Biology subjects

Tagami, S.

Publications and source records attributed to Tagami, S..

9 recordsLinked to original sources

bioRxiv2026

Prebiotic cationic amino acids support formation of an ancient protein fold

The evolutionary basis for the selection of the standard proteinogenic amino acids remains elusive, particularly for the long cationic amino acids, lysine and arginine, which were likely scarce in the prebiotic environment. In contrast, shorter cationic amino acids, such as ornithine (Orn), and 2,4-diaminobutyric acid (Dab), are thought to have been more abundant. Here, we investigated whether these prebiotic cationic amino acids can support protein tertiary structure formation, using computational protein design, biophysical and crystallographic analyses, and molecular dynamics (MD) simulations. We designed sequences for an ancient protein fold, the double-{Psi} {beta}-barrel (DPBB), with ornithine and plausible prebiotic amino acid sets. Although all the designed sequences were unfolded under standard dilute aqueous conditions, one Orn-containing variant folded in highly concentrated conditions. Crystallographic analysis revealed that both this peptide and its Dab-substituted derivative adopted the double-Z {beta}-barrel (DZBB) fold, a likely evolutionary intermediate between extant {beta}-barrel folds. Therefore, Orn and Dab might have supported the foldability of primitive proteins before the incorporation of lysine and arginine into the genetic code.

biochemistry↗

bioRxiv2026

RPL38 controls 60S ribosomal subunit homeostasis to regulate start-codon stringency and frame selection during C9ORF72 RAN translation

Translation initiation depends on stringent start-codon recognition, yet how ribosomal states modulate initiation stringency remains incompletely defined. Repeat-associated non-AUG (RAN) translation is a non-canonical initiation pathway that generates toxic dipeptide repeat proteins in C9ORF72-associated frontotemporal dementia and amyotrophic lateral sclerosis (FTD/ALS). Here, we performed a dual-luciferase reporter-based siRNA screen to identify factors that differentially regulate canonical AUG-initiated and C9ORF72 RAN translation. We identified the 60S ribosomal protein RPL38 as a key determinant of translational output. RPL38 depletion selectively suppressed AUG-dependent translation while relatively preserving near-cognate-initiated RAN translation, resulting in a dose-dependent increase in the RAN-to-AUG translation ratio. Polysome profiling showed a selective reduction in large ribosomal subunit abundance without impaired subunit joining, consistent with defective 60S subunit homeostasis. Substitution of a near-cognate CUG start codon with AUG rendered translation sensitive to RPL38 depletion, supporting a role for ribosomal subunit availability in modulating initiation stringency. Graded RPL38 depletion altered frame usage within C9ORF72 repeat RNA. Consistent with these findings, RPL38 knockdown in the Drosophila eye enhanced GR-frame RAN translation while suppressing canonical AUG-dependent translation. Collectively, these findings identify 60S ribosomal subunit availability as a key determinant of start-codon stringency and frame selection in pathological C9ORF72 RAN translation.

molecular biology↗

bioRxiv2026

Emergence of a potentially ancestral ATP-synthesizing activity under prebiotic amino acid constraints

