Fumiyo Ikeda

The ubiquitin ligase HOIL-1 regulates the formation of Met1-linked (linear) ubiquitin chains through its coordination with the E3 ligase HOIP within the Linear Ubiquitin Chain Assembly Complex (LUBAC). While HOIP-dependent Met1-linked ubiquitination is well established in inflammation and immunity, the physiological importance of its quantitative control remains unclear. Here, we show that cells expressing catalytically inactive HOIL-1 accumulate increased α-synuclein, tau, and amyloid-β aggregates. This is associated with defective late-stage autophagic flux, characterized by impaired delivery of p62-positive aggregates to lysosomes. In parallel, p62 bodies undergo a biophysical transition from dynamic, liquid-like condensates to rigid, solid-like structures. Elevation of Met1-linked ubiquitin chains, either through HOIL-1 inactivation or depletion of the Met1-specific deubiquitinase OTULIN, phenocopies these defects. Together, our findings identify HOIL-1 as a key regulator of aggregate clearance and proteostasis through quantitative control of Met1-linked ubiquitination.

The ubiquitin ligase HOIL-1 has a unique role in controlling quantity of Met1-linked/ linear ubiquitin chains in cells by coordinating action with the ubiquitin ligase HOIP. Both ligases are components of the Linear UBiquitin chain Assembly Complex (LUBAC), the only known ligase complex that is able to generate Met1-linked ubiquitin chains. Although importance of Met1-linked ubiquitin chains in inflammation and immunity is well established, physiological relevance of quantity of these chains remain unknown. Here, we demonstrate that cells expressing catalytically inactive HOIL-1 exhibited significantly higher numbers of α-Synuclein, tau, and amyloid beta aggregates. This phenotype is associated with a disruption in late-stage autophagic flux, wherein p62-positive aggregates fail to colocalize with lysosomal markers, leading to impaired clearance. Additionally, a biophysical transition in aggregate properties was observed in vitro , with mutant cells forming more rigid solid-like inclusions, shifting from dynamic and liquid-like condensates. Elevated Met1-linked ubiquitin chains, either through HOIL-1 catalytic inactivation or knockdown of the Met1-linked chain-specific deubiquitinase OTULIN, phenocopied the defects in aggregate clearance. These findings reveal a critical role of HOIL-1 catalytic activity in modulating aggregate clearance through autophagy and maintaining the quantity of Met1-ubiquitin chains, highlighting HOIL-1 as a key factor in proteostasis in neurodegenerative diseases. Highlights: HOIL-1 catalytic activity prevents neurodegenerative protein aggregate accumulation Inactive HOIL-1 impairs late-stage autophagic clearance of protein aggregates Loss of HOIL-1 shifts aggregates to rigid, solid-like states Excess Met1-ubiquitin chains drive aggregate solidification

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.

Cells use a specialized process called mitophagy to degrade mitochondria, and the conditions that induce mitophagy can endanger mitochondrial DNA (mtDNA). Here, we report a novel mechanism by which cells undergoing hypoxia response (HR)-induced mitophagy protect their mtDNA from destruction by segregating it into large mitochondria (Mito-L). Using time-lapse imaging, we found that Mito-L are formed when mitochondria undergo tubular-to-spherical transitions and then fuse together. Under HR-activating conditions, we found the mitophagy process itself is essential for Mito-L formation. When we fragmented mitochondria prior to inducing the HR, we observed fewer Mito-L and more mtDNA degradation. Thus, mammalian cells protect mtDNA against excessive mitophagy induced by HR, thereby preventing the loss of the genetic information. Hypoxia response-induced mitophagy triggers cells to shelter mitochondrial DNA (70 characters)

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.

Ubiquitin regulates various cellular functions by posttranslationally modifying substrates with diverse ubiquitin codes. Recent discoveries of new ubiquitin chain topologies, types of bonds, and non-protein substrates have substantially expanded the complexity of the ubiquitin code. Here, we describe the ubiquitin system covering the basic principles and recent discoveries related to mechanisms, technologies, and biological importance.

Ubiquitin regulates a wide variety of biological functions by modifying diverse substrates, via many different conjugation types. Classically, the C-terminus of ubiquitin conjugates to protein substrates via an isopeptide or peptide bond. Recent studies revealed that ubiquitin can form an atypical oxyester bond, which can target protein and even nonproteinaceous substrates, including sugars and lipids. How nonprotein ubiquitination affects substrate and cellular functions is incompletely understood. This review covers recent discoveries in ubiquitination and its potential impacts on biology.

Supplementary Figures (Fig.S1-S4) and Supplementary Materials and Methods. Supplementary Figure S1. Loss of Hace1 results in increased lung tumorigenesis independent of RIPK3. Supplementary Figure S2. Analysis of RAC, NRF2 and 4HNE expression. Supplementary Figure S3. Analysis of tumor microenvironment. Supplementary Figure S4. Treatment of KRasG12D;Hace1-/- mice with a ROS scavenger diminishes lung tumorigenesis and Hace1-deficient cells are sensitive to exogenous induction of oxidative stress.

HACE1 mutational landscape in human cancer.

HACE1 mutational landscape in human cancer.