Kay Hofmann

Intracellular bacteria are largely shielded from humoral immunity but remain vulnerable to cell-autonomous defense mechanisms, many of which depend on the ubiquitin system. To evade autophagy and lysosomal targeting, numerous bacteria have evolved secreted effectors that disrupt or manipulate the host ubiquitin machinery to their advantage. Bacterial deubiquitinases can protect bacterial surfaces or their enclosing vacuoles from ubiquitin coating and interfere with host inflammatory and cell death pathways. Conversely, bacterial ubiquitin ligases may induce the degradation of host proteins, engaging in an evolutionary arms race with host ligases. In addition, ubiquitin-modifying and ubiquitin-crosslinking effectors can disable major host defense signaling pathways. In the present overview, we summarize currently known ubiquitin-directed effectors, their targets, and their roles in bacteria–host interaction.

TIR domains are found in diverse immune signaling proteins and, in several systems, function as NAD+-cleaving enzymes causing or signaling cell death. In plants, TIR domains occur as effector domains in TNL immune receptors, where they are coupled to a central STAND ATPase and a C-terminal repeat domain. Here, we identify TeTIR (TEP1 extended TIR), a previously unrecognized family of TIR-like domains associated with NACHT-type ATPases and C-terminal WD40 or TPR repeat domains. The TeTIR domain of human TEP1 exhibits robust NADase activity comparable to SARM1-TIR, making TEP1 the only other NADase-active TIR-like protein identified in mammals. NADase activity is conserved in TEP1 homologs from diverse eukaryotes. The crystal structure of TEP1 TeTIR reveals an extended TIR-like fold with additional secondary-structure elements and a distinctive RWG-containing loop. Molecular dynamics simulations indicate that the catalytic glutamate contributes to catalysis and the conformational equilibrium of the active-site region, promoting formation of a substrate-accessible state. Structure-guided mutagenesis identifies conserved residues contributing to NAD+ recognition and catalysis, including features distinct from canonical TIR NADases. Expression of isolated TEP1 TeTIR causes NAD+ depletion and cell death in a catalytic-glutamate-dependent manner. TeTIRs thus define a distinct family of animal TIR-like NADases embedded in NACHT-type STAND architectures.

Gram-negative bacteria use a plethora of virulence factors to infect eukaryotic cells. CE-clan protease-related virulence factors were reported to act as deubiquitinases/ubiquitin-like specific proteases. Some have an additional acetyl-transferase activity. The molecular mechanisms underlying this dual activity and the physiological consequences are only marginally understood. Here, we report crystal structures for the Simkania negevensis virulence factor SnCE1 in apo-states and in complex with SUMO1. We confirm SnCE1 acting as an efficient deSUMOylase and discover an intrinsic autoacetyltransferase activity. Acetylation impairs SnCE1 tetramer formation structurally being incompatible with SUMO1 binding. We provide a model for regulation of SnCE1-mediated virulence by lysine acetylation modulating autoproteolytic processing and its subcellular distribution in the host cell. SnCE1 localizes to the endoplasmic reticulum in human cells and increases fragmentation of mitochondria. Our data provide mechanistic insights into how lysine acetylation of virulence factors is used to reprogram virulence adjusting it to the host cells’ metabolic state.

Ubiquitin signalling covers a vast realm of protein modifications, yet may still be underestimated due to non-proteinaceous substrates, such as sugars, lipids, and nucleotides¹ . The breadth of ubiquitinated non-protein substrates, their abundance, and cellular roles are currently unclear, since current ubiquitinomic and proteomic techniques are blind to non-proteinaceous modifications. We report Non-Protein Ub-clipping (NoPro-clipping) as a mass-spectrometry-based technique that combines ubiquitin clippases with sortase labelling. Targeted and untargeted workflows unveil a vast new canvas of ubiquitin modifications in mammalian cells, and in mouse and human tissues. We find ubiquitinated glycogen in any glycogen-containing tissue in mice, with highest abundance in liver and skeletal muscle. Ubiquitination can deliver glycogen to lysosomes, and leads to reduced glycogen levels. Glycogen ubiquitination is modulated in glycogen storage diseases and regulated by the Met1-polyubiquitin machinery. Strikingly, glycogen depletion in the liver during fasting coincides with elevated glycogen ubiquitination, suggesting that ubiquitin is a previously unknown component of physiological glycogen catabolism. We also reveal ubiquitination of endogenous glycerol and spermine in cells and tissues. NoPro-clipping hence unveils unexpected endogenous non-proteinaceous targets of ubiquitination, broadening the role of ubiquitin from a protein modifier to a general modifier of biomolecules.

SUMMARY: Modification by ubiquitination governs the half-lives of thousands of proteins that are fated for elimination by either the proteasome or autophagy pathways, depending on the intricate architectures of ubiquitin modification. This system mediates quality control for individual proteins, protein complexes, and organelles, as well as myriad purely regulatory functions. Here we provide a comprehensive survey of the ubiquitin-proteasome system (UPS), the scope of which is at present poorly defined. The UPS, with the inclusion of pathways involving ubiquitin-like modifiers, comprises in our estimate over 1400 distinct proteins in humans, a vast set of activities whose collective impact on the biology of the cell is pervasive. The UPS is an integral component of the proteostasis network (PN), the remainder of which we have also surveyed in recent studies. With the addition of molecular chaperones, proteins from autophagy-lysosome pathway, and related activities, the PN includes in total over 3100 components by our estimates. Comprehensive and systematic definition of these pathways should support a range of ongoing investigations in the areas of genomics, proteomics, biochemistry, cell biology, and disease research.

