Abstract Trimethylation of lysine 4 of histone H3 (H3K4me3) is a post-translational modification (PTM) enriched at promoters of actively transcribed genes. H3K4me3 is removed by the human histone demethylases of the KDM5 family. KDM5 demethylases act as transcriptional repressors through their catalytic activity in addition to more complex roles that depend on their interactions with other chromatin regulators and may be independent of demethylase activity. To better understand the mechanistic differences of the closely related paralogs KDM5A and KDM5B as well as their interactions with Retinoblastoma protein (RB), we systematically analyzed and compared their demethylase activities, nucleosome engagement, and RB binding. We used recombinant nucleosome binding and demethylase activity assays, as well as an integrative structural biology approach using negative-stain electron microscopy (EM), AlphaFold predictions, and cross-linking mass spectrometry for a comprehensive in vitro analysis of these critical and largely non-redundant enzymes. KDM5A and KDM5B showed differences in enzyme kinetics using peptide substrates, as well as in nucleosome binding. Furthermore, KDM5A interacts with RB, mainly mediated by its canonical LxCxE RB binding motif. KDM5B, on the other hand, lacks an LxCxE binding motif and does not stably bind to RB under the conditions tested here. RB directly interacts with nucleosomes, and its nucleosome binding does not measurably affect KDM5A demethylase activity or nucleosome interactions. Our findings provide a biochemical framework for the differences between KDM5A and KDM5B regarding RB interactions and nucleosome engagement.

Cell migration is a fundamental biological process essential for embryonal development, immune function, and cancer metastasis, with migration velocity representing a key parameter of this behaviour. Today, cell migration velocity can be measured in high-throughput assays that generate complex, hierarchically structured datasets with technical noise, batch effects, and biological variability, introducing significant uncertainty in velocity estimates. Current statistical approaches often fail to rigorously quantify this uncertainty, limiting reproducibility and comparisons across independent experimental datasets. Here, we present cellmig, a specialized computational tool that addresses this challenge. It implements established Bayesian hierarchical modeling within an accessible workflow tailored for high-throughput live cell migration assays, to separate biological signals from technical variation while explicitly quantifying uncertainty in migration velocity. cellmig provides a robust framework for analyzing cell migration assays, including dose-response studies and large-scale screens with multiple biological and technical replicates. By modeling biological variability (e.g., compound-dependent effects) and technical confounders (e.g., batch variability) within a unified Bayesian framework, cellmig estimates condition-specific effects on cell velocity with probabilistic uncertainty intervals, avoiding common pitfalls associated with null-hypothesis testing. Through exhaustive benchmarking against commonly used approaches in the field, we demonstrate that cellmig achieves improved sensitivity in detecting subtle migration effects and enhanced robustness against technical variability. Additionally, its generative models enable simulation of migration velocities under various assumptions, aiding experimental planning. We validated cellmig through a tiered strategy: (1) benchmarking on two independent experimental datasets and (2) deployment on a large-scale high-throughput screen that discovered new chemical biology. Our results demonstrate that cellmig can detect subtle dose-dependent velocity changes, maintain robustness against systematic variability and batch effects, and facilitate reliable integration of multi-experiment datasets. In summary, cellmig enhances reproducibility, reliability, and biological insight in high-throughput migration studies, facilitating quantitative inter-dataset comparisons. cellmig is implemented as an open-source R package and is freely available on Bioconductor (https://bioconductor.org/packages/cellmig).

Activity-based profiling with fluorescent probes is a powerful tool for the functional characterization of whole enzyme classes in crude proteomes. Here, we discovered that probe cocktails consisting of the E-64 warhead carrying different fluorophores via short linkers distinguishes the labeling of papain-like cysteine proteases and their isoforms because of their differential affinity to these probes. This causes polychrome labeling with specific apparent colors for different protease families and isoforms. Polychrome labeling revealed differential labeling of isoforms of RD21-like proteases carrying C-terminal granulin domains. Molecular modeling of the RD21 isoforms revealed that the C-terminal granulin may create a fluorophore-binding pocket with the substrate-binding groove, implying that the granulin domain influences substrate selectivity. The concept of polychrome labeling may be widely applicable to other chemical probes for the characterization of protein families in all kingdoms of life.

