Search

Abstract Proteogenomics is becoming a powerful tool in personalized medicine by linking genomics, transcriptomics and mass spectrometry (MS)-based proteomics. Due to increasing evidence of alternative open reading frame-encoded proteins (AltProts), proteogenomics has a high potential to unravel the characteristics, variants, expression levels of the alternative proteome, in addition to already annotated proteins (RefProts). To obtain a broader view of the proteome of ovarian cancer cells compared to ovarian epithelial cells, cell-specific total RNA-sequencing profiles and customized protein databases were generated. In total, 128 RefProts and 30 AltProts were identified exclusively in SKOV-3 and PEO-4 cells. Among them, an AltProt variant of IP_715944, translated from DHX8, was found mutated (p.Leu44Pro). We show high variation in protein expression levels of RefProts and AltProts in different subcellular compartments. The presence of 117 RefProt and two AltProt variants was described, along with their possible implications in the different physiological/pathological characteristics. To identify the possible involvement of AltProts in cellular processes, cross-linking-MS (XL-MS) was performed in each cell line to identify AltProt-RefProt interactions. This approach revealed an interaction between POLD3 and the AltProt IP_183088, which after molecular docking, was placed between POLD3-POLD2 binding sites, highlighting its possibility of the involvement in DNA replication and repair.

Plants express an array of receptor-like kinases (RLKs) to control development and communicate with their environment. Many RLKs are uncharacterized and some of them are expected to regulate plant responses to plant growth-promoting rhizobacteria (PGPR). Despite documented effects induced by Caulobacter RHG1, the underlying signaling pathways and the involved RLKs remain uncharted. Through a targeted RLK mutant screening, we aimed to decipher the receptors that steer the Caulobacter RHG1-induced growth promotion in Arabidopsis thaliana. We identified four RLKs that are pivotal in the RHG1-Arabidopsis interaction, including the coreceptors SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) and BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1/SERK3), which act redundantly in the RHG1-Arabidopsis interaction, possibly by interplaying with the unknown RLK AT3G28040 and the immunity-related ELONGATION FACTOR-TU RECEPTOR (EFR). These results shed new light on the molecular dynamics orchestrating plant responses to PGPR, and concurrently contribute a crucial piece to the intricate puzzle of RLK interactions.Key policy highlights Four RLKs; BAK1, SERK1, EFR, and AT3G28040 (RRHG) are involved in the RHG1-Arabidopsis interaction.BAK1 and SERK1, two well-described co-receptors, act redundantly and play a pivotal role in the RHG1-driven growth promotion, possibly by interplaying with the unknown RLK AT3G28040 and the immunity-related RLK EFR.Most known development – and immunity-related RLKs barely influence RHG1-driven plant growth promotion in Arabidopsis.

Abstract To survive extreme desiccation, seeds enter a period of quiescence that can last millennia. Seed quiescence involves the accumulation of protective storage proteins and lipids through unknown adjustments in protein homeostasis (proteostasis). Here, we show that mutation of all six type–II metacaspase (MCA–II) proteases in Arabidopsis thaliana disturbs proteostasis in seeds. MCA–II mutant seeds fail to restrict the AAA ATPase CELL DIVISION CYCLE 48 (CDC48) at the endoplasmic reticulum to discard misfolded proteins, compromising seed storability. Endoplasmic reticulum (ER) localization of CDC48 relies on the MCA–IIs-dependent cleavage of PUX10 (ubiquitination regulatory X domain–containing 10), the adaptor protein responsible for titrating CDC48 to lipid droplets. PUX10 cleavage enables the shuttling of CDC48 between lipid droplets and the ER, providing an important regulatory mechanism sustaining spatiotemporal proteolysis, lipid droplet dynamics, and protein homeostasis. In turn, the removal of the PUX10 adaptor in MCA–II mutant seeds partially restores proteostasis, CDC48 localization, and lipid droplet dynamics prolonging seed lifespan. Taken together, we uncover a proteolytic module conferring seed longevity.

