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. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

BacPROTACs mediate targeted protein degradation in bacteria.

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.

Ubiquitin-Independent Disassembly by a p97 AAA-ATPase Complex Drives PP1 Holoenzyme Formation.

The functional diversity of protein phosphatase-1 (PP1), with its countless substrates, relies on the ordered assembly of alternative PP1 holoenzymes. Here, we show that newly synthesized PP1 is first held by its partners SDS22 and inhibitor-3 (I3) in an inactive complex, which needs to be disassembled by the p97 AAA-ATPase to promote exchange to substrate specifiers. Unlike p97-mediated degradative processes that require the Ufd1-Npl4 ubiquitin adapters, p97 is targeted to PP1 by p37 and related adapter proteins. Reconstitution with purified components revealed direct interaction of the p37 SEP domain with I3 without the need for ubiquitination, and ATP-driven pulling of I3 into the central channel of the p97 hexamer, which triggers dissociation of I3 and SDS22. Thus, we establish regulatory ubiquitin-independent protein complex disassembly as part of the functional arsenal of p97 and define an unanticipated essential step in PP1 biogenesis that illustrates the molecular challenges of ordered subunit exchange.

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. Under UV irradiation, this probe quickly and robustly labels GSTs in crude protein extracts of different plant species. Purification and mass spectrometry (MS) analysis of labeled proteins from Arabidopsis identified 10 enriched GSTs from the Phi(F) and Tau(U) classes. Photoaffinity labeling of GSTs demonstrated GST induction in wheat seedlings upon treatment with safeners and in Arabidopsis leaves upon infection with avirulent bacteria. Treatment of Arabidopsis with salicylic acid (SA) analog benzothiadiazole (BTH) induces GST labeling independent of NPR1, the master regulator of SA. Six Phi- and Tau-class GSTs that are induced upon BTH treatment were identified, and their labeling was confirmed upon transient overexpression. These data demonstrate that GST photoaffinity labeling is a useful approach to studying GST induction in crude extracts of different plant species upon different types of stress.

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. Using activity-based proteomics with a cocktail of biotinylated probes, we simultaneously monitored 171 active hydrolases, including 109 serine hydrolases (SHs), 49 glycosidases (GHs) and 13 cysteine proteases (CPs). The activity of 82 of these hydrolases (mostly SHs) increases during infection, while the activity of 60 hydrolases (mostly GHs and CPs) is suppressed during infection. Active ß-galactosidase-1 (BGAL1) is amongst the suppressed hydrolases, consistent with production of the BGAL1 inhibitor by P. syringae. One of the other suppressed hydrolases, the pathogenesis-related NbPR3, decreases bacterial growth when transiently overexpressed. This is dependent on its active site, revealing a role for NbPR3 activity in antibacterial immunity. Despite being annotated as a chitinase, NbPR3 does not possess chitinase activity and contains an E112Q active site substitution that is essential for antibacterial activity and is present only in Nicotiana species. This study introduces a powerful approach to reveal novel components of extracellular immunity, exemplified by the discovery of the suppression of neo-functionalised Nicotiana-specific antibacterial NbPR3.

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. These data uncovered that 60% of the detected proteins have proteoforms that migrate at lower than predicted molecular weights, implicating extensive proteolytic processing. This analysis confirms the proteolytic removal and degradation of autoinhibitory prodomains of most but not all proteases, and revealed differential processing within pectinemethylesterase and lipase families. This analysis also uncovered intricate processing of glycosidases and uncovered that ectodomain shedding might be common for a diverse range of receptor-like kinases. Transient expression of double-tagged candidate proteins confirmed processing events in vivo. This large proteomic dataset implicates an elaborate proteolytic machinery shaping the proteome of N. benthamiana.

VCP/p97 Extracts Sterically Trapped Ku70/80 Rings from DNA in Double-Strand Break Repair.

