Biology subjects

Ikenoue, T.

Publications and source records attributed to Ikenoue, T..

4 recordsLinked to original sources

bioRxiv2026

Macrocyclic Peptide Tools for Huntingtin-bound HAP40

Huntingtons disease is a fatal neurodegenerative disorder caused by expansion of a cytosine-adenosine-guanine repeat in exon 1 of the Huntingtin (HTT) gene, resulting in a polyglutamine-expanded HTT protein. Although the genetic cause of Huntingtons is well defined, the molecular functions of HTT and the mechanisms linking polyglutamine expansion to neurodegeneration remain incompletely understood. Huntingtin Associated Protein 40 kDa (HAP40) is a key HTT interaction partner that forms a stable complex with HTT and is increasingly recognized as an important player in the HTT structure-function paradigm. However, investigation of HAP40 biology has been limited by a lack of tools capable of directly targeting endogenous protein. Here, we report the discovery and characterization of a panel of nanomolar-affinity macrocyclic peptides targeting HAP40 identified using Random nonstandard Peptide Integrated discovery platform. We characterized macrocycle binding in vitro using surface plasmon resonance, fluorescence polarization, and hydrogen-deuterium exchange mass spectrometry, revealing selective, high-affinity engagement of distinct epitopes on HAP40. We further demonstrate that these macrocycles engage endogenous HAP40 in cellular lysates, enable selective isolation of HAP40-containing protein complexes using macrocycle precipitations and insights into interaction partners of distinct HTT and HAP40 proteoforms. Together, these macrocycles establish a new toolkit for investigating HTT-HAP40 biology and provide a framework for dissecting HAP40-specific functions relevant to Huntingtons disease pathogenesis.

molecular biology↗

bioRxiv2026

Fine-Tuning α-Synuclein Phase Separation through Sequence-Optimized Peptide Modulators

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins underlies the formation of biomolecular condensates that regulate diverse cellular processes, while its dysregulation contributes to protein aggregation and disease. Despite its importance, molecularly defined and target-specific strategies to control LLPS remain limited. Here, we present a systematic framework for designing de novo peptides that induce and modulate LLPS of -synuclein. By integrating deep mutational scanning with peptide screening, we identified sequence features that govern condensate formation and enabled the creation of optimized peptides with high efficiency and specificity. Biophysical analyses revealed that LLPS efficiency is dictated by the interplay of solubility, multivalency, and cooperative interactions, resulting in a distinctive bell-shaped phase diagram. Thermodynamic measurements and imaging-based analyses further demonstrated that condensate stability and material properties can be rationally tuned through peptide optimization. Together, these findings establish generalizable design principles for engineering LLPS modulators in biologically and pathologically relevant protein systems.

biophysics↗

bioRxiv2025

High-Affinity, Structure-Validated and Selective Macrocyclic Peptide Tools for Chemical Biology Studies of Huntingtin

Huntingtons disease (HD) is a fatal neurodegenerative disorder caused by a CAG repeat expansion in the Huntingtin (HTT) gene, with no disease-modifying therapies currently available. The precise molecular function of the HTT protein is unclear, and the lack of selective chemical tools has limited functional studies. We have identified and characterized macrocyclic peptide binders targeting HTT. These binders exhibit low-nanomolar affinity in vitro and engage distinct HTT and HTT-HAP40 interfaces, as revealed by hydrogen-deuterium exchange mass spectrometry and cryo-electron microscopy. Chemoproteomics confirmed selective binding in cell extracts from wildtype but not HTT-null cell lines. HAP40 consistently and stoichiometrically co-purified with HTT across cell lines, including with HTT variants containing different CAG repeat lengths, highlighting the broad presence of the HTT-HAP40 complex. Significance StatementHuntingtin (HTT) is a large, essential protein with conserved roles in development, intracellular trafficking, and protein homeostasis, yet its precise molecular functions remain incompletely defined. Here, we report the first high-affinity, selective, and structurally validated macrocyclic peptide ligands for HTT. These chemical tools bind HTT and its complex with HAP40 across polyglutamine repeat lengths, enabling direct interrogation of HTT structure and function in health and disease contexts. By overcoming longstanding barriers to studying HTT at the molecular level, these ligands open new avenues for discovery in neurobiology, cell biology, and offer opportunities for therapeutic development. This study delivers urgently needed tools to both the Huntingtons disease field and the broader scientific community seeking to understand this elusive and biologically fundamental protein.

biochemistry↗

bioRxiv2021

Potent macrocycle inhibitors of the human SAGA deubiquitinating module

The SAGA transcriptional coactivator contains a four-protein subcomplex called the DUB module that removes ubiquitin from histone H2B-K120. The human DUB module contains the catalytic subunit, USP22, which is overexpressed in a number of cancers that are resistant to available therapies. We screened a massive combinatorial library of cyclic peptides and identified potent inhibitors of USP22. The top hit was highly specific for USP22 as compared to a panel of 44 other human DUBs. Cells treated with peptide had increased levels of H2B monoubiquitination, demonstrating the ability of the cyclic peptides to enter human cells and inhibit H2B deubiquitination. These macrocycle inhibitors are, to our knowledge, the first reported inhibitors of USP22/SAGA DUB module and show promise for development.

biochemistry↗