Abstract Background European beech (Fagus sylvatica L.) trees produce seeds irregularly; therefore, it is necessary to store beech seeds for forestation. Despite the acquisition of desiccation tolerance during development, beech seeds are classified as intermediate because they lose viability during long-term storage faster than typical orthodox seeds. In this study, beech seeds stored for short (3 years) or long (20 years) periods under optimal conditions and displaying 92 and 30% germination capacity, respectively, were compared. Results Aged seeds displayed increased membrane damage, manifested as electrolyte leakage and lipid peroxidation levels. Analyses have been based on embryonic axes, which contained higher levels of reactive oxygen species (ROS) and higher levels of protein-bound methionine sulfoxide (MetO) in aged seeds. Using label-free quantitative proteomics, 3,949 proteins were identified, of which 2,442 were reliably quantified pointing to 24 more abundant proteins and 35 less abundant proteins in beech seeds under long-term storage conditions. Functional analyses based on gene ontology annotations revealed that nucleic acid binding activity (molecular function), ribosome organization or biogenesis and transmembrane transport (cellular processes), translational proteins (protein class) and membranous anatomical entities (cellular compartment) were affected in aged seeds. To verify whether MetO, the oxidative posttranslational modification of proteins that can be reversed via the action of methionine sulfoxide reductase (Msr) enzymes, is involved in the aging of beech seeds, we identified and quantified 226 MetO-containing proteins, among which 9 and 19 exhibited significantly up- and downregulated MetO levels, respectively, in beech seeds under long-term storage conditions. Several Msr isoforms were identified and recognized as MsrA1-like, MsrA4, MsrB5 and MsrB5-like in beech seeds. Only MsrA1-like displayed decreased abundance in aged seeds. Conclusions We demonstrated that the loss of membrane integrity reflected in the elevated abundance of membrane proteins had a higher impact on seed aging progress than the MetO/Msr system. Proteome analyses enabled us to propose protein Sec61 and glyceraldehyde-3-phosphate dehydrogenase as potential longevity modulators in beech seeds.

Abstract Transglutaminase 1 (TGM1) plays an essential role in skin barrier formation by cross-linking proteins in differentiated keratinocytes. Here, we established a protocol for the antibody-dependent detection of TGM1 protein and the parallel detection of TGM activity. TGM1 immunoreactivity initially increased and co-localized with membrane-associated TGM activity during keratinocyte differentiation. TGM activity persisted upon further differentiation of keratinocytes, whereas TGM1 immunoreactivity was lost under standard assay conditions. Pretreatment of tissue sections with the proteases trypsin or proteinase K enabled immunodetection of TGM1 in cornified keratinocytes, indicating that removal of other proteins was a prerequisite for TGM1 immunolabeling after cornification. The increase of TGM activity and subsequent loss of TGM1 immunoreactivity could be replicated in HEK293T cells transfected with TGM1, suggesting that protein cross-linking mediated by TGM1 itself may lead to reduced recognition of TGM1 by antibodies. To screen for proteins potentially regulating TGM1, we performed Virotrap experiments and identified the CAPNS1 subunit of calpain as an interaction partner of TGM1. Treatment of keratinocytes and TGM1-transfected HEK293T cells with chemical inhibitors of calpain suppressed transglutamination. Our findings suggest that calpain contributes to the control of TGM1-mediated transglutamination and proteins cross-linked by transglutamination mask epitopes of TGM1.

Abstract Public proteomics data often lack essential metadata, limiting its potential. To address this, we present lesSDRF, a tool to simplify the process of metadata annotation, thereby ensuring that data leave a lasting, impactful legacy well beyond its initial publication.

Abstract Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

Abstract 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

Abstract Addressing the elusive specificity of cysteine cathepsins, which in contrast to caspases and trypsin-like proteases lack strict specificity determining P1 pocket, calls for innovative approaches. Proteomic analysis of cell lysates with human cathepsins K, V, B, L, S, and F identified 30,000 cleavage sites, which we analyzed by software platform SAPS-ESI (Statistical Approach to Peptidyl Substrate-Enzyme Specific Interactions). SAPS-ESI is used to generate clusters and training sets for support vector machine learning. Cleavage site predictions on the SARS-CoV-2 S protein, confirmed experimentally, expose the most probable first cut under physiological conditions and suggested furin-like behavior of cathepsins. Crystal structure analysis of representative peptides in complex with cathepsin V reveals rigid and flexible sites consistent with analysis of proteomics data by SAPS-ESI that correspond to positions with heterogeneous and homogeneous distribution of residues. Thereby support for design of selective cleavable linkers of drug conjugates and drug discovery studies is provided.