During DNA double-strand break (DSB) repair, the ring-shaped Ku70/80 complex becomes trapped on DNA and needs to be actively extracted, but it has remained unclear what provides the required energy. By means of reconstitution of DSB repair on beads, we demonstrate here that DNA-locked Ku rings are released by the AAA-ATPase p97. To achieve this, p97 requires ATP hydrolysis, cooperates with the Ufd1-Npl4 ubiquitin-adaptor complex, and specifically targets Ku80 that is modified by K48-linked ubiquitin chains. In U2OS cells, chemical inhibition of p97 or siRNA-mediated depletion of p97 or its adapters impairs Ku80 removal after non-homologous end joining of DSBs. Moreover, this inhibition attenuates early steps in homologous recombination, consistent with p97-driven Ku release also affecting repair pathway choice. Thus, our data answer a central question regarding regulation of Ku in DSB repair and illustrate the ability of p97 to segregate even tightly bound protein complexes for release from DNA.

HTRA proteases: regulated proteolysis in protein quality control.

Controlled proteolysis underlies a vast diversity of protective and regulatory processes that are of key importance to cell fate. The unique molecular architecture of the widely conserved high temperature requirement A (HTRA) proteases has evolved to mediate critical aspects of ATP-independent protein quality control. The simple combination of a classic Ser protease domain and a carboxy-terminal peptide-binding domain produces cellular factors of remarkable structural and functional plasticity that allow cells to rapidly respond to the presence of misfolded or mislocalized polypeptides.

Activation by substoichiometric inhibition.

Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.

The chemical compound 'Heatin' stimulates hypocotyl elongation and interferes with the Arabidopsis NIT1-subfamily of nitrilases.

Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth-based adaptations that enhance leaf-cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4-2-deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound 'Heatin', containing 1-iminomethyl-2-naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin-mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1-subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin-mediated hypocotyl elongation requires the NITRILASE1-subfamily members, NIT1 and NIT2. Heatin inhibits NIT1-subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1-subfamily substrate indole-3-acetonitrile in vivo. However, levels of the NIT1-subfamily product, bioactive auxin (indole-3-acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1-subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole-3-acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Chemoproteomics-Enabled Identification of 4-Oxo-ß-Lactams as Inhibitors of Dipeptidyl Peptidases 8 and 9.

Dipeptidyl peptidases 8 and 9 (DPP8/9) have gathered interest as drug targets due to their important roles in biological processes like immunity and tumorigenesis. Elucidation of their distinct individual functions remains an ongoing task and could benefit from the availability of novel, chemically diverse and selective chemical tools. Here, we report the activity-based protein profiling (ABPP)-mediated discovery of 4-oxo-ß-lactams as potent, non-substrate-like nanomolar DPP8/9 inhibitors. X-ray crystallographic structures revealed different ligand binding modes for DPP8 and DPP9, including an unprecedented targeting of an extended S2' (eS2') subsite in DPP8. Biological assays confirmed inhibition at both target and cellular levels. Altogether, our integrated chemical proteomics and structure-guided small molecule design approach led to novel DPP8/9 inhibitors with alternative molecular inhibition mechanisms, delivering the highest selectivity index reported to date.

Cathepsin L Regulates Metabolic Networks Controlling Rapid Cell Growth and Proliferation.

Rapidly proliferating cells reshape their metabolism to satisfy their ever-lasting need for cellular building blocks. This phenomenon is exemplified in certain malignant conditions such as cancer but also during embryonic development when cells rely heavily on glycolytic metabolism to exploit its metabolic intermediates for biosynthetic processes. How cells reshape their metabolism is not fully understood. Here we report that loss of cathepsin L (Cts L) is associated with a fast proliferation rate and enhanced glycolytic metabolism that depend on lactate dehydrogenase A (LDHA) activity. Using mass spectrometry analysis of cells treated with a pan cathepsin inhibitor, we observed an increased abundance of proteins involved in central carbon metabolism. Further inspection of putative Cts L targets revealed an enrichment for glycolytic metabolism that was independently confirmed by metabolomic and biochemical analyses. Moreover, proteomic analysis of Cts L-knockout cells identified LDHA overexpression that was demonstrated to be a key metabolic junction in these cells. Lastly, we show that Cts L inhibition led to increased LDHA protein expression, suggesting a causal relationship between LDHA expression and function. In conclusion, we propose that Cts L regulates this metabolic circuit to keep cell division under control, suggesting the therapeutic potential of targeting this protein and its networks in cancer.

Leaf Apoplast of Field-Grown Potato Analyzed by Quantitative Proteomics and Activity-Based Protein Profiling.

Multiple biotic and abiotic stresses challenge plants growing in agricultural fields. Most molecular studies have aimed to understand plant responses to challenges under controlled conditions. However, studies on field-grown plants are scarce, limiting application of the findings in agricultural conditions. In this study, we investigated the composition of apoplastic proteomes of potato cultivar Bintje grown under field conditions, i.e., two field sites in June-August across two years and fungicide treated and untreated, using quantitative proteomics, as well as its activity using activity-based protein profiling (ABPP). Samples were clustered and some proteins showed significant intensity and activity differences, based on their field site and sampling time (June-August), indicating differential regulation of certain proteins in response to environmental or developmental factors. Peroxidases, class II chitinases, pectinesterases, and osmotins were among the proteins more abundant later in the growing season (July-August) as compared to early in the season (June). We did not detect significant differences between fungicide Shirlan treated and untreated field samples in two growing seasons. Using ABPP, we showed differential activity of serine hydrolases and ß-glycosidases under greenhouse and field conditions and across a growing season. Furthermore, the activity of serine hydrolases and ß-glycosidases, including proteins related to biotic stress tolerance, decreased as the season progressed. The generated proteomics data would facilitate further studies aiming at understanding mechanisms of molecular plant physiology in agricultural fields and help applying effective strategies to mitigate biotic and abiotic stresses.

From dolastatin 13 to cyanopeptolins, micropeptins, and lyngbyastatins: the chemical biology of Ahp-cyclodepsipeptides.

Covering: 1989 up to 2019 Ahp-cyclodepsipeptides (also known as Ahp-containing cyclodepsipeptides, cyanopeptolins, micropeptins, microginines, and lyngbyastatins, and by many other names) are a family of non-ribosomal peptide synthesis (NRPS)-derived natural products with potent serine protease inhibitory properties. Here, we review their isolation and structural elucidation from natural sources as well as studies of their biosynthesis, molecular mode of action, and use in drug discovery efforts. Accordingly, this summary aims to provide a comprehensive overview of the current state-of-the-art Ahp-cyclodepsipeptide research.

Targeting of parvulin interactors by diazirine mediated cross-linking discloses a cellular role of human Par14/17 in actin polymerization.

The peptidyl-prolyl cis/trans isomerases (PPIases) Parvulin 14 (Par14) and Parvulin 17 (Par17) result from alternative transcription initiation of the PIN4 gene. Whereas Par14 is present in all metazoan, Par17 is only expressed in Hominidae. Par14 resides mainly within the cellular nucleus, while Par17 is translocated into mitochondria. Using photo-affinity labeling, cross-linking and mass spectrometry (MS) we identified binding partners for both enzymes from HeLa lysates and disentangled their cellular roles. Par14 is involved in biogenesis of ribonucleoprotein (RNP)-complexes, RNA processing and DNA repair. Its elongated isoform Par17 participates in protein transport/translocation and in cytoskeleton organization. Nuclear magnetic resonance (NMR) spectroscopy reveals that Par17 binds to ß-actin with its N-terminal region, while both parvulins initiate actin polymerization depending on their PPIase activity as monitored by fluorescence spectroscopy. The knockdown (KD) of Par17 in HCT116 cells results in a defect in cell motility and migration.

Triazine Probes Target Ascorbate Peroxidases in Plants.

Though they are rare in nature, anthropogenic 1,3,5-triazines have been used in herbicides as chemically stable scaffolds. Here, we show that small 1,3,5-triazines selectively target ascorbate peroxidases (APXs) in Arabidopsis (Arabidopsis thaliana), tomato (Solanum lycopersicum), rice (Oryza sativa), maize (Zea mays), liverwort (Marchantia polymorpha), and other plant species. The alkyne-tagged 2-chloro-4-methyl-1,3,5-triazine probe KSC-3 selectively binds APX enzymes, both in crude extracts and in living cells. KSC-3 blocks APX activity, thereby reducing photosynthetic activity under moderate light stress, even in apx1 mutant plants. This suggests that APX enzymes in addition to APX1 protect the photosystem against reactive oxygen species. Profiling APX1 with KCS-3 revealed that the catabolic products of atrazine (a 1,3,5-triazine herbicide), which are common soil pollutants, also target APX1. Thus, KSC-3 is a powerful chemical probe to study APX enzymes in the plant kingdom.