For targeted covalent protein modification at low-reactivity aspartates and glutamates, new methods are in high demand. We report a technique inspired by the HaloTag technology, which employs nucleophilic substitution at chloroalkane-functionalised ligands by a specific aspartate residue. Embedding of alkyl bromide warheads into non-covalent inhibitors enables covalent modification of a glutamate in the lipoprotein binding chaperone - phosphodiesterase of retinal rod subunit delta (PDEδ), which shuttles prenylated lipoproteins between cellular membranes and thereby mediates their activity. Its hydrophobic ligand-binding pocket contains p.E88 as the only accessible nucleophile for covalent targeting. We show that a covalent inhibitor, termed DeltaTag, overcomes limitations of non-covalent inhibitors. DeltaTag labels PDEδ at its p.E88 under biologically relevant conditions, modulates mammalian target of rapamycin (mTOR) signalling by disrupting the PDEδ-Rheb (Ras homologue enriched in brain)-mTORC1 (mTOR complex 1) axis and inhibits cancer cell proliferation. This proof-of-concept study demonstrates that the design strategy holds promise for the covalent modification of proteins with lipophilic binding sites that lack accessible reactive amino acids but contain specific carboxylates. New methods for targeted covalent protein modification at low reactivity aspartates and glutamates are of high interest. Here, the authors report a technique inspired by the HaloTag technology, which employs a covalent conjugation reaction between ligands with a reactive chloroalkane linker and a specific aspartic acid, and use it to covalently modify lipoprotein chaperone PDEδ at a binding site glutamic acid.

RD21-like proteases are papain-like cysteine proteases with a C-terminal granulin domain that are abundant and ubiquitous in angiosperms and have often been implicated in immunity. We previously found that the activity of RD21 in Nicotiana benthamiana (NbRD21) is suppressed during infection with Pseudomonas syringae. Here, we studied the role of NbRD21 in immunity and proteome processing. NbRD21 was disrupted by genome editing and rd21 mutants were subjected to disease assays and shotgun proteomics. Dipeptide substrate zLR-AMC was used in protease assays and agroinfiltration was used to transiently express NbRD21 and candidate substrates. Genome-edited lines lacking NbRD21 develop normally but have drastically reduced zLRase activity and are significantly more susceptible to P. syringae. Shotgun proteomics revealed an increased accumulation of c. 20 diverse receptor-like kinases (RLKs) in untreated rd21 knockout lines, but their transcript levels are unaltered when compared to wild-type plants. 35S-driven GFP-tagged RLKs accumulate more upon transient expression in rd21 plants than in wild-type plants. These data indicate that NbRD21 post-translationally controls RLK homeostasis, either by directly degrading RLKs or indirectly by regulating endocytic RLK recycling.

Recent advances in mitochondrial network dynamic and signalling highlight mitochondria as key therapeutic targets across diverse diseases. Yet, high drug development failure rates reflect an incomplete understanding of upstream molecular regulators of mitochondrial fate. Here, we address this gap by reverse engineering of the BH3-only protein BNIP3. Structural modelling and sequence-function analyses of its N-terminus identify a critical functional domain and amino acid hotspots that directly activate BCL-2 executioner proteins, triggering mitochondrial cell death. Leveraging these insights, we develop a BNIP3 antagonist peptide (B-017) that disrupts interactions between BNIP3 and BCL-2 executioner proteins, preserving mitochondrial integrity. B-017 demonstrates target specificity, a favourable safety profile, and robust suppression of cell death signalling in human cells. In clinically relevant animal models, it reduces tissue damage in the heart, brain, and liver. Together, these findings position B-017 as a promising therapeutic candidate targeting mitochondrial dysfunction.

