Biology subjects

Hofmann, K.

Publications and source records attributed to Hofmann, K..

17 recordsLinked to original sources

bioRxiv2026

TeTIR domains define a new class of animal TIR-like NADase effectors in STAND proteins

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.

biochemistry↗

bioRxiv2026

Molecular basis for C-degron recognition by the SCFDas1 ubiquitin ligase

Selective protein degradation by the ubiquitin-proteasome system frequently involves substrate recognition via short linear motifs known as degradation signals or degrons. Whereas degrons located at protein N-termini (N-degrons) have been extensively studied, our understanding of C-terminal degrons (C-degrons) is comparatively limited. Previously, we showed that the yeast SCF ubiquitin ligase and one of its substrate receptor subunits, the F-box protein Das1, target a broad range of C-degrons and implicated SCFDas1 in orphan quality control. Here, we sought to determine how Das1 recognizes its substrates. By combining in vivo competition assays with structural modeling and mutational analysis, we demonstrate that distinct C-degrons compete for a common site on Das1, indicating a shared mode of recognition. We identify a positively charged pocket within the Das1 leucine-rich repeat domain as the C-degron binding site. Three basic residues at the base of this pocket are essential for Das1 function, likely mediating electrostatic interactions with the C-terminal carboxyl group of the degron, while additional residues contribute to substrate specificity. Comparative analysis reveals that this pocket and its function are conserved across the Saccharomycetaceae family, supporting a conserved role for Das1 in protein quality control.

biochemistry↗

bioRxiv2026

Survey of the human proteostasis network: the ubiquitin-proteasome system

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 1430 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 3150 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.

bioinformatics↗

bioRxiv2025

Human liver organoids uncover ductular reaction in Wolman disease and inform gene therapy design

Wolman disease (WD), the severe infantile form of lysosomal acid lipase deficiency, is a rare metabolic disorder caused by inactivating mutations in the LIPA gene. Although WD is characterized by profound hepatic dysfunction, experimental human systems capable of modelling multicellular liver pathology and supporting therapeutic testing remain limited. Here, we generated an isogenic human model of WD by introducing LIPA loss-of-function mutations into induced pluripotent stem cells and differentiating them into multicellular human liver organoids (HLO). LIPA-deficient HLO preserved hepatic lineage specification while recapitulating key biochemical and cellular features of WD, including loss of LIPA activity, lysosomal expansion, lipid accumulation, and activation of inflammatory and fibrogenic programs. Single-cell RNA sequencing resolved cell-type-specific disease states across hepatocyte-, stromal-, and biliary-like populations, revealing the emergence of a reactive biliary program consistent with ductular reaction, a complex tissue response associated with chronic liver injury. Importantly, this reactive biliary phenotype was supported by targeted gene-expression analysis in WD liver organoids and independently validated in liver tissue from mouse models and WD patients. Isolated LIPA-deficient cholangiocyte organoids failed to reproduce the DR-associated program, indicating that this response depends on multicellular interactions within the hepatic microenvironment rather than on biliary cell-autonomous dysfunction alone. Consistently, hepatocyte-directed AAV-mediated restoration of LIPA expression attenuated metabolic stress, inflammatory and fibrogenic programs, and suppressed ductular reaction both in organoids and in vivo. Together, these findings establish multicellular human liver organoids as a physiologically relevant platform for modelling emergent tissue-level responses in WD and for evaluating therapeutic rescue strategies in a human context.

pathology↗

bioRxiv2025

DUCs are C2 domain containing plant-specific deubiquitinases stabilizing endocytic cargo at the plasma membrane

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.

biochemistry↗

bioRxiv2025

The importance of UBQLN2 ubiquitylation for its turnover and localization

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.

biochemistry↗

bioRxiv2024

Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia-like bacterium Simkania negevensis

In the arms race between a pathogen and the host, many bacteria have acquired a sizable armory to counteract or change the defense mechanisms of the host cell, including the eukaryotic ubiquitin system. Simkania negevensis, an obligate intracellular Chlamydia-like bacterium, is one such example. S. negevensis has a biphasic developmental cycle, similar to other members of the Chlamydiales order. The bacterium develops inside a tubular membranous compartment called Simkania-containing vacuole, which is in close contact with the host endoplasmic reticulum (ER) and mitochondria. It can infect a wide range of hosts, with a long infection cycle lasting up to 15 days, and is associated with respiratory tract diseases. Recently, S. negevensis has been discovered to possess an unusually large number of deubiquitinating enzymes, but not much is known about the variety of other ubiquitin-modifying enzymes in this bacterium. Our study provides an initial description of the activity of a so far uncharacterized S. negevensis ubiquitin E3 RING-ligase (SNE_A12920 or SneRING). We report that SneRING primarily generates K63- and K-11 linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 ubiquitin ligases among the ones we tested. Bacteria express SneRING upon infection of various human cell lines, as well as amoeba. In addition, we demonstrate that a portion of the expressed SneRING co-localizes with mitochondria and ER. Mass spectrometry studies of the SneRING interactome show enrichment in mitochondrial and ER proteins containing a prohibitin domain and involved in organelle morphology, respiration, and stress response. Our work offers the first insights into the function of an S. negevensis RING ligase, an enzyme potentially involved in organelle remodeling to accommodate the unique lifestyle of this intracellular bacterium.

