Biomedical subjects

Paul Schimmel

Publications and source records attributed to Paul Schimmel.

At least 19 recordsLinked to original sources

PubMed2003

Structure-specific tRNA determinants for editing a mischarged amino acid.

Alanyl-tRNA synthetase efficiently aminoacylates tRNAAla and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNAAla using a specialized domain in its C-terminal half. In contrast to full-length tRNAAla, minihelixAla was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelixAla. To understand these differences between minihelixAla and tRNAAla, several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNAAla. The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.

Alanine-tRNA Ligase↗

PubMed2003

Peptide synthesis with a template-like RNA guide and aminoacyl phosphate adaptors.

The genetic code is thought to have developed from an early system of RNA-dependent peptide synthesis. To investigate one kind of template-like peptide synthesis that might emerge from an RNA world, we constructed highly reactive aminoacyl phosphate oligonucleotides as adaptors that bound to RNA guide sequences. The reactive aminoacyl groups mimic a chemistry found in modern protein biosynthesis. Guide sequence interactions with adaptors were borrowed in part from universal contacts seen between tRNAs and rRNA. With these constructions, di- and tripeptides formed in a single guide sequence-dependent reaction. The order of amino acids was not random but directional in a way consistent with substrate reactivities. No ribosomes or ribozymes were required. Thus, aminoacyl phosphate adaptors and RNA guides could, in principle, have been intermediates in the transition from the RNA world to modern template-dependent protein synthesis.

Amino Acid Sequence↗

PubMed2003

RNA recognition by designed peptide fusion creates "artificial" tRNA synthetase.

The genetic code was established through aminoacylations of RNA substrates that emerged as tRNAs. The 20 aminoacyl-tRNA synthetases (one for each amino acid) are ancient proteins, the active-site domain of which catalyzes formation of an aminoacyl adenylate that subsequently reacts with the 3' end of bound tRNA. Binding of tRNA depends on idiosyncratic (to the particular synthetase) domains and motifs that are fused to or inserted into the conserved active-site domain. Here we take the domain for synthesis of alanyl adenylate and fuse it to "artificial" peptide sequences (28 aa) that were shown previously to bind to the acceptor arm of tRNAAla. Certain fusions confer aminoacylation activity on tRNAAla and on hairpin microhelices modeled after its acceptor stem. Aminoacylation was sensitive to the presence of a specific G:U base pair known to be a major determinant of tRNAAla identity. Aminoacylation efficiency and specificity also depended on the specific peptide sequence. The results demonstrate that barriers to RNA-specific aminoacylations are low and can be achieved by relatively simple peptide fusions. They also suggest a paradigm for rationally designed specific aminoacylations based on peptide fusions.

Amino Acid Motifs↗

PubMed2003

tRNA structure goes from L to lambda.

In this issue of Cell, Ishitani et al. (2003) report, in a crystal, a new L-like structure of tRNA designated as lambda-form, where disruption of universal tertiary interactions is compensated by interactions with an enzyme that makes a base modification at the corner of the L.

Base Sequence↗

PubMed2003

Mitochondrial form of a tRNA synthetase can be made bifunctional by manipulating its leader peptide.

Previous studies showed that yeast VAS1 encodes both the cytoplasmic and mitochondrial forms of valyl-tRNA synthetase (ValRS), using alternative transcription and translation. The ValRS isoforms have identical polypeptide sequences, except for a 46-amino acid leader peptide that functions as a mitochondrial targeting signal. Although the two forms of the enzyme exhibit indistinguishable tRNA specificities in vitro, they cannot substitute for each other in vivo because of their different localizations. Here we show that the 46-residue leader sequence can be divided into two nonoverlapping peptides, each of which retains the ability to target the enzyme into mitochondria. The engineered proteins (with truncated leader sequences) are dual-targeted, rescuing both the cytoplasmic and mitochondrial defects of a vas1 knockout strain. Thus, in addition to alternative splicing and alternative translation initiation as mechanisms by which a single gene can encode cytoplasmic and mitochondrial activities, the inherent characteristics of a single polypeptide may enable it to be distributed simultaneously between two cellular compartments. This mechanism may explain how certain other single genes in Saccharomyces cerevisiae provide dual functions.

Cloning, Molecular↗

PubMed2003

Elucidation of tRNA-dependent editing by a class II tRNA synthetase and significance for cell viability.

Editing of misactivated amino acids by class I tRNA synthetases is encoded by a specialized internal domain specific to class I enzymes. In contrast, little is known about editing activities of the structurally distinct class II enzymes. Here we show that the class II alanyl-tRNA synthetase (AlaRS) has a specialized internal domain that appears weakly related to an appended domain of threonyl-tRNA synthetase (ThrRS), but is unrelated to that found in class I enzymes. Editing of misactivated glycine or serine was shown to require a tRNA cofactor. Specific mutations in the aforementioned domain disrupt editing and lead to production of mischarged tRNA. This class-specific editing domain was found to be essential for cell growth, in the presence of elevated concentrations of glycine or serine. In contrast to ThrRS, where the editing domain is not found in all three kingdoms of living organisms, it was incorporated early into AlaRSs and is present throughout evolution. Thus, tRNA-dependent editing by AlaRS may have been critical for making the genetic code sufficiently accurate to generate the tree of life.

Adenosine Triphosphate↗

PubMed2002

Trbp111 selectively binds a noncovalently assembled tRNA-like structure.

Transfer RNAs are key components of the genetic code by virtue of aminoacylation reactions whereby each amino acid is linked to the tRNA that bears the anticodon for the attached amino acid. The L-shaped tRNA structure contains two domains connected at right angles through a corner formed from tertiary interactions involving loops of each domain. Some evidence suggests that the domains arose separately and eventually were fused into a single covalent structure. In this scenario, the present-day tRNA possibly developed through a noncovalently assembled heterodimeric intermediate. Trbp111 is an ancient structure-specific tRNA binding protein that interacts specifically with the outside corner of the L-shaped molecule. Plausibly, this protein could act as a chaperone to cover and protect the fragile corner and thereby have a historical role in the development of tRNA. Here we show that Trbp111 interacts with a noncovalently assembled tRNA-like structure, under conditions where it does not interact with individual tRNA domains. Trbp111 binding specifically requires formation of the tRNA-like corner. In a mixture of RNA domains, it selects those that can make the L-like structure. Thus, cofactors such as Trbp111 have the capacity to help assemble and stabilize RNA dimers that are tRNA-like.

Bacterial Proteins↗

PubMed2002

Mutational switching of a yeast tRNA synthetase into a mammalian-like synthetase cytokine.

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs. A link was recently established between protein biosynthesis and cytokine signal transduction. Human tyrosyl-tRNA synthetase can be split into two fragments, each of which has a distinct cytokine function. This activity is specific to the human enzyme. It is absent in the enzymes from lower organisms such as bacteria and yeast. Here, yeast tyrosyl-tRNA synthetase (TyrRS), which lacks cytokine activity, was used as a model to explore how a human tyrosyl-tRNA synthetase during evolution acquired novel functions beyond aminoacylation. We found that a rationally designed mutant yeast TyrRS(ELR) gained cytokine function. The mutant yeast enzyme gained this function without sacrifice of aminoacylation activity. Therefore, relatively simple alteration of a basic structural motif imparts cytokine activity to a tRNA synthetase while preserving its canonical function. Further work established that mutational switching of a yeast protein to a mammalian-like cytokine was specific to this synthetase and not to just any yeast ortholog of a mammalian cytokine.

Acylation↗

PubMed2002

Activation of angiogenic signaling pathways by two human tRNA synthetases.

Aminoacyl-tRNA synthetases establish the rules of the genetic code by joining amino acids to tRNAs that bear the anticodon triplets corresponding to the attached amino acids. The enzymes are thought to be among the earliest proteins to appear, in the transition from a putative RNA world to the theater of proteins. Over their long evolution, the enzymes have acquired additional functions that typically require specialized insertions or domain fusions. Recently, fragments of the closely related human tyrosyl- and tryptophanyl-tRNA synthetases were discovered to be active in angiogenesis signaling pathways. One synthetase fragment has proangiogenic activity, while the other is antiangiogenic. Activity was demonstrated in cell-based assays in vitro and in vivo in the chick embryo, and in the neonatal and adult mouse. The full-length, native enzymes are inactive in these same assays. Activation of angiogenesis activity requires fragment production from the native enzymes by protease cleavage or by translation of alternatively spliced pre-mRNA. Thus, these tRNA synthetases link translation to a major cell-signaling pathway in mammalian cells. The results with animals suggest that therapeutic applications are possible with these tRNA synthetases.

Animals↗

PubMed2002

Crystal structure of a human aminoacyl-tRNA synthetase cytokine.

The 20 aminoacyl-tRNA synthetases catalyze the first step of protein synthesis and establish the rules of the genetic code through aminoacylation reactions. Biological fragments of two human enzymes, tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, connect protein synthesis to cell-signaling pathways including angiogenesis. Alternative splicing or proteolysis produces these fragments. The proangiogenic N-terminal fragment mini-TyrRS has IL-8-like cytokine activity that, like other CXC cytokines, depends on a Glu-Leu-Arg motif. Point mutations in this motif abolish cytokine activity. The full-length native TyrRS lacks cytokine activity. No structure has been available for any mammalian tRNA synthetase that, in turn, might give insight into why mini-TyrRS and not TyrRS has cytokine activities. Here, the structure of human mini-TyrRS, which contains both the catalytic and the anticodon recognition domain, is reported to a resolution of 1.18 A. The critical Glu-Leu-Arg motif is located on an internal alpha-helix of the catalytic domain, where the guanidino side chain of R is part of a hydrogen-bonding network tethering the anticodon-recognition domain back to the catalytic site. Whereas the catalytic domains of the human and bacterial enzymes superimpose, the spatial disposition of the anticodon recognition domain relative to the catalytic domain is unique in mini-TyrRS relative to the bacterial orthologs. This unique orientation of the anticodon-recognition domain can explain why the fragment mini-TyrRS, and not full-length native TyrRS, is active in cytokine-signaling pathways.

Alternative Splicing↗

PubMed2002

Genetic code ambiguity. Cell viability related to the severity of editing defects in mutant tRNA synthetases.

The rules of the genetic code are established in reactions that aminoacylate tRNAs with specific amino acids. Ambiguity in the code is prevented by editing activities whereby incorrect aminoacylations are cleared by specialized hydrolytic reactions of aminoacyl tRNA synthetases. Whereas editing reactions have long been known, their significance for cell viability is still poorly understood. Here we investigated in vitro and in vivo four different mutations in the center for editing that diminish the proofreading activity of valyl-tRNA synthetase (ValRS). The four mutant enzymes were shown to differ quantitatively in the severity of the defect in their ability to clear mischarged tRNA in vitro. Strikingly, in the presence of excess concentrations of alpha-aminobutyrate, one of the amino acids that is misactivated by ValRS, growth of bacterial strains bearing these mutant alleles is arrested. The concentration of misactivated amino acid required for growth arrest correlates inversely in a rank order with the degree of the editing defect seen in vitro. Thus, cell viability depends directly on the suppression of genetic code ambiguity by these specific editing reactions and is finely tuned to any perturbation of these reactions.

Amino Acid Sequence↗

PubMed2002

Bone marrow-derived stem cells target retinal astrocytes and can promote or inhibit retinal angiogenesis.

Adult bone marrow (BM) contains cells capable of differentiating along hematopoietic (Lin(+)) or non-hematopoietic (Lin(-)) lineages. Lin(-) hematopoietic stem cells (HSCs) have recently been shown to contain a population of endothelial precursor cells (EPCs) capable of forming blood vessels. Here we show that intravitreally injected Lin(-) BM cells selectively target retinal astrocytes, cells that serve as a template for both developmental and injury-associated retinal angiogenesis. When Lin(-) BM cells were injected into neonatal mouse eyes, they extensively and stably incorporated into forming retinal vasculature. When EPC-enriched HSCs were injected into the eyes of neonatal rd/rd mice, whose vasculature ordinarily degenerates with age, they rescued and maintained a normal vasculature. In contrast, normal retinal angiogenesis was inhibited when EPCs expressing a potent angiostatic protein were injected. We have demonstrated that Lin(-) BM cells and astrocytes specifically interact with one another during normal angiogenesis and pathological vascular degeneration in the retina. Selective targeting with Lin(-) HSC may be a useful therapeutic approach for the treatment of many ocular diseases.

Animals↗

PubMed2002

Identification of the tRNA-dihydrouridine synthase family.

5,6-Dihydrouridine (D) is a modified base found abundantly in the D-loops of tRNA from Archaea, Bacteria, and Eukarya. D is thought to be formed post-transcriptionally by the reduction of uridines in tRNA transcripts. Despite its abundance, no enzymes that catalyze D-formation have been identified. Using comparative genomics and computational methods we have identified members of the cluster of orthologous genes, COG0042, as putative dihydrouridine synthase encoding genes. Escherichia coli contains three COG0042 family members (yjbN, yhdG, and yohI). Strains were created where one, two, or all three of the COG0042 genes were deleted. Purified tRNA samples were investigated from the three single and the three double knockout strains, as well as from the triple deletion strain. The results showed that the COG0042 gene family is responsible for tRNA-dihydrouridine synthase activity in E. coli. They also suggest that the COG0042-encoded family members act site-specifically on the tRNA D-loop and contain non-redundant catalytic functions in vivo.

Escherichia coli↗

PubMed2002

Induction of angiogenesis by a fragment of human tyrosyl-tRNA synthetase.

The first step of protein synthesis is catalyzed by aminoacyl-tRNA synthetases. In addition, certain mammalian tRNA synthetases link protein synthesis to cytokine signaling pathways. In particular, human tyrosyl-tRNA synthetase (TyrRS) can be split by proteolysis into two fragments having distinct cytokine activities. One of the TyrRS fragments (mini TyrRS) contains features identical to those in CXC chemokines (like interleukin-8) that also act as angiogenic factors. Here mini TyrRS (but not full-length TyrRS) is shown to stimulate chemotaxis of endothelial cells in vitro and stimulate angiogenesis in each of two in vivo animal models. The angiogenic activity of mini TyrRS can be opposed by anti-angiogenic chemokines like IP-10. Thus, a biological fragment of human tyrosyl-tRNA synthetase links protein synthesis to regulation of angiogenesis.

Allantoin↗

PubMed2002

Plasticity of recognition of the 3'-end of mischarged tRNA by class I aminoacyl-tRNA synthetases.

Certain aminoacyl-tRNA synthetases prevent potential errors in protein synthesis through deacylation of mischarged tRNAs. For example, the close homologs isoleucyl-tRNA synthetase (IleRS) and valyl-tRNA synthetase (ValRS) deacylate Val-tRNA(Ile) and Thr-tRNA(Val), respectively. Here we examined the chemical requirements at the 3'-end of the tRNA for these hydrolysis reactions. Single atom substitutions at the 2'- and 3'-hydroxyls of a variety of mischarged RNAs revealed that, while acylation is at the 2'-OH for both enzymes, IleRS catalyzes deacylation specifically from the 3'-OH and not from the 2'-OH. In contrast, ValRS can deacylate non-cognate amino acids from the 2'-OH. Moreover, for IleRS the specificity for a 3'-O location of the scissile ester bond could be forced to the 2'-position by introduction of a 3'-O-methyl moiety. Cumulatively, these and other results suggest that the editing sites of these class I aminoacyl-tRNA synthetases have a degree of inherent plasticity for substrate recognition. The ability to adapt to subtle differences in mischarged RNAs may be important for the high accuracy of aminoacylation.

Acylation↗

PubMed2002

Blocking site-to-site translocation of a misactivated amino acid by mutation of a class I tRNA synthetase.

The genetic code is established by the aminoacylation reactions of tRNA synthetases. Its accuracy depends on editing reactions that prevent amino acids from being assigned to incorrect codons. A group of class I synthetases share a common insertion that encodes a distinct site for editing that is about 30 A from the active site. Both misactivated aminoacyl adenylates and mischarged amino acids attached to tRNA are translocated to this site, which, in turn, is divided into subsites--one for the adenylate and one for the aminoacyl moiety attached to tRNA. Here we report that a specific mutation in isoleucyl-tRNA synthetase prevents editing by blocking translocation. The mutation alters a widely conserved residue that is believed to tether the amino group of mischarged tRNA to its subsite for editing. These and other data support a model where editing is initiated by translocation of the misacylated amino acid attached to tRNA to create an "editing complex" that facilitates subsequent rounds of editing by translocation of the misactivated adenylate.

Amino Acid Sequence↗

PubMed2002

A fragment of human TrpRS as a potent antagonist of ocular angiogenesis.

Pathological angiogenesis contributes directly to profound loss of vision associated with many diseases of the eye. Recent work suggests that human tyrosyl- and tryptophanyl-tRNA synthetases (TrpRS) link protein synthesis to signal transduction pathways including angiogenesis. In this study, we show that a recombinant form of a COOH-terminal fragment of TrpRS is a potent antagonist of vascular endothelial growth factor-induced angiogenesis in a mouse model and of naturally occurring retinal angiogenesis in the neonatal mouse. The angiostatic activity is dose-dependent in both systems. The recombinant fragment is similar in size to one generated naturally by alternative splicing and can be produced by proteolysis of the full-length protein. In contrast, the full-length protein is inactive as an antagonist of angiogenesis. These results suggest that fragments of TrpRS, as naturally occurring and potentially nonimmunogenic anti-angiogenics, can be used for the treatment of neovascular eye diseases.

Alternative Splicing↗

PubMed2002

A human aminoacyl-tRNA synthetase as a regulator of angiogenesis.

Aminoacyl-tRNA synthetases catalyze the first step of protein synthesis. It was shown recently that human tyrosyl-tRNA synthetase (TyrRS) can be split into two fragments having distinct cytokine activities, thereby linking protein synthesis to cytokine signaling pathways. Tryptophanyl-tRNA synthetase (TrpRS) is a close homologue of TyrRS. A natural fragment, herein designated as mini TrpRS, was shown by others to be produced by alternative splicing. Production of this fragment is reported to be stimulated by IFN-gamma, a cytokine that also stimulates production of angiostatic factors. Mini TrpRS is shown here to be angiostatic in a mammalian cell culture system, the chicken embryo, and two independent angiogenesis assays in the mouse. The full-length enzyme is inactive in the same assays. Thus, protein synthesis may be linked to the regulation of angiogenesis by a natural fragment of TrpRS.

Animals↗