Subtilase SBT5.2 inactivates flagellin immunogenicity in the plant apoplast.

Most angiosperm plants recognise the 22-residue flagellin (flg22) epitope in bacterial flagellin via homologs of cell surface receptor FLS2 (flagellin sensitive-2) and mount pattern-triggered immune responses. However, flg22 is buried within the flagellin protein indicating that proteases might be required for flg22 release. Here, we demonstrate the extracellular subtilase SBT5.

Induced degradation of SNAP-fusion proteins.

Self-labeling protein tags are an efficient means to visualize, manipulate, and isolate engineered fusion proteins with suitable chemical probes. The SNAP-tag, which covalently conjugates to benzyl-guanine and -chloropyrimidine derivatives is used extensively in fluorescence microscopy, given the availability of suitable SNAP-ligand-based probes. Here, we extend the applicability of the SNAP-tag to targeted protein degradation.

High resolution analysis of proteolytic substrate processing.

Members of the widely conserved high temperature requirement A (HtrA) family of serine proteases are involved in multiple aspects of protein quality control. In this context, they have been shown to efficiently degrade misfolded proteins or protein fragments. However, recent reports suggest that folded proteins can also be native substrates.

Activation of automethylated PRC2 by dimerization on chromatin.

Polycomb repressive complex 2 (PRC2) is an epigenetic regulator that trimethylates lysine 27 of histone 3 (H3K27me3) and is essential for embryonic development and cellular differentiation. H3K27me3 is associated with transcriptionally repressed chromatin and is established when PRC2 is allosterically activated upon methyl-lysine binding by the regulatory subunit EED. Automethylation of the catalytic subunit enhancer of zeste homolog 2 (EZH2) stimulates its activity by an unknown mechanism.

Characterization of 2-phenanthroate:CoA ligase from the sulfate-reducing, phenanthrene-degrading enrichment culture TRIP.

Unlabelled: Polycyclic aromatic hydrocarbons (PAHs) are chemically stable pollutants that are poorly degraded by microorganisms in anoxic sediments. The anaerobic degradation pathway of PAHs such as phenanthrene starts with a carboxylation reaction forming phenanthroic acid. In this study, we identified and characterized the next enzyme in the pathway, the 2-phenanthroate:CoA ligase involved in the ATP-dependent formation of 2-phenanthroyl-CoA from cell-free extracts of the sulfate-reducing enrichment culture TRIP grown anaerobically with phenanthrene.

Factors governing attachment of to legume roots at acid, neutral, and alkaline pHs.

Unlabelled: Rhizobial attachment to host legume roots is the first physical interaction of bacteria and plants in symbiotic nitrogen fixation. The pH-dependent primary attachment of biovar viciae 3841 to (pea) roots was investigated by genome-wide insertion sequencing, luminescence-based attachment assays, and proteomic analysis. Under acid, neutral, or alkaline pH, a total of 115 genes are needed for primary attachment under one or more environmental pH, with 22 genes required for all.

The proteome of Nicotiana benthamiana is shaped by extensive protein processing.

Processing by proteases irreversibly regulates the fate of plant proteins and hampers the production of recombinant proteins in plants, yet only few processing events have been described in agroinfiltrated Nicotiana benthamiana, which has emerged as the main transient protein expression platform in plant science and molecular pharming. Here, we used in-gel digests and mass spectrometry to monitor the migration and topography of 5040 plant proteins within a protein gel. By plotting the peptides over the gel slices, we generated peptographs that reveal where which part of each protein was detected within the protein gel.

Environmental activity-based protein profiling for function-driven enzyme discovery from natural communities.

Background: Microbial communities are important drivers of global biogeochemical cycles, xenobiotic detoxification, as well as organic matter decomposition. Their major metabolic role in ecosystem functioning is ensured by a unique set of enzymes, providing a tremendous yet mostly hidden enzymatic potential. Exploring this enzymatic repertoire is therefore not only relevant for a better understanding of how microorganisms function in their natural environment, and thus for ecological research, but further turns microbial communities, in particular from extreme habitats, into a valuable resource for the discovery of novel enzymes with potential applications in biotechnology.

Microtubule end-on attachment maturation regulates Mps1 association with its kinetochore receptor.

Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear.

Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.

Eukaryotes produce a large number of cytochrome P450s that mediate the synthesis and degradation of diverse endogenous and exogenous metabolites. Yet, most of these P450s are uncharacterized and global tools to study these challenging, membrane-resident enzymes remain to be exploited. Here, we applied activity profiling of plant, mouse and fungal P450s with chemical probes that become reactive when oxidized by P450 enzymes.

Differences in the Synovial Fluid Proteome of Septic and Aseptic Implant Failure.

Implant loosening is a severe complication after total joint replacement. Here, differential diagnosis between septic and aseptic cases is crucial for further surgical treatment, but low-grade periprosthetic joint infections (PJIs) in particular remain a challenge. In this study, we analyzed the synovial fluid proteome of 21 patients undergoing revision surgery for septic (eight cases) or aseptic (thirteen cases) implant failure using LC-MS/MS to identify potential new biomarkers as future diagnostic tools.

TopBP1 utilises a bipartite GINS binding mode to support genome replication.

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1.

Glutathione Transferase Photoaffinity Labeling Displays GST Induction by Safeners and Pathogen Infection.

Glutathione transferases (GSTs) represent a large and diverse enzyme family involved in the detoxification of small molecules by glutathione conjugation in crops, weeds and model plants. In this study, we introduce an easy and quick assay for photoaffinity labeling of GSTs to study GSTs globally in various plant species. The small-molecule probe contains glutathione, a photoreactive group and a minitag for coupling to reporter tags via click chemistry.

Structure of the peroxisomal Pex1/Pex6 ATPase complex bound to a substrate.

The double-ring AAA+ ATPase Pex1/Pex6 is required for peroxisomal receptor recycling and is essential for peroxisome formation. Pex1/Pex6 mutations cause severe peroxisome associated developmental disorders. Despite its pathophysiological importance, mechanistic details of the heterohexamer are not yet available.

The UBX domain in UBXD1 organizes ubiquitin binding at the C-terminus of the VCP/p97 AAA-ATPase.

The AAA+ ATPase p97/VCP together with different sets of substrate-delivery adapters and accessory cofactor proteins unfolds ubiquitinated substrates to facilitate degradation by the proteasome. The UBXD1 cofactor is connected to p97-associated multisystem proteinopathy but its biochemical function and structural organization on p97 has remained largely elusive. Using a combination of crosslinking mass spectrometry and biochemical assays, we identify an extended UBX (eUBX) module in UBXD1 related to a lariat in another cofactor, ASPL.

Clp-targeting BacPROTACs impair mycobacterial proteostasis and survival.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3.

IFNα primes cancer cells for Fusicoccin-induced cell death via 14-3-3 PPI stabilization.

The natural product family of the fusicoccanes (FCs) has been shown to display anti-cancer activity, especially when combined with established therapeutic agents. FCs stabilize 14-3-3 protein-protein interactions (PPIs). Here, we tested combinations of a small library of FCs with interferon α (IFNα) on different cancer cell lines and report a proteomics approach to identify the specific 14-3-3 PPIs that are induced by IFNα and stabilized by FCs in OVCAR-3 cells.

Chemoproteomics Reveals the Pan-HER Kinase Inhibitor Neratinib To Target an Epoxide Hydrolase Related to Phytohormone Signaling.

Plant phytohormone pathways are regulated by an intricate network of signaling components and modulators, many of which still remain unknown. Here, we report a forward chemical genetics approach for the identification of functional SA agonists in that revealed Neratinib (), a covalent pan-HER kinase inhibitor drug in humans, as a modulator of SA signaling. Instead of a protein kinase, chemoproteomics unveiled that covalently modifies a surface-exposed cysteine residue of epoxide hydrolase isoform 7 (AtEH7), thereby triggering its allosteric inhibition.

The root pathogen secretes modular proteases in pea apoplast during host infection.

To successfully colonize the host, phytopathogens have developed a large repertoire of components to both combat the host plant defense mechanisms and to survive in adverse environmental conditions. Microbial proteases are predicted to be crucial components of these systems. In the present work, we aimed to identify active secreted proteases from the oomycete , which causes root rot diseases on legumes.

Activity-based proteomics uncovers suppressed hydrolases and a neo-functionalised antibacterial enzyme at the plant-pathogen interface.

The extracellular space of plant tissues contains hundreds of hydrolases that might harm colonising microbes. Successful pathogens may suppress these hydrolases to enable disease. Here, we report the dynamics of extracellular hydrolases in Nicotiana benthamiana upon infection with Pseudomonas syringae.

Taspase1 Facilitates Topoisomerase IIβ-Mediated DNA Double-Strand Breaks Driving Estrogen-Induced Transcription.

The human protease Taspase1 plays a pivotal role in developmental processes and cancerous diseases by processing critical regulators, such as the leukemia proto-oncoprotein MLL. Despite almost two decades of intense research, Taspase1's biology is, however, still poorly understood, and so far its cellular function was not assigned to a superordinate biological pathway or a specific signaling cascade. Our data, gained by methods such as co-immunoprecipitation, LC-MS/MS and Topoisomerase II DNA cleavage assays, now functionally link Taspase1 and hormone-induced, Topoisomerase IIβ-mediated transient DNA double-strand breaks, leading to active transcription.

Identification of fungal lignocellulose-degrading biocatalysts secreted by Phanerochaete chrysosporium via activity-based protein profiling.

Activity-based protein profiling (ABPP) has emerged as a versatile biochemical method for studying enzyme activity under various physiological conditions, with applications so far mainly in biomedicine. Here, we show the potential of ABPP in the discovery of biocatalysts from the thermophilic and lignocellulose-degrading white rot fungus Phanerochaete chrysosporium. By employing a comparative ABPP-based functional screen, including a direct profiling of wood substrate-bound enzymes, we identify those lignocellulose-degrading carbohydrate esterase (CE1 and CE15) and glycoside hydrolase (GH3, GH5, GH16, GH17, GH18, GH25, GH30, GH74 and GH79) enzymes specifically active in presence of the substrate.