Harunori Yoshikawa

Autophagy, a highly conserved cellular degradation process, plays essential roles in various physiological processes including aging. Though autophagy is required for lifespan extension in multiple longevity paradigms, the tissue‐specific roles of autophagy‐related genes ( atgs ) in longevity remain incompletely understood. Here, we investigate the tissue‐specific requirements of atgs to promote longevity conferred by germline ablation (called gonadal longevity) using C. elegans . Remarkably, we discovered that neuronal or intestinal knockdown of atg‐18 , but not other atgs , specifically abolished gonadal longevity, although knockdown of all tested atgs effectively inhibited autophagic activity in these targeted tissues, implying the presence of an autophagy‐independent function of ATG‐18 in gonadal longevity. We demonstrated that germline deficiency triggered significant upregulation of ATG‐18 in neurons and the intestine. From the proteomics analysis and subsequent screening, we found ATG‐18 interacts with PCK‐2, a phosphoenolpyruvate carboxykinase. PCK‐2 is upregulated within the intestine of germline‐deficient animals, but this depends on the non‐autophagic function of ATG‐18. Consistently, we showed that PCK‐2 overexpression mediated longevity required ATG‐18 but not its potential interacting partner to regulate autophagy, ATG‐2. These findings reveal a previously unrecognized autophagy‐independent role for ATG‐18 in regulating lifespan in response to germline signals, expanding our understanding of how this evolutionarily conserved protein coordinates organism‐wide responses to promote longevity.

Transverse tubules (T-tubules) are invaginations of the muscle plasma membrane that facilitate rapid transmission of action potentials, ensuring synchronized muscle contraction. Despite their essential role in muscle physiology, the mechanisms underlying T-tubule formation remain elusive. Here, we identify LUBEL/RNF31, a ubiquitin E3 ligase responsible for linear (M1-linked) ubiquitination, as a key regulator of T-tubule biogenesis in Drosophila . Loss of LUBEL leads to Amphiphysin (Amph)–positive membrane sheets instead of tubular networks. The ubiquitin ligase activity of LUBEL and direct interaction with Amph, a BAR domain protein involved in membrane tubulation, are crucial for proper T-tubule morphology. LUBEL and M1-linked ubiquitin chains assemble into puncta on membranes through multivalent interactions, facilitating Amph-mediated tubulation. Notably, the Amph-LUBEL/RNF31 interaction is evolutionarily conserved across species, underscoring a fundamental role for linear ubiquitination in membrane remodeling. Our findings uncover an unexpected function of linear ubiquitination in membrane deformation driven by BAR proteins.

Molecular targeted therapies targeting KRAS signaling have significantly improved patient outcomes, but they have not achieved sufficient therapeutic efficacy in colorectal cancer (CRC). Here, we demonstrate that a subset of KRAS-mutant CRC cells transitions to a cellular state characterized by enhanced ribosome biogenesis upon KRAS signaling inhibition. The mitogen-activated protein kinase kinase inhibitor, trametinib, and AMG510 induce a cellular state characterized by a gene expression profile highly enriched for ribosome biogenesis. We find that they are vulnerable to the inhibition of RNA polymerase I, and they exhibit synergistic anti-tumor effects with trametinib in an autochthonous mouse model of intestinal tumors and human patient-derived organoids (PDOs). These observations demonstrate that high ribosome biogenesis induced by KRAS inhibition is indispensable to maintain this cellular state and is a potential therapeutic target. Overall, this study reveals novel mechanisms of drug tolerance to KRAS inhibition, thereby facilitating the development of new therapeutic strategies.

T-tubules are specialized invaginations of the plasma membrane essential for muscle contraction. While their physiological importance is well established, the mechanisms underlying T-tubule formation remain elusive. Here, we identify LUBEL/RNF31, a ubiquitin E3 ligase responsible for linear (M1-linked) ubiquitination, as a key regulator of T-tubule biogenesis through proximity proteomics and RNAi screening in Drosophila . Loss of LUBEL leads to the formation of Amphiphysin (Amph)-positive membrane sheets instead of tubular networks in muscle cells. Mechanistically, the ubiquitin ligase activity of LUBEL, and direct interaction with Amph, a BAR domain protein involved in membrane tubule extension, are crucial for proper T-tubule morphology. LUBEL and M1-linked ubiquitin chains assemble into condensates on membranes, facilitating Amph-mediated membrane tubulation. Notably, the Amph-LUBEL/RNF31 interaction is evolutionarily conserved across a broad range of species, underscoring a fundamental role for linear ubiquitination in membrane remodeling. Our findings uncover a previously unrecognized role for linear ubiquitination in membrane deformation driven by BAR domain proteins.