Abstract Heparan sulfates (HS) proteoglycans are commonly found on the cell surface and mediate many processes. Binding of HS ligands is determined by the sulfation code on the HS chain that can be N-/2-O/6-O- or 3-O-sulfated, generating heterogenous sulfation patterns. 3-O sulfated HS (3S-HS) play a role in several (patho)physiological processes such as blood coagulation, viral pathogenesis and binding and internalization of tau in Alzheimer’s disease. However, few 3S-HS-specific interactors are known. Thus, our insight into the role of 3S-HS in health and disease is limited, especially in the central nervous system. Using human CSF, we determined the interactome of synthetic HS with defined sulfation patterns. Our affinity-enrichment mass spectrometry studies expand the repertoire of proteins that may interact with (3S-)HS. Validating our approach, ATIII, a known 3S-HS interactor, was found to require GlcA-GlcNS6S3S for binding, similar to what has been reported. Our dataset holds novel, potential HS and 3S-HS protein ligands, that can be explored in future studies focusing on molecular mechanisms that depend on 3S-HS in (patho)physiological conditions.

Feeding an increasing world population in the context of climate change is one of the grand challenges faced by our generation. Here, the authors show that external application of synthetic complementary peptides can increase the abundance of target proteins to modulate plant growth or stress resistance.

Protein cysteinyl thiols are susceptible to reduction-oxidation reactions that can influence protein function. Accurate quantification of cysteine oxidation is therefore crucial for decoding protein redox regulation. Here, we present CysQuant, a novel approach for simultaneous quantification of cysteine oxidation degrees and protein abundancies. CysQuant involves light/heavy iodoacetamide isotopologues for differential labeling of reduced and reversibly oxidized cysteines analyzed by data-dependent acquisition (DDA) or data-independent acquisition mass spectrometry (DIA-MS). Using plexDIA with in silico predicted spectral libraries, we quantified an average of 18% cysteine oxidation in Arabidopsis thaliana by DIA-MS, including a subset of highly oxidized cysteines forming disulfide bridges in AlphaFold2 predicted structures. Applying CysQuant to Arabidopsis seedlings exposed to excessive light, we successfully quantified the well-established increased reduction of Calvin-Benson cycle enzymes and discovered yet uncharacterized redox-sensitive disulfides in chloroplastic enzymes. Overall, CysQuant is a highly versatile tool for assessing the cysteine modification status that can be widely applied across various mass spectrometry platforms and organisms.

Summary: Eukaryotic mRNA has long been considered monocistronic, but nowadays, alternative proteins (AltProts) challenge this tenet. The alternative or ghost proteome has largely been neglected and the involvement of AltProts in biological processes. Here, we used subcellular fractionation to increase the information about AltProts and facilitate the detection of protein-protein interactions by the identification of crosslinked peptides. In total, 112 unique AltProts were identified, and we were able to identify 220 crosslinks without peptide enrichment. Among these, 16 crosslinks between AltProts and Referenced Proteins (RefProts) were identified. We further focused on specific examples such as the interaction between IP_2292176 (AltFAM227B) and HLA-B, in which this protein could be a potential new immunopeptide, and the interactions between HIST1H4F and several AltProts which can play a role in mRNA transcription. Thanks to the study of the interactome and the localization of AltProts, we can reveal more of the importance of the ghost proteome.

Abstract Increasing evidence indicates that extracellular vesicles (EVs) play an important role in the pathogenesis of Alzheimer’s disease (AD). We previously reported that the blood–cerebrospinal fluid (CSF) interface, formed by the choroid plexus epithelial (CPE) cells, releases an increased amount of EVs into the CSF in response to peripheral inflammation. Here, we studied the importance of CP-mediated EV release in AD pathogenesis. We observed increased EV levels in the CSF of young transgenic APP/PS1 mice which correlated with high amyloid beta (Aβ) CSF levels at this age. The intracerebroventricular (icv) injection of Aβ oligomers (AβO) in wild-type mice revealed a significant increase of EVs in the CSF, signifying that the presence of CSF-AβO is sufficient to induce increased EV secretion. Using in vivo, in vitro and ex vivo approaches, we identified the CP as a major source of the CSF-EVs. Interestingly, AβO-induced, CP-derived EVs induced pro-inflammatory effects in mixed cortical cultures. Proteome analysis of these EVs revealed the presence of several pro-inflammatory proteins, including the complement protein C3. Strikingly, inhibition of EV production using GW4869 resulted in protection against acute AβO-induced cognitive decline. Further research into the underlying mechanisms of this EV secretion might open up novel therapeutic strategies to impact the pathogenesis and progression of AD.