biochemistry↗

bioRxiv2024

A family of bacterial Josephin-like deubiquitinases with an unusual cleavage mode

Many intracellular bacteria secrete deubiquitinase (DUB) effectors into eukaryotic host cells to keep the bacterial surface or the enclosing vesicle membrane free of ubiquitin marks. Here, we describe a new family of bacterial DUBs that is structurally related to eukaryotic Josephins, but contains members that catalyze a unique destructive substrate deubiquitination. These ubiquitin C-terminal clippases (UCCs) cleave ubiquitin before the C-terminal diGly motif, thereby truncating the modifier and leaving a remnant on the substrate. By comparing the crystal structures of substrate-bound clippases and a closely related conventional DUB, we identified the factors causing the shift and found them conserved in other clippases, including one highly specific for M1-linked ubiquitin chains. This new enzyme class has great potential as tools to study the ubiquitin system, in particular aspects involving branched chains.

biochemistry↗

bioRxiv2024

PEX39 facilitates the peroxisomal import of PTS2 proteins

Peroxisomes are metabolic organelles essential for human health. Defects in peroxisomal biogenesis proteins (peroxins/PEXs) cause devastating disease. PEX7 binds newly synthesized proteins containing a type 2 peroxisomal targeting signal (PTS2) to enable their import from the cytosol into peroxisomes, although many aspects of this import pathway remain enigmatic. Utilizing in vitro assays, yeast, and human cells, we show that PEX39, a previously uncharacterized protein, is a cytosolic peroxin that facilitates PTS2-protein import by binding PEX7 and stabilizing its interaction with PTS2 cargo. PEX39 and PEX13, a peroxisomal membrane translocon protein, both possess a KPWE motif necessary for PEX7 binding. Sequential binding of PEX7 to this motif in PEX39 and PEX13 provides a novel paradigm for how PTS2 cargo engage the translocation machinery. Collectively, our work uncovers an ancient and functionally important relationship among PEX39, PEX7, and PEX13, offering insights that will advance our understanding of peroxisomal biogenesis and disease.

cell biology↗

bioRxiv2024

ERLIN1/2 scaffolds bridge TMUB1 and RNF170 and restrict cholesterol esterification to regulate the secretory pathway

Complexes of ERLIN1 and ERLIN2 form large ring-like cup-shaped structures on the endoplasmic reticulum (ER) membrane and serve as platforms to bind cholesterol and E3-ubiquitin ligases, potentially defining functional nanodomains. Here, we show that ERLIN scaffolds mediate the interaction between the full-length isoform of TMUB1 and RNF170. We identify a luminal N-terminal conserved region in TMUB1 and RNF170 required for this interaction. Three-dimensional modelling shows that this conserved motif binds the SPFH domain of two adjacent ERLIN subunits at different interfaces. Protein variants that preclude these interactions have been previously linked to hereditary spastic paraplegia (HSP). By using omics approaches in combination with phenotypic characterisation of cells lacking both ERLINs, we demonstrate a role for ERLIN scaffolds in maintaining cholesterol levels in the ER by favouring transport to the Golgi over esterification, thereby regulating Golgi morphology and the secretory pathway.

cell biology↗

bioRxiv2023

The structural basis for deubiquitination by the fingerless USP-type effector TssM

Intracellular bacteria are threatened by ubiquitin-mediated autophagy, whenever the bacterial surface or enclosing membrane structures become targets of host ubiquitin ligases. As a countermeasure, many intracellular pathogens encode deubiquitinase (DUB) effectors to keep their surfaces free of ubiquitin modifications. Most bacterial DUBs belong to the OTU or CE-clan families. The betaproteobacteria B. pseudomallei and B. mallei, causative agents of melioidosis and glanders, respectively, encode the TssM effector, the only known bacterial DUB belonging to the USP class. TssM is much shorter than typical eukaryotic USP enzymes and lacks the canonical ubiquitin-recognition region. By solving the crystal structures of isolated TssM and its complex with ubiquitin, we found that TssM lacks the entire Fingers subdomain of the USP fold. Instead, the TssM family has evolved the functionally analog Littlefinger loop, which is located towards the end of the USP domain and recognizes different ubiquitin interfaces than those used by canonical USPs. The structures also revealed the presence of an N-terminal immunoglobulin-fold domain, which is able to form a strand-exchange dimer and might mediate TssM localization to the bacterial surface.

biochemistry↗

bioRxiv2023

FAM104 proteins promote the nuclear localization of p97/VCP

The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis and DNA damage repair, and its de-regulation is linked to neuro-muscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97-cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. FAM104 proteins bind p97 directly via a novel, alpha-helical motif and associate with the p97- UFD1-NPL4 complex in cells. FAM104 proteins localize to the nucleus and promote both the nuclear import and chromatin binding of p97. Loss of FAM104 proteins results in slow growth and hypersensitivity to p97 inhibition in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.