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling, thereby avoiding many of the ethical issues arising from the use of embryo-derived cells. However, despite clear similarities between the two cell types, it is likely they are not identical. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors. We find that while hESCs and hiPSCs express a near identical set of proteins, they show consistent quantitative differences in the expression levels of a wide subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs. The proteomic data show hiPSCs have significantly increased abundance of vital cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins, which correlated with phenotypic differences between hiPSCs and hESCs. Thus, higher levels of glutamine transporters correlated with increased glutamine uptake, while higher levels of proteins involved in lipid synthesis correlated with increased lipid droplet formation. Some of the biggest metabolic changes were seen in proteins involved in mitochondrial metabolism, with corresponding enhanced mitochondrial potential, shown experimentally using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including ECM components and growth factors, some with known tumorigenic properties, as well as proteins involved in the inhibition of the immune system. Our data indicate that reprogramming of human fibroblasts to iPSCs effectively restores protein expression in cell nuclei to a state comparable to hESCs, but does not similarly restore the profile of cytoplasmic and mitochondrial proteins, with consequences for cell phenotypes affecting growth and metabolism. The data improve understanding of the molecular differences between induced and embryonic stem cells, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.

A method was developed that enables the magnesium chloride (MgCl₂)‐activated S‐acetamidomethyl cysteine sulfoxide (Cys(Acm)(O)) to induce the sp²(C−H) sulfenylation of the indole of Trp residues. The reaction operates under mild acidic conditions using acetic acid or an ionic liquid to give the Trp‐sulfenylated products. Other than Trp, all other proteinogenic amino acids are unreactive to the sulfenylation conditions. We demonstrated the successful application of this reaction to various peptides, including lysozyme. Furthermore, we achieved the Trp‐modification of a monoclonal antibody by a MgCl₂‐mediated reaction in an acidic ionic liquid. The resulting antibody exhibited antibody performance comparable to the parent protein. The amide moiety in the Acm group contributes to the difference in chemical behaviors between S‐Acm and S‐p‐methoxybenzyl (MBzl)‐protected cysteine sulfoxides. This is because the S‐Acm sulfoxide is converted to S‐chlorocysteine responsible for Trp‐sulfenylation under less acidic conditions than those required for the reaction of S‐MBzl sulfoxide. Based on this rationale, we prepared a linker possessing S‐Acm and S‐MBzl oxide moieties and subjected the linker to heterodimerization of DNA‐binding MYC and MAX peptides containing a Trp handle. The one‐pot/stepwise Cys‐Trp conjugation between the linker and DNA‐binding peptides allowed the generation of a heterodimeric MYC/MAX DNA binder.

A method was developed that enables the magnesium chloride (MgCl2)-activated S-acetamidomethyl cysteine sulfoxide (Cys(Acm)(O)) to induce the sp2(C−H) sulfenylation of the indole ring of Trp residues. The reaction operates under mild acidic conditions using acetic acid or an ionic liquid in a highly Trp-selective manner to give the Trp-sulfenylated products. Other than Trp, all other proteinogenic amino acids are unreactive to the sulfenylation conditions. We demonstrated the successful application of this reaction to a variety of peptides, including lysozyme protein. Furthermore, we achieved the Trp-modification of a monoclonal antibody (trastuzumab®) by a MgCl2-mediated reaction in an acidic ionic liquid. The resulting modified antibody exhibited antibody performance comparable to the parent protein. The presence of an amide moiety in the Acm group contributes to the difference in chemical behavior between S-Acm and S-p-methoxybenzyl (MBzl)-protected cysteine sulfoxide. This is because the S-Acm sulfoxide is converted to S-chlorocysteine responsible for Trp-sulfenylation under less acidic conditions than those required for the reaction of S-MBzl sulfoxide. Based on this rationale, we prepared a linker possessing S-Acm and S-MBzl oxide moieties and subjected the linker to hetero dimerization of DNA-binding Myc and Max peptides containing a Trp handle. The one-pot/stepwise Cys-Trp conjugation between the linker and DNA-binding peptides allowed the generation of a heterodimeric Myc/Max DNA binder.

UPF1-like helicases play roles in telomeric heterochromatin formation and X-chromosome inactivation, and also in monogenic variant surface glycoprotein (VSG) expression via VSG exclusion-factor-2 (VEX2), a UPF1-related protein in the African trypanosome. We show that VEX2 associates with chromatin specifically at the single active VSG expression site on chromosome 6, forming an allele-selective connection, via VEX1, to the trans-splicing locus on chromosome 9, physically bridging two chromosomes and the VSG transcription and splicing compartments. We further show that the VEX-complex is multimeric and self-regulates turnover to tightly control its abundance. Using single cell transcriptomics following VEX2-depletion, we observed simultaneous derepression of many other telomeric VSGs and multi-allelic VSG expression in individual cells. Thus, an allele-selective, inter-chromosomal, and self-limiting VEX1-2 bridge supports monogenic VSG expression and multi-allelic VSG exclusion.