Plants respond to warmth via growth processes termed thermomorphogenesis. Here, via a phosphoproteomics approach, the authors show that the mitogen activated protein kinase TOT3 regulates thermomorphogenesis in both wheat and Arabidopsis and modifies brassinosteroid signaling in Arabidopsis.

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.

There is no universal reference material to develop extracellular vesicle (EV) separation methods and carry out calibration and normalization. Here the authors use HIV-derived gag proteins to assemble recombinant fluorescent EV as a trackable reference material resembling the physical and biochemical properties of sample EV.

Abstract Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size‐exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context‐dependent and time‐dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context‐dependent and/or time‐dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.

Summary: M2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Serving a robust platform for reverse genetics enabling the in vivo study of gene functions primarily in enterobacteriaceae, recombineering -or recombination-mediated genetic engineering-represents a powerful and relative straightforward genetic engineering tool. Catalyzed by components of bacteriophage-encoded homologous recombination systems and only requiring short ∼40–50 base homologies, the targeted and precise introduction of modifications (e.g., deletions, knockouts, insertions and point mutations) into the chromosome and other episomal replicons is empowered. Furthermore, by its ability to make use of both double- and single-stranded linear DNA editing substrates (e.g., PCR products or oligonucleotides, respectively), lengthy subcloning of specific DNA sequences is circumvented. Further, the more recent implementation of CRISPR-associated endonucleases has allowed for more efficient screening of successful recombinants by the selective purging of non-edited cells, as well as the creation of markerless and scarless mutants. In this review we discuss various recombineering strategies to promote different types of gene modifications, how they are best applied, and their possible pitfalls.

Exogenous glucocorticoids are widely used in the clinic for the treatment of inflammatory disorders and hematological cancers. Unfortunately, their use is associated with debilitating side effects, including hyperglycemia, osteoporosis, mood swings, and weight gain. Despite the continued efforts of pharma as well as academia, the search for so-called selective glucocorticoid receptor modulators (SEGRMs), compounds with strong anti-inflammatory or anti-cancer properties but a reduced number or level of side effects, has had limited success so far. Although monoclonal antibody therapies have been successfully introduced for the treatment of certain disorders (such as anti-TNF for rheumatoid arthritis), glucocorticoids remain the first-in-line option for many other chronic diseases including asthma, multiple sclerosis, and multiple myeloma. This perspective offers our opinion on why a continued search for SEGRMs remains highly relevant in an era where small molecules are sometimes unrightfully considered old-fashioned. Besides a discussion on which bottlenecks and pitfalls might have been overlooked in the past, we elaborate on potential solutions and recent developments that may push future research in the right direction.

Protein ubiquitination is a very diverse post-translational modification leading to protein degradation or delocalization, or altering protein activity. In Arabidopsis thaliana, two E3 ligases, BIG BROTHER (BB) and DA2, activate the latent peptidases DA1, DAR1 and DAR2 by mono-ubiquitination at multiple sites. Subsequently, these activated peptidases destabilize various positive growth regulators. Here, we show that two ubiquitin-specific proteases, UBP12 and UBP13, deubiquitinate DA1, DAR1 and DAR2, hence reducing their peptidase activity. Overexpression of UBP12 or UBP13 strongly decreased leaf size and cell area, and resulted in lower ploidy levels. Mutants in which UBP12 and UBP13 were downregulated produced smaller leaves that contained fewer and smaller cells. Remarkably, neither UBP12 nor UBP13 were found to be cleavage substrates of the activated DA1. Our results therefore suggest that UBP12 and UBP13 work upstream of DA1, DAR1 and DAR2 to restrict their protease activity and hence fine-tune plant growth and development.