cell biology↗

bioRxiv2022

Lysine deserts prevent adventitious ubiquitylation of ubiquitin-proteasome components

In terms of its relative frequency, lysine is a common amino acid in the human proteome. However, by bioinformatics we find hundreds of proteins that contain long and evolutionarily conserved stretches completely devoid of lysine residues. These so-called lysine deserts show a high prevalence in intrinsically disordered proteins with known or predicted functions within the ubiquitin-proteasome system (UPS), including many E3 ubiquitin-protein ligases and UBL domain proteasome substrate shuttles, such as BAG6, RAD23A, UBQLN1 and UBQLN2. We show that introduction of lysine residues into the deserts leads to a striking increase in ubiquitylation of some of these proteins. In case of BAG6, we show that ubiquitylation is catalyzed by the E3 RNF126, while RAD23A is ubiquitylated by E6AP. Despite the elevated ubiquitylation, mutant RAD23A appears stable, but displays a partial loss of function phenotype in fission yeast. In case of UBQLN1 and BAG6, introducing lysine leads to a reduced abundance due to proteasomal degradation of the proteins. For UBQLN1 we show that arginine residues within the lysine depleted region are critical for its ability to form cytosolic inclusions. We propose that selective pressure to avoid lysine residues may be a common evolutionary mechanism to prevent unwarranted ubiquitylation and/or perhaps other lysine post-translational modifications. This may be particularly relevant for UPS components as they closely and frequently encounter the ubiquitylation machinery and are thus more susceptible to non-specific ubiquitylation.

biochemistry↗

bioRxiv2021

N4BP1 is dimerization-dependent linear ubiquitin reader regulating TNFR1 signalling through linear ubiquitin binding and Caspase-8-mediated processing

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) was recently identified as a critical suppressor of proinflammatory cytokine production1, whose mode of action remained unknown. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NF{kappa}B signalling by its unique dimerizationdependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure exclusive recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNF-induced cell death by increasing complex II assembly. Under proapoptotic conditions, Caspase-8 mediates proteolytic processing of N4BP1 and the resulting cleavage fragment of N4BP1, which retains the ability to bind linear ubiquitin, is rapidly degraded by the 26S proteasome, accelerating apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a unique linear ubiquitin reader that ensures timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

biochemistry↗

bioRxiv2020

An evolutionary approach to systematic discovery of novel deubiquitinases, applied to Legionella

Deubiquitinating enzymes (DUBs) are important regulators of the posttranslational protein ubiquitination system. Mammalian genomes encode about hundred different DUBs, which can be grouped into seven different classes. Members of other DUB classes are found in pathogenic bacteria, which use them to target the host defense. By combining bioinformatical and experimental approaches, we address the question if the known DUB families have a common evolutionary ancestry and share conserved features that set them apart from other proteases. By systematically comparing family-specific Hidden-Markov-Models, we uncovered distant relationships between established DUBs and other cysteine protease families. Most DUB families share a conserved aromatic residue linked to the active site, which restricts the cleavage of substrates with sidechains at the S2 position, corresponding to Gly-75 in ubiquitin. By applying these criteria to Legionella pneumophila ORFs, we identified lpg1621 and lpg1148 as deubiquitinases, characterized their cleavage specificities, and confirmed the importance of the aromatic gatekeeper motif for substrate selection.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗

bioRxiv2020

Identification and characterization of diverse OTU deubiquitinases in bacteria

Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The Ovarian Tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co-opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin-bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and to recognize a ubiquitin substrate. With these new examples, we perform the first cross-kingdom structural analysis of the OTU fold that highlights commonalities among distantly-related OTU deubiquitinases.

biochemistry↗

bioRxiv2019

Plant mixed lineage kinase domain-like proteins limit biotrophic pathogen growth

Mixed lineage kinase domain-like (MLKL) protein mediates necroptotic cell death in vertebrates. We report here the discovery of a conserved protein family across seed plants that is structurally homologous to vertebrate MLKL. The Arabidopsis thaliana genome encodes three MLKLs with overlapping functions in limiting growth of obligate biotrophic fungal and oomycete pathogens. Although displaying a cell death activity mediated by N-terminal helical bundles, termed HeLo domain, AtMLKL-dependent immunity can be separated from host cell death. Cryo-electron microscopy structures of AtMLKLs reveal a tetrameric configuration, in which the pseudokinase domain and brace region bury the HeLo-domains, indicative of an auto-repressed complex. We also show the association of two AtMLKLs with microtubules. These findings, coupled with resistance-enhancing activity and altered microtubule association of a phosphomimetic mutation in the pseudokinase domain of AtMLKL1, point to a cell death-independent immunity mechanism.\n\nOne Sentence SummaryPlants have a protein family that is structurally homologous to vertebrate mixed lineage kinase domain-like protein, which induces necroptotic cell death, but these plant proteins can confer immunity without host cell death.

plant biology↗