掲載種別：研究論文（学術雑誌）  

DOI： 10.1073/pnas.2606606123

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Microsatellite repeat expansions contribute to the pathogenesis of many neurodegenerative disorders. In spinocerebellar ataxia type 8 (SCA8), abnormal expansion of CTA/CTG repeats in the 3' untranslated region of the ATXN8OS (ATXN8 Opposite Strand) gene has been implicated in disease pathology. Although the occurrence of repeat-associated non-AUG (RAN) translation from the ATXN8 transcript has been reported, whether and how RAN translation occurs from the ATXN8OS transcript has remained unexplored. Here, using a cell-free translation system and cultured cells, we showed that ATXN8OS undergoes robust AUG-independent translation in a repeat length-dependent manner. Mechanistic analyses revealed that translation of the poly L (0) frame initiates at a non-AUG codon located upstream of the repeats. Moreover, using live-cell imaging at a single messenger RNA level, we directly visualized ribosomal -1 frameshifting from the poly L (0) frame to the poly T-poly A (+2) frame during translation elongation. We further showed that ATXN8OS translation was enhanced upon activation of the integrated stress response. Together, these findings establish both the occurrence and the molecular mechanisms of ATXN8OS translation from expanded CTA/CTG repeats and provide insights into the pathogenic processes underlying SCA8.

DOI： 10.1093/nar/gkag730

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Bacteriophages exhibit strict host specificity, primarily determined by adsorption to bacterial surface receptors. However, the molecular basis underlying this specificity remains incompletely understood. Here, we investigate the interaction between outer membrane protein C (OmpC) of Escherichia coli O157 and gp38, the receptor-binding protein located at the tip of the long tail fibers of phage PP01. We determined the crystal structure of the receptor-binding domain (RBD) of gp38PP01 at 2.1 Å resolution. The structure reveals a lattice of poly-glycine type II helices with protruding receptor recognition loops, resembling that of gp38 from Salmonella phage S16. To identify interaction sites, we performed site-specific photo-crosslinking using p-benzoyl-L-phenylalanine (pBPA), followed by liquid chromatography-tandem mass spectrometry. Two critical contacts were identified: Gly208 in loop-D of gp38PP01 crosslinked to Ser225 and Pro226 in extracellular loop-5 of OmpCO157, and Tyr230 in loop-E of gp38PP01 to the Val304-Arg308 region in loop-7 of OmpCO157. A structural model of the gp38PP01-OmpCO157 complex was constructed using distance-constrained prediction and validated by targeted mutagenesis. Our findings demonstrate that PP01 phage specificity is governed by loop-E of gp38PP01 engaging a cleft formed by loops -5 and -7 of OmpCO157. These structural and functional insights enhance our understanding of phage-host recognition and may inform the rational design of engineered bacteriophages with altered host ranges.IMPORTANCEBacteriophages must precisely recognize and bind to specific molecules on the surface of their bacterial hosts to initiate infection, but the details of these interactions are often unclear. In this study, we examined how phage PP01 targets Escherichia coli O157. Using structural analysis of the phage tail fiber and a technique to capture contact points between the phage and a bacterial surface protein, we mapped the molecular basis of host recognition. We also developed a simple test system using a modified phage to identify which parts of the tail fiber are essential for binding. These methods can be broadly applied to other phages to better understand how they select their hosts. This work provides valuable insights and tools that could aid the design of phages with customized host specificity for therapeutic or biotechnological applications.

DOI： 10.1128/mbio.02110-25

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Post-translational modifications are an essential process for proper protein function and localization. In particular, lipid modification plays a crucial role in the spatial regulation of proteins functioning on a lipid membrane surface. While cell-free protein synthesis allows rapid protein production, technical advances in lipidation modification are behind. Here, we developed a cell-free system for the myristoylation and palmitoylation of proteins. Based on our previous study, we improved myristoylation efficiency by trimming a precursor nascent peptide, which undergoes lipidation at the N-terminal glycine. We also found that N-myristoyltransferase (NMT) catalyzes both myristoylation and palmitoylation. The localization of lipidated proteins onto liposomes is further aided by the insertion of polyarginine residues downstream of the NMT recognition site. Finally, we demonstrated that lipidation of VHH antibodies and localization onto liposomes resulted in target-specific binding to cancer cells. This system offers a platform for displaying soluble proteins on lipid membranes, with potential applications in developing liposomes for targeted cell binding.

DOI： 10.1021/acssynbio.5c00155

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Eukaryotic ribosomes exhibit higher mRNA translation fidelity than prokaryotic ribosomes, partly due to eukaryote-specific ribosomal RNA (rRNA) insertions. Among these, expansion segment 27L (ES27L) on the 60S subunit enhances fidelity by anchoring methionine aminopeptidase (MetAP) at the nascent protein exit tunnel, accelerating co-translational N-terminal initiator methionine (iMet) processing. However, the mechanisms by which iMet processing influences translation fidelity remain unknown. Using yeast in vitro translation (IVT) systems, we found that inhibiting co-translational iMet processing does not impact ribosome decoding of ongoing peptide synthesis. Instead, our novel method to monitor iMet processing in vivo revealed that ribosomes purified from strains lacking MetAP ribosomal association (ES27L Δb1-4) or major yeast MetAP (Δmap1) increase iMet retention on ribosomal proteins (RPs). Given the densely packed structure of ribosomes, iMet retention on RPs may distort ribosomal structure and impair its function. Indeed, reconstituted IVT systems containing iMet-retaining ribosome subunits from ES27L Δb1-4 strain, combined with translation factors from wild-type strains, elucidated that iMet retention on the 40S ribosomal subunit causes translation errors. Our study demonstrated the critical role of ES27L in adjusting ribosome association of universally conserved MetAP enzyme to fine-tune iMet processing of key RPs, thereby ensuring the structural integrity and functional accuracy of eukaryotic ribosomes.

DOI： 10.1093/nar/gkaf448

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