Agroinfiltration of Nicotiana benthamiana is widely used for recombinant protein production in plant science and molecular pharming, but enzymatic browning and native protein crosslinking during extraction may limit protein integrity and purification efficiency. We generated genome-edited N. benthamiana lines lacking two polyphenol oxidases (PPOs) and analyzed protein integrity, enzymatic activity profiles, and recombinant protein purification under non-denaturing extraction conditions. PPO-deficient plants showed reduced browning and native protein crosslinking, preserved endogenous proteins at their predicted molecular weights, displayed increased detectable enzyme activities, and achieved a significantly higher recovery and improved purity of a transiently expressed recombinant protein. These findings identify PPO-mediated oxidation as a major bottleneck during protein extraction and demonstrate that PPO depletion enhances recombinant protein purification while preserving native protein integrity.

Cellular stress response pathways support cell survival under stress and are often leveraged by cancer cells to gain advantageous traits. How cells respond to Golgi stress is incompletely understood, limiting insights into the role of Golgi stress in cancer. Here, we combined small-molecule stress models and proteomic analyses to elucidate stress-induced changes at the Golgi. Our data establish the depletion of Golgi transport proteins as a common response to different Golgi stressors, including ionophores and oxysterol-binding protein (OSBP) inhibitors. Ionophores further induce de novo expression of the stress response protein FAM129A (also known as NIBAN1), which localizes to the remodeled secretory pathway. In a group of melanoma cells, displaying a dedifferentiated epithelialto-mesenchymal-transition (EMT)-like phenotype, FAM129A is constitutively expressed. In these cells, stress-induced localization of FAM129A to the secretory pathway is achieved by relocalization from the plasma membrane. Collectively, our data highlight the Golgi-endosome interface as a critical hub of the cellular response to Golgi stress and reveal cancer cell-specific effects of this response.

To maintain cellular homeostasis, protein quality control (PQC) machineries target damaged proteins for degradation. However, the PQC machinery, operating at the major protein sorting and processing site of mammalian cells, the Golgi, has remained largely elusive. Here we used a chemical biology tool to induce misfolding and ubiquitination of Golgi-localized model substrates. Our tool recruits a Cullin-RING ubiquitin ligase complex by exposure of a degron, recognized by the substrate receptor KLHDC2. Using this tool, we found that ubiquitinated Golgi membrane proteins are targeted for proteasomal degradation, a process that is critical to avoid Golgi fragmentation. This process is facilitated by the p97-unfoldase, which assembles with its adaptors UFD1-NPL4 and FAF2 at the Golgi. We further identified the rhomboid pseudo-protease RHBDD2 as an important contributor to Golgi homeostasis as it binds misfolded, ubiquitinated Golgi membrane proteins and regulates Golgi morphology. Our findings establish a molecular framework for Golgi-PQC in mammalian cells. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, https://ror.org/018mejw64, SFB1430, Project-ID 424228829, Project-ID 219183055, Project-ID 397277702 Alexander von Humboldt Foundation, https://ror.org/012kf4317, Sofja Kovalevskaja Award

Proteolysis-targeting chimeras (PROTACs) co-op the ubiquitin system for targeted protein degradation, creating opportunities to interrogate cellular functions of proteins through "chemical knockdown". However, matched pairs of protein degraders and inhibitors, that possess high specificity and chemical complementarity, for individual components of the ubiquitin system have remained scarce. This includes reagents to modulate activity and abundance of deubiquitinases (DUBs). Here, using an integrated chemical biology approach, we explore cellular functions of the DUB USP7 as a case study by comparing inhibition and degradation in melanoma and pancreatic cancer cells. Through the synthesis of a degrader library, we identify and characterize potent USP7 PROTACs for each cancer type. Proteomic and cellular analyses reveal that selective USP7 degradation modulates both shared and distinct protein sets across both cancers without affecting cell growth. In contrast, prolonged inhibitor treatment induces USP7-independent proteomic and metabolic dysregulation, highlighting important caveats for the cellular use of hydroxypiperidine-based USP7 inhibitors. Collectively, our work provides a comprehensively characterized chemical toolbox to distinguish on-target phenotypes which will aid the understanding of USP7 in malignant diseases. More broadly, our data emphasize the importance of increased specificity via PROTAC-mediated degradation and the potential of this modality to elucidate cell-line specific functions of DUBs.