Abstract The multi-domain transcriptional coactivators CBP/p300 integrate a multitude of signaling inputs, interacting with more than 400 proteins via one or more of their globular domains. While CBP/p300 function is typically considered in terms of these structured domains, about half of the protein consists of intrinsically disordered regions (IDRs) of varying length. However, these IDRs have only been thought of as linkers that allow flexible spatial arrangement of the structured domains, but recent studies have shown that similar IDRs mediate specific and critical interactions in other proteins. To examine the roles of IDRs in CBP, we performed yeast-two-hybrid screenings of placenta and lung cancer cDNA libraries, which demonstrated that the long IDR linking the KIX domain and bromodomain of CBP (termed ID3) can potentially bind to several proteins. The RNA-binding Zinc-finger protein 106 (ZFP106) detected in both libraries was identified as a novel substrate for CBP-mediated acetylation. Nuclear magnetic resonance (NMR) spectroscopy combined with cross-linking experiments and competition-binding assays showed that the fully disordered isolated ID3 transiently interacts with an IDR of ZFP106 in a fashion that disorder of both regions is maintained. These findings demonstrate that beside the linking function, ID3 can also interact with acetylation substrates of CBP.

Abstract Identification and validation of extracellular vesicle (EV)-associated biomarkers requires robust isolation and characterization protocols. We assessed the impact of some commonly implemented pre-analytical, analytical and post-analytical variables in EV research. Centrifugal filters with different membrane types and pore sizes are used to reduce large volume biofluids prior to EV isolation or to concentrate EVs. We compared five commonly reported filters for their efficiency when using plasma, urine and EV-spiked PBS. Regenerated cellulose membranes with pore size of 10 kDa recovered EVs the most efficient. Less than 40% recovery was achieved with other filters. Next, we analyzed the effect of the type of protein assays to measure EV protein in colorimetric and fluorometric kits. The fluorometric assay Qubit measured low concentration EV and BSA samples the most accurately with the lowest variation among technical and biological replicates. Lastly, we quantified Optiprep remnants in EV samples from density gradient ultracentrifugation and demonstrate that size-exclusion chromatography efficiently removes Optiprep from EVs. In conclusion, choice of centrifugal filters and protein assays confound EV analysis and should be carefully considered to increase efficiency towards biomarker discovery. SEC-based removal of Optiprep remnants from EVs can be considered for downstream applications.

The plant circadian clock regulates numerous developmental processes such as seasonal growth and flowering time. Here Ritteret al. identify two transcription factors, FRS7 and FRS12, which interact to form a repressor complex that regulates clock output partially by controlling the expression of GIGANTEA and PIF4.

The serine/threonine protein kinase AKT is frequently over-activated in cancer and is associated with poor prognosis. As a central node in the PI3K/AKT/mTOR pathway, which regulates various processes considered to be hallmarks of cancer, this kinase has become a prime target for cancer therapy. However, AKT has proven to be a highly complex target as it comes in three isoforms (AKT1, AKT2 and AKT3) which are highly homologous, yet non-redundant. The isoform-specific functions of the AKT kinases can be dependent on context (i.e. different types of cancer) and even opposed to one another. To date, there is no isoform-specific inhibitor available and no alternative to genetic approaches to study the function of a single AKT isoform. We have developed and characterized nanobodies that specifically interact with the AKT1 or AKT2 isoforms. These new tools should enable future studies of AKT1 and AKT2 isoform-specific functions. Furthermore, for both isoforms we obtained a nanobody that interferes with the AKT-PIP3-interaction, an essential step in the activation of the kinase. The nanobodies characterized in this study are a new stepping stone towards unravelling AKT isoform-specific signalling.

BackgroundSalmonella enterica subsp. enterica contains more than 2,600 serovars of which four are of major medical relevance for humans. While the typhoidal serovars (Typhi and Paratyphi A) are human-restricted and cause enteric fever, non-typhoidal Salmonella serovars (Typhimurium and Enteritidis) have a broad host range and predominantly cause gastroenteritis.Methodology/principle findingsWe compared the core proteomes of Salmonella Typhi, Paratyphi A, Typhimurium and Enteritidis using contemporary proteomics. For each serovar, five clinical isolates (covering different geographical origins) and one reference strain were grown in vitro to the exponential phase. Levels of orthologous proteins quantified in all four serovars and within the typhoidal and non-typhoidal groups were compared and subjected to gene ontology term enrