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• The N-end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. • Here we explored the role of the N-end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. • We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln-specific amino-terminal (Nt)-amidase (NTAQ1) controls the expression of specific defence-response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt-Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen-induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. • We propose that that separate branches of the N-end rule pathway act as distinct components of the plant immune response in flowering plants.

Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

Extracellular vesicles (EVs), nanoscale vesicles that are secreted by cells, are critical mediators of intercellular communication and play a crucial role in diverse pathologies such as cancer development. Therefore, EVs are regarded as having high potential in the clinic, both for diagnostic and therapeutic applications. Unfortunately, EVs reside in complex biofluids and their consistent preparation at sufficient purity for mass spectrometry‐based proteomics has proven to be challenging, especially when increased high‐throughput is required. Here, we describe the incorporation of our previously reported filter‐aided EV enrichment (FAEVEr) strategy for the separation of EVs from conditioned medium, from harvest to proteomic analysis completely to a streamlined 96‐well format. We compared our approach with ultracentrifugation, the most widely used method for EV enrichment, in terms of protein identifications, consistency, reproducibility and overall performance, including the invested time, resources and required expertise. In addition, our results show that including relative high percentages of Tween‐20, a mild detergent, markedly improves the final purity of the EV proteome by removing the bulk of non‐EV proteins (e.g., serum proteins) and significantly increases the number of identified transmembrane proteins. Moreover, our FAEVEr 96‐well strategy improves the overall reproducibility with a consistent number of protein identifications and decreased number of missing values across replicates. This promotes the validity and comparability between results, which is essential in both a clinical and research setting, where consistency is paramount. Schematic overview of the FAEVEr 96‐well workflow: Small volumes of conditioned medium are pre‐cleared, followed by enrichment and purification of extracellular vesicles (EVs) for either EV proteome preparation or recovery for downstream EV characterization, all within a streamlined 96‐well format.

Background Long-term drug evaluation heavily relies upon rodent models. Drug discovery methods to reduce animal models in oncology may include three-dimensional (3D) cellular systems that take into account tumor microenvironment (TME) cell types and biomechanical properties. Methods In this study we reconstructed a 3D tumor using an elastic polymer (acrylate-endcapped urethane-based poly(ethylene glycol) (AUPPEG)) with clinical relevant stiffness. Single cell suspensions from low-grade serous ovarian cancer (LGSOC) patient-derived early passage cultures of cancer cells and cancer-associated fibroblasts (CAF) embedded in a collagen gel were introduced to the AUPPEG scaffold. After self-organization in to a 3D tumor, this model was evaluated by a long-term (> 40 days) exposure to a drug combination of MEK and HSP90 inhibitors. The drug-response results from this long-term in vitro model are compared with drug responses in an orthotopic LGSOC xenograft mouse model. Results The in vitro 3D scaffold LGSOC model mimics the growth ratio and spatial organization of the LGSOC. The AUPPEG scaffold approach allows to test new targeted treatments and monitor long-term drug responses. The results correlate with those of the orthotopic LGSOC xenograft mouse model. Conclusions The mechanically-tunable scaffolds colonized by a three-dimensional LGSOC allow long-term drug evaluation and can be considered as a valid alternative to reduce, replace and refine animal models in drug discovery. Graphical Abstract

In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that induction of a heat shock-related stress response mediated by Foldlin, a small-molecule tool compound, reduces the protein levels of misfolded/aggregated mutant p53, while contact mutants or wild-type p53 remain largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. Despite our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems to be an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.

Despite the growing interest in using chemical genetics in plant research, small molecule target identification remains a major challenge. The cellular thermal shift assay coupled with high-resolution mass spectrometry (CETSA MS) that monitors changes in the thermal stability of proteins caused by their interactions with small molecules, other proteins, or posttranslational modifications, allows the discovery of drug targets or the study of protein–metabolite and protein–protein interactions mainly in mammalian cells. To showcase the applicability of this method in plants, we applied CETSA MS to intact Arabidopsis thaliana cells and identified the thermal proteome of the plant-specific glycogen synthase kinase 3 (GSK3) inhibitor, bikinin. A comparison between the thermal and the phosphoproteomes of bikinin revealed the auxin efflux carrier PIN-FORMED1 (PIN1) as a substrate of the Arabidopsis GSK3s that negatively regulate the brassinosteroid signaling. We established that PIN1 phosphorylation by the GSK3s is essential for maintaining its intracellular polarity that is required for auxin-mediated regulation of vascular patterning in the leaf, thus revealing cross-talk between brassinosteroid and auxin signaling.

The N‐end rule pathway is a highly conserved constituent of the ubiquitin proteasome system, yet little is known about its biological roles. Here we explored the role of the N‐end rule pathway in the plant immune response. We investigated the genetic influences of components of the pathway and known protein substrates on physiological, biochemical and metabolic responses to pathogen infection. We show that the glutamine (Gln) deamidation and cysteine (Cys) oxidation branches are both components of the plant immune system, through the E3 ligase PROTEOLYSIS (PRT)6. In Arabidopsis thaliana Gln‐specific amino‐terminal (Nt)‐amidase (NTAQ1) controls the expression of specific defence‐response genes, activates the synthesis pathway for the phytoalexin camalexin and influences basal resistance to the hemibiotroph pathogen Pseudomonas syringae pv tomato (Pst). The Nt‐Cys ETHYLENE RESPONSE FACTOR VII transcription factor substrates enhance pathogen‐induced stomatal closure. Transgenic barley with reduced HvPRT6 expression showed enhanced resistance to Ps. japonica and Blumeria graminis f. sp. hordei, indicating a conserved role of the pathway. We propose that that separate branches of the N‐end rule pathway act as distinct components of the plant immune response in flowering plants.

Purpose: To identify a molecular signature of macrophages exposed to clinically relevant ionizing radiation (IR) doses, mirroring radiotherapy sessions. Methods: Human monocyte-derived macrophages were exposed to 2 Gy/ fraction/ day for 5 days, mimicking one week of cancer patient’s radiotherapy. Protein expression profile by proteomics was performed. Results: A gene ontology analysis revealed that radiation-induced protein changes are associated with metabolic alterations, which were further supported by a reduction of both cellular ATP levels and glucose uptake. Most of the radiation-induced deregulated targets exhibited a decreased expression, as was the case of cathepsin D, a lysosomal protease associated with cell death, which was validated by Western blot. We also found that irradiated macrophages exhibited an increased expression of the transferrin receptor 1 (TfR1), which is responsible for the uptake of transferrin-bound iron. TfR1 upregulation was also found in tumor-associated mouse macrophages upon tumor irradiation. In vitro irradiated macrophages also presented a trend for increased divalent metal transporter 1 (DMT1), which transports iron from the endosome to the cytosol, and a significant increase in iron release. Conclusions: Irradiated macrophages present lower ATP levels and glucose uptake, and exhibit decreased cathepsin D expression, while increasing TfR1 expression and altering iron metabolism.

Extracellular vesicles (EVs), nanoscale vesicles that are secreted by cells, are critical mediators of intercellular communication and play a crucial role in diverse pathologies such as cancer development. Therefore, EVs are regarded as having high potential in the clinic, both for diagnostic and therapeutic applications. Unfortunately, EVs reside in complex biofluids and their consistent isolation at sufficient purity for mass spectrometry-based proteomics has proven to be challenging, especially when increased high-throughput is required. Here, we describe the incorporation of our previously reported filter-aided EV enrichment (FAEVEr) strategy for the isolation of EVs from conditioned medium, from harvest to proteomic analysis completely to a streamlined 96well format. We compared our approach with ultracentrifugation, the most widely used method for EV enrichment, in terms of protein identifications, consistency, reproducibility and overall performance, including the invested time, resources and required expertise. In addition, our results show that including relative high percentages of TWEEN-20, a mild detergent, markedly improves the final purity of the EV proteome by removing the bulk of non-EV proteins (e.g. serum proteins) and significantly increases the number of identified transmembrane proteins. Moreover, our FAEVEr 96well strategy improves the overall reproducibility with a consistent number of protein identifications and decreased number of missing values across replicates. This promotes the validity and comparability between results, which is essential in both a clinical and research setting, where consistency is paramount.Competing Interest StatementThe authors have declared no competing interest.

Positively charged repeat peptides are emerging as key players in neurodegenerative diseases. These peptides can perturb diverse cellular pathways but a unifying framework for how such promiscuous toxicity arises has remained elusive. We used mass-spectrometry-based proteomics to define the protein targets of these neurotoxic peptides and found that they all share similar sequence features that drive their aberrant condensation with these positively charged peptides. We trained a machine learning algorithm to detect such sequence features and unexpectedly discovered that this mode of toxicity is not limited to human repeat expansion disorders but has evolved countless times across the tree of life in the form of cationic antimicrobial and venom peptides. We demonstrate that an excess in positive charge is necessary and sufficient for this killer activity, which we name 'polycation poisoning'. These findings reveal an ancient and conserved mechanism and inform ways to leverage its design rules for new generations of bioactive peptides.