The extracellular space (apoplast) of plants is an important molecular battleground during infection by many pathogens. We previously found that a plant-secreted β-galactosidase BGAL1 acts in immunity by facilitating the release of immunogenic peptides from bacterial flagellin and that Pseudomonas syringae suppresses this enzyme by producing a small molecule inhibitor called galactosyrin. Here, we elucidated the structure and biosynthesis of galactosyrin and uncovered its multifunctional roles during infection. Structural elucidation by cryo-EM and chemical synthesis revealed that galactosyrin is an iminosugar featuring a unique geminal diol attached to the pyrrolidine moiety that mimics galactose binding to the β-galactosidase active site. Galactosyrin biosynthesis branches off from purine biosynthesis and involves three enzymes of which the first is a reductase that is unique in iminosugar biosynthesis. Besides inhibiting BGAL1 to avoid detection, galactosyrin also changes the glycoproteome and metabolome of the apoplast. The manipulation of host glycobiology may be common to plant-associated bacteria that carry putative iminosugar biosynthesis clusters.

Protein adsorption on orthopedic biomaterials during the initial intraoperative contact critically influences biological responses and osseointegration. Osteoconductive grafts such as β-tricalcium phosphate (β-TCP) and poly (ε-caprolactone)-β-TCP (PCL-TCP) can be functionally activated by exposure to autologous tissue. However, the composition and relevance of the resulting protein layer still remain unclear. In this study involving 10 patients undergoing primary total hip arthroplasty, β-TCP and PCL-TCP samples were incubated both in the femoral medullary cavity and within a surgical tissue collector harvesting autologous tissue (blood, bone fragments, muscle, and fat). Surface morphology was assessed microscopically, and protein adsorption was characterized via high-resolution LC-MS/MS with subsequent bioinformatics and statistical analysis. Both materials adsorbed over 2000 different autologous proteins. β-TCP showed higher overall protein concentrations, while PCL-TCP demonstrated greater proteomic diversity and incubation method-dependent shifts in protein profiles, influenced by surface roughness and wettability. Samples incubated in the tissue collector exhibited less protein variability and smaller material-specific differences compared to incubation in the femoral cavity, particularly for PCL-TCP. Predominant proteins were linked to immune regulation, stress response, and protein metabolism. These findings emphasize the impact of material properties and incubation environment on protein adsorption, with ex vivo incubation leading to more consistent protein adsorption patterns.

Covalent chemical probes and drugs combine unique pharmacologic properties with the availability of straightforward compound profiling technologies via chemoproteomic platforms. These advantages have fostered the development of suitable electrophilic "warheads" for systematic covalent chemical probe discovery. Despite undisputable advances in the last years, the targeted development of proteome-wide selective covalent probes remains a challenge for dipeptidyl peptidase (DPP) 8 and 9 (DPP8/9), intracellular serine hydrolases of the pharmacologically relevant dipeptidyl peptidase 4 activity/structure homologues (DASH) family. Here, we show the exploration of the natural product Sulphostin, a DPP4 inhibitor, as a starting point for DPP8/9 inhibitor development. The generation of Sulphostin-inspired N-phosphonopiperidones leads to derivatives with improved DPP8/9 inhibitory potency, an enhanced proteome-wide selectivity and confirmed DPP8/9 engagement in cells, thereby representing that structural fine-tuning of the warhead's leaving group may represent a straightforward strategy for achieving target selectivity in exoproteases such as DPPs.