Zelkovamycin is an OXPHOS Inhibitory Member of the Argyrin Natural Product Family.

Natural products (NPs) are an important inspirational source for developing drugs and chemical probes. In 1999, the group of Omura reported the constitutional elucidation of zelkovamycin. Although largely unrecognized so far, this NP displays structural similarities as well as differences to the argyrin NP family, a class of peptidic NPs with promising anticancer activities and diverse mode-of-action at the molecular level. By a combination of structure elucidation experiments, the first total synthesis of zelkovamycin and bioassays, the zelkovamycin configuration was determined and its previously proposed molecular structure was revised. The full structure assignment proves zelkovamycin as an additional member of the argyrins with however unique OXPHOS inhibitory properties. Zelkovamycin may therefore not only serve as a new starting point for chemical inhibitors of the OXPHOS system, but also guide customized argyrin NP isolation and biosynthesis studies.

Natural brominated phenoxyphenols kill persistent and biofilm-incorporated cells of MRSA and other pathogenic bacteria.

Due to a high unresponsiveness to chemotherapy, biofilm formation is an important medical problem that frequently occurs during infection with many bacterial pathogens. In this study, the marine sponge-derived natural compounds 4,6-dibromo-2-(2',4'-dibromophenoxy)phenol and 3,4,6-tribromo-2-(2',4'-dibromophenoxy)phenol were found to exhibit broad antibacterial activity against medically relevant gram-positive and gram-negative pathogens. The compounds were not only bactericidal against both replicating and stationary phase-persistent planktonic cells of methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa; they also killed biofilm-incorporated cells of both species while not affecting biofilm structural integrity. Moreover, these compounds were active against carbapenemase-producing Enterobacter sp. This simultaneous activity of compounds against different growth forms of both gram-positive and gram-negative bacteria is rare. Genome sequencing of spontaneous resistant mutants and proteome analysis suggest that resistance is mediated by downregulation of the bacterial EIIBC phosphotransferase components scrA and mtlA in MRSA likely leading to a lower uptake of the molecules. Due to their only moderate cytotoxicity against human cell lines, phenoxyphenols provide an interesting new scaffold for development of antimicrobial agents with activity against planktonic cells, persisters and biofilm-incoporated cells of ESKAPE pathogens. KEY POINTS: • Brominated phenoxyphenols kill actively replicating and biofilm-incorporated bacteria. • Phosphotransferase systems mediate uptake of brominated phenoxyphenols. • Downregulation of phosphotransferase systems mediate resistance.

Protease Activities Triggered by Ralstonia solanacearum Infection in Susceptible and Tolerant Tomato Lines.

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum.

Dynamic hydrolase labelling as a marker for seed quality in Arabidopsis seeds.

Seed quality is affected by different constituents of the seed. In general, seed lots are considered to be of high quality when they exhibit fast and homogeneous germination. When seeds are stored, they undergo different degrees of damage that have detrimental effects on their quality. Therefore, accurate prediction of the seed quality and viability levels of a seed lot is of high importance in the seed-producing industry. Here, we describe the use of activity-based protein profiling of proteases to evaluate the quality of artificially and naturally aged seeds of Arabidopsis thaliana Using this approach, we have identified two protease activities with opposite behaviours in aged seeds of Arabidopsis that correlate with the quality status of the seeds. We show that vacuolar processing enzymes (VPEs) become more active during the ageing process, in both artificial and natural ageing treatments. Secondly, we demonstrate that serine hydrolases are active at the beginning of our artificial ageing treatment, but their labelling decreases along with seed viability. We present a list of candidate hydrolases active during seed germination and propose that these protease activities can be used in combination with VPEs to develop novel markers of seed quality.