is the only organism known so far which is able to serve as a host for 'Nanoarchaeum equitans', the only cultivated member of the 'Nanoarchaeota'. Therefore, the new isolate represents a novel species of the genus Ignicoccus, which we name Ignicoccus hospitalis sp. nov. (type strain KIN4/I(T)=DSM 18386(T)=JCM 14125(T)).

The molecular basis of the genetic code manifests itself in the interaction of the aminoacyl-tRNA synthetases and their cognate tRNAs. The fundamental biological question regarding these enzymes' role in the evolution of the genetic code remains open. Here we probe this question in a system in which the same tRNA species is aminoacylated by two unrelated synthetases. Should this tRNA possess major identity elements common to both enzymes, this would favor a scenario where the aminoacyl-tRNA synthetases evolved in the context of preestablished tRNA identity, i.e., after the universal genetic code emerged. An experimental system is provided by the recently discovered O-phosphoseryl-tRNA synthetase (SepRS), which acylates tRNA(Cys) with phosphoserine (Sep), and the well known cysteinyl-tRNA synthetase, which charges the same tRNA with cysteine. We determined the identity elements of Methanocaldococcus jannaschii tRNA(Cys) in the aminoacylation reaction for the two Methanococcus maripaludis synthetases SepRS (forming Sep-tRNA(Cys)) and cysteinyl-tRNA synthetase (forming Cys-tRNA(Cys)). The major elements, the discriminator base and the three anticodon bases, are shared by both tRNA synthetases. An evolutionary analysis of archaeal, bacterial, and eukaryotic tRNA(Cys) sequences predicted additional SepRS-specific minor identity elements (G37, A47, and A59) and suggested the dominance of vertical inheritance for tRNA(Cys) from a single common ancestor. Transplantation of the identified identity elements into the Escherichia coli tRNA(Gly) scaffold endowed facile phosphoserylation activity on the resulting chimera. Thus, tRNA(Cys) identity is an ancient RNA record that depicts the emergence of the universal genetic code before the evolution of the modern aminoacylation systems.