The cellular response to lysosomal damage involves fine-tuned mechanisms of membrane repair, lysosome regeneration and lysophagy, but how these different processes are coordinated is unclear. Here we show in human cells that the deubiquitinating enzyme ATXN3 helps restore integrity of the lysosomal system after damage by targeting K48-K63-branched ubiquitin chains on regenerating lysosomes. We find that ATXN3 is required for lysophagic flux after lysosomal damage but is not involved in the initial phagophore formation on terminally damaged lysosomes. Instead, ATXN3 is recruited to a distinct subset of lysosomes that are decorated with phosphatidylinositol-(4,5)-bisphosphate and that are not yet fully reacidified. There, ATXN3, along with its partner VCP/p97, targets and turns over K48-K63-branched ubiquitin conjugates. ATXN3 thus facilitates degradation of a fraction of LAMP2 via microautophagy to regenerate the lysosomal membrane and to thereby reestablish degradative capacity needed also for completion of lysophagy. Our findings identify a key role of ATXN3 in restoring lysosomal function after lysosomal membrane damage and uncover K48-K63-branched ubiquitin chain-regulated regeneration as a critical element of the lysosomal damage stress response.

The human peptidyl-prolyl-cis/trans isomerases (PPIases), Parvulin 14 and Parvulin 17, accelerate the cis/trans isomerization of Xaa-Pro moieties within protein sequences. By modulating the respective binding interfaces of their target proteins, they play a crucial role in determining the fate of their substrates within the cell. Although both enzymes share the same amino acid sequence, they have different cellular functions. This difference is due to a 25 residue N-terminal extension present in Par17 but absent in Par14. Using activity assays, NMR spectroscopy, and mass spectrometry, we demonstrate that the N-terminal extension of Par17 determines substrate selectivity by an intramolecular allosteric mechanism and exhibits a target-binding motif that interacts with actin.

The Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-responsive transcription factor (TF) with two major splice isoforms (α and β). In chronic hyperglycemia and glucolipotoxicity, ChREBPα-mediated ChREBPβ expression surges, leading to insulin-secreting β-cell dedifferentiation and death. 14-3-3 binding to ChREBPα results in cytoplasmic retention and suppression of transcriptional activity. Thus, small molecule-mediated stabilization of this protein-protein interaction (PPI) may be of therapeutic value. Here, we show that structure-based optimizations of a 'molecular glue' compound led to potent ChREBPα/14-3-3 PPI stabilizers with cellular activity. In primary human β-cells, the most active compound retained ChREBPα in the cytoplasm, and efficiently protected β-cells from glucolipotoxicity while maintaining β-cell identity. This study may thus not only provide the basis for the development of a unique class of compounds for the treatment of Type 2 Diabetes but also showcases an alternative 'molecular glue' approach for achieving small molecule control of notoriously difficult to target TFs.

Glycerol is highly abundant in natural ecosystems and serves as both an important carbon source for microorganisms as well as a promising feedstock for industrial applications. However, the pathways involved in glycerol degradation in Archaea remain unclear. Here, we show that the thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius can grow with glycerol as its sole carbon source and characterize the mechanisms involved in glycerol utilization. We show that after uptake involving facilitated diffusion, glycerol is phosphorylated to glycerol-3-phosphate by glycerol kinase (GK), followed by oxidation to dihydroxyacetone phosphate catalyzed by an unusual glycerol-3-phosphate dehydrogenase (G3PDH) with a previously undescribed type of membrane anchoring via a CoxG-like protein. Furthermore, we show that while S. acidocaldarius has two paralogous GK/G3PDH copies (saci_1117-1119, saci_2031-2033) with similar biochemical activity, only saci_2031-2033 is highly upregulated and essential on glycerol, suggesting that distinct enzyme pairs may be regulated by different environmental conditions. Finally, we explore the diversity of glycerol metabolism enzymes across the Archaea domain, revealing a high versatility of G3PDHs with respect to interacting proteins, electron transfer mechanisms, and modes of membrane anchoring. Our findings help to elucidate the mechanisms involved in glycerol utilization in Archaea, highlighting unique evolutionary strategies that likely enabled adaptation to different lifestyles.

Hsp70 and Hsp90 chaperones and their regulatory cochaperones are critical for maintaining protein homeostasis. Glucose-regulated protein 94 (GRP94), the sole Hsp90 chaperone in the secretory pathway of mammalian cells, is essential for the maturation of important secretory and transmembrane proteins. Without the requirement of cochaperones, the Hsp70 protein BiP controls regulatory conformational changes of GRP94, the structural basis of which has remained elusive. Here we biochemically and structurally characterize the formation of a BiP-GRP94 chaperone complex and its transition to a conformation expected to support the loading of substrate proteins from BiP onto GRP94. BiP initially binds to the open GRP94 dimer through an interaction interface that is conserved among Hsp70 and Hsp90 paralogs. Subsequently, binding of a second BiP protein stabilizes a semiclosed GRP94 dimer, thereby advancing the chaperone cycle. Our findings highlight a fundamental mechanism of direct Hsp70-Hsp90 cooperation, independent of cochaperones.

Bacterial wilt caused by Ralstonia solanacearum is one of the most destructive bacterial diseases for which no effective treatment exists. There is an urgent need to understand the basis of resistance against this pathogen in order to engineer efficient strategies in the field. We previously demonstrated that resistant tomato plants limit bacterial movement in the apoplast and the xylem. As a first step to dissect the underlying mechanisms, we analysed the apoplast proteome upon challenge with R. solanacearum in the susceptible tomato cultivar Marmande and the resistant cultivar Hawaii 7996. Here, we described the xylem proteome in these same cultivars and compared it with the apoplastic proteome, revealing variety-dependent and infection-dependent changes. This proteomic analysis led to the identification of pathogenesis-related 1 (PR1) proteins as highly induced upon infection. Since PR1b was the most abundant PR1 protein in both the apoplast and the xylem, we concentrated on this family member to study the role of PR1s in the interaction between tomato and R. solanacearum. Surprisingly, lack of PR1b resulted in enhanced resistance to R. solanacearum in tomato, which could be due to an up-regulation of homologous genes in a compensatory effect as has been reported before. PR1 processing by an unknown protease in tomato results in the generation of the CAPE peptide. Treatment of tomato plants with the CAPE1 peptide resulted in restriction of R. solanacearum growth, via defence gene reprogramming. Future work in the lab will help determine which tomato secreted proteases cleave PR1s to generate CAPEs.

Reactivity-based protein profiling (RBPP) is a powerful chemical proteomics technique that uses chemical probes to gain new insights into the surface exposure of amino acid residues within native proteomes. While RBPP has primarily been employed to identify proteins that could be potential targets for novel small molecule drugs, its applications in biological research are limited, especially in plant science. In this study, we introduce RBPP in plants by identifying over 15,000 labelling sites in native protein extracts from Arabidopsis cell cultures using an amine-reactive N-hydroxysuccinimide (NHS) ester probe linked to biotin. We demonstrate the utility of this approach by examining differential labelling in the Arabidopsis proteome in response to ATP. Pre-treatment with ATP results in decreased biotin-NHS labelling at residues near known ATP-binding sites and also revealed several novel ATP-responsive proteins. Lastly, we established RBPP with phosphonic acid-based NHS probes and immobilised metal affinity chromatography (IMAC) enrichment, revealing over 5,700 additional labelling sites. Collectively, these findings highlight that RBPP is a powerful and adaptable tool that can address key questions in plant biology. ### Competing Interest Statement The authors have declared no competing interest. European Research CouncilEuropean Research Council, , 101019324 Biotechnology and Biological Sciences Research CouncilBiotechnology and Biological Sciences Research Council, , DDT00230