How amino acid composition shapes enzymatic function remains a central question in molecular evolution. Early proteins were likely composed of a limited set of amino acids, yet the catalytic properties of proteins under such compositional constraints are still poorly understood. Here we show that restricting amino acid repertoires can replace native enzymatic activity with an alternative phosphoryl-transfer reaction that disproportionates ADP into ATP and AMP. Using a reconstructed ancestral nucleoside diphosphate kinase (NDK) variant composed of a restricted amino acid set, we demonstrate this reaction, which is not observed in extant NDKs but is chemically analogous to that catalyzed by adenylate kinase. Although modest in catalytic rate, X-ray crystallography, molecular dynamics, and mutational analyses reveal a distinct active-site organization in which aspartate and arginine residues cooperatively coordinate Mg2+ and support phosphoryl transfer through a noncanonical mechanism. Lysine can substitute for arginine under these constraints while retaining activity. Together, these findings show that restricting amino acid diversity can remodel active sites and promote alternative phosphoryl-transfer reactions, illustrating how limitations in amino acid availability could influence catalytic functions in early enzyme evolution. Significant StatementThe first enzymes arose on early Earth, where only a limited set of amino acids was likely available. Whether such limitations reduce catalytic efficiency or reshape enzyme function is still unclear. We reconstructed a model of an ancestral enzyme using a simplified amino acid set approximating those available on early Earth and found that restricting amino acid diversity replaced its original catalytic activity with an alternative function that produces ATP from two ADP molecules. Structural and mutational analyses indicate that this shift involves a reorganization of the active site. These results provide experimental evidence that limiting amino acid diversity can reshape catalytic function and may have influenced the emergence of catalytic activities during enzyme evolution.

evolutionary biology↗

bioRxiv2023

Missing-link fold reveals the evolutionary pathway between RNA polymerase and ribosomal proteins

Numerous molecular machines are required to drive the central dogma of molecular biology. However, the means by which such numerous proteins emerged in the early evolutionary stage of life remains enigmatic. Many of them possess small {beta}-barrel folds with different topologies, represented by DPBB conserved in DNA and RNA polymerases, and RIFT, OB, and SH3 in ribosomal proteins. Here, we discovered that the previously reconstructed ancient DPBB sequence could also adopt a novel {beta}-barrel fold named DZBB, which shares similarities with RIFT and OB. Indeed, DZBB could be transformed into them through simple engineering experiments. Furthermore, the OB designs could be converted into SH3 by circular-permutation. These results indicate that these {beta}-barrels diversified quickly from a common ancestor at the beginning of the central dogma evolution.

biochemistry↗

bioRxiv2022

Tail-tape-fused virion and non-virion RNA polymerases of a thermophilic virus with an extremely long tail

Thermus thermophilus bacteriophage P23-45 encodes a giant 5,002-residue tail tape measure protein (TMP)1 that defines the length of its extraordinarily long 800 nm tail2,3. We found that the N-terminal portion of P23-45 TMP is an unusual RNA polymerase (RNAP) homologous to cellular and viral two-barrel RNAPs. The TMP-fused virion RNAP transcribes pre-early phage genes, including a gene that encodes another, non-virion RNAP, that transcribes early and some middle phage genes. We determined the crystal structures of both P23-45 RNAPs. The non-virion RNAP has a crab claw-like architecture similar to previously reported two-barrel RNAPs. The virion RNAP adopts a unique flat structure without a clamp, which likely reflects the requirement for its extrusion through the narrow channel in the phage tail for delivery into the cell. Structure and sequence comparisons of the P23-45 RNAPs with other phage and cellular RNAPs suggest that, despite the extensive functional differences, the two P23-45 RNAPs originate from an ancient gene duplication in an ancestral phage. Our findings demonstrate remarkable adaptability of two-barrel RNAPs that can be attained within a single virus species.

molecular biology↗

bioRxiv2022

Cell-free mutant analysis combined with structure prediction of a lasso peptide biosynthetic protein B2

Biochemical and structural analyses of purified proteins are essential for the understanding of their properties. However, many proteins are unstable and difficult to purify, hindering their characterization. The B2 proteins of the lasso peptide biosynthetic pathways are cysteine proteases that cleave precursor peptides during the maturation process. The B2 proteins are poorly soluble and no experimentally-solved structures are available. Here, we performed a rabid semi-comprehensive mutational analysis of the B2 protein from the thermophilic actinobacterium, Thermobifida fusca (TfuB2) using a cell-free transcription/translation system, and compared the results with the structure prediction by AlphaFold2. Analysis of 34 TfuB2 mutants with substitutions of hydrophobic residues confirmed the accuracy of the predicted structure, and revealed a hydrophobic patch on the protein surface, which likely serves as the binding site of the partner protein, TfuB1. Our results suggest that the combination of rapid cell-free mutant analyses with precise structure predictions can greatly accelerate structure-function research of proteins for which no structures are available.

biochemistry↗

bioRxiv2021

Seven amino acid types suffice to reconstruct the core fold of RNA polymerase

AbstractThe extant complex proteins must have evolved from ancient short and simple ancestors. Nevertheless, how such prototype proteins emerged on the primitive earth remains enigmatic. The double-psi beta-barrel (DPBB) is one of the oldest protein folds and conserved in various fundamental enzymes, such as the core domain of RNA polymerase. Here, by reverse engineering a modern DPBB domain, we reconstructed its evolutionary pathway started by "interlacing homo- dimerization" of a half-size peptide, followed by gene duplication and fusion. Furthermore, by simplifying the amino acid repertoire of the peptide, we successfully created the DPBB fold with only seven amino acid types (Ala, Asp, Glu, Gly, Lys, Arg, and Val), which can be coded by only GNN and ARR (R = A or G) codons in the modern translation system. Thus, the DPBB fold could have been materialized by the early translation system and genetic code.

biochemistry↗

bioRxiv2021

Hydrophobic-cationic peptides enhance RNA polymerase ribozyme activity by accretion

Accretion and the resulting increase in local concentration to enhance target stability and function is a widespread mechanism in biology (for example in the liquid-liquid demixing phases and coacervates). It is widely believed that such macromolecular aggregates (formed through ionic and hydrophobic interactions) may have played a role in the origin of life. Here, we report on the behaviour of a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) that forms insoluble aggregates, accrete RNA on their surfaces in a size-dependent manner, and thus enhance the activities of various ribozymes. At low Mg2+ concentrations ([Mg2+]: 25 mM MgCl2), the activity of a small ribozyme (hammerhead ribozyme) was enhanced by P43, while larger ribozymes (RNA polymerase ribozyme (RPR), RNase P, F1* ligase) were inhibited. In contrast, at high [Mg2+] ([≥]200 mM), the RPR activity was enhanced. Another hydrophobic-cationic peptide with a simpler sequence (K2V6: KKVVVVVV) also exhibited similar regulatory effects on the RPR activity. Furthermore, inactive RPR captured on P43 aggregates at low [Mg2+] could be reactivated in a high [Mg2+] buffer. Therefore, in marked contrast to previously studied purely cationic peptides (like K10) that enhance RPR only at low ionic strength, hydrophobic-cationic peptides can reversibly concentrate RNA and enhance the RPR activity even at high ionic strength conditions such as in eutectic ice phases. Such peptides could have aided the emergence of longer and functional RNAs in a fluctuating environment (e.g., dry-wet / freeze-thaw cycles) on the prebiotic earth.

biochemistry↗

bioRxiv2020

Efficient Development of Platform Cell Lines Using CRISPR-Cas9 1 and Transcriptomics Analysis

Antibody-drug conjugates offers many advantages as a drug delivery platform that allows for highly specific targeting of cell types and genes. Ideally, testing the efficacy of these systems requires two cell types to be different only in the gene targeted by the drug, with the rest of the cellular machinery unchanged, in order to minimize other potential differences from obscuring the effects of the drug. In this study, we created multiple variants of U87MG cells with targeted mutation in the TP53 gene using the CRISPR-Cas9 system, and determined that their major transcriptional differences stem from the loss of p53 function. Using the transcriptome data, we predicted which mutant clones would have less divergent phenotypes from the wild type and thereby serve as the best candidates to be used as drug delivery testing platforms. Further in vitro and in vivo assays of cell morphology, proliferation rate and target antigen-mediated uptake supported our predictions. Based on the combined analysis results, we successfully selected the best qualifying mutant clone. This study serves as proof-of-principle of the approach and paves the way for extending to additional cell types and target genes.

bioengineering↗