Oomycetes of the genus Phytophthora are devastating plant pathogens that affect many commercially important plants. Considerable efforts have been made to investigate the transcriptional response of individual plant species to Phytophthora infection, often showing a concerted upregulation of pathogen-response (PR) gene families, which are also induced upon infection by fungi and other biotic and even non-biotic stressors. For many of the upregulated factors, a biological role has not been firmly established. Here, we performed a large-scale exploratory bioinformatical function prediction of protein-coding genes, which are consistently upregulated by Phytophthora infection in multiple hosts. By integrating four very different transcriptomics datasets derived from three different plants ( Arabidopsis, soybean, and cocoa), a core set of upregulated sequence clusters was derived, which represents a multi-gene/multispecies response to Phytophthora infections. We annotated 309 upregulated multi-gene clusters and subjected the encoded proteins to bioinformatical analysis. Besides the expected PR genes, several novel gene families without known links to biotic stress were found to be strongly induced in all tested datasets. Among the most prominent response genes are two families of putatively secreted peptides and a family of predicted mitochondrial complex IV-associated proteins. Interestingly, the latter sequences are related to the mammalian NDUFA4 family, which also contains members with constitutive and pathogen-induced expression. This recurrent functional diversification points toward an important role of complex IV regulation within the biotic defense response in multiple kingdoms. [ ] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .

Deubiquitinases (DUBs) remove ubiquitin modifications from proteins in a substrate- or linkage-selective manner and regulate numerous cell-biological processes, including endocytosis. By performing homology-agnostic bioinformatical screens for undescribed deubiquitinase classes, we identified the plant-specific DUC (deubiquitinase with C2) family, whose members are highly selective for cleaving K63-linked ubiquitin chains. The crystal structure of the Arabidopsis member AtDUC1 reveals that DUC enzymes display a papain-like fold with a characteristic DUB-like active site, despite the apparent absence of sequence similarity to other deubiquitinase families. The K63 linkage specificity is attributed to the recognition of both distal and proximal ubiquitin units via highly conserved contact residues. Arabidopsis DUC members localize to the plasma membrane by virtue of their lipid-binding C2-like domains. In protoplast experiments, all three Arabidopsis DUC enzymes demonstrate the capacity to stabilize an endocytotic model cargo at the plasma membrane, a process that requires both the C2-mediated membrane association and catalytic DUB activity.

In mammals, ubiquitylation is orchestrated by the canonical ubiquitin-activating E1 enzyme UBA1 and the orthogonal E1 UBA6. Growing evidence underscores the essentiality of both E1s, which differentiate between 29 active ubiquitin-conjugating enzymes (E2s). The mechanisms governing this distinction have remained unclear. Here we establish a framework for ubiquitin E1–E2 specificity. Focusing on UBA6-controlled ubiquitylation cascades, we reveal that BIRC6, a UBA6-exclusive E2, gains priority over all other UBA6-competent E2s, underpinning the functional importance of defined UBA6–BIRC6 ubiquitylation events in regulating cell death, embryogenesis and autophagy. By capturing BIRC6 receiving ubiquitin from UBA6 in different states, we observe BIRC6 engaging with the UBA6 ubiquitin fold domain, driving an exceptionally high-affinity interaction that is modulated by the UBA6 Cys-Cap loop. Using this interaction as a template, we demonstrate how to confer activity between E2s and their noncognate E1, providing a tool to delineate E1–E2-dependent pathways. Lastly, we explain how BIRC6 priority does not lead to inhibition of UBA6, through a bespoke thioester switch mechanism that disengages BIRC6 upon receiving ubiquitin. Our findings propose a concept of hierarchy of E2 activity with cognate E1s, which may explain how ubiquitin E1s can each function with over a dozen E2s and orchestrate E2-specific cellular functions.

UBQLN2 is a member of the UBL-UBA domain protein family that functions as extrinsic substrate receptors for the 26S proteasome. UBQLN2 has been shown to undergo phase separation in vitro . In cells, UBQLN2 forms condensates that may be of importance for tuning protein degradation via the ubiquitin-proteasome system and potentially of relevance for UBQLN2 -linked amyotrophic lateral sclerosis (ALS). Here we show that UBQLN2 is ubiquitylated on lysine residues in the N-terminal UBL domain. The C-terminal region of UBQLN2 is lysine-depleted, and we show that introducing lysine residues in this region leads to its E6AP-dependent degradation. The UBL domain critically stabilizes UBQLN2 and protects it from proteasomal degradation. Fusion of ubiquitin to the UBQLN2 N-terminus stabilizes UBQLN2 and increases its propensity for locating in puncta, indicating that ubiquitylation of the UBQLN2 UBL domain regulates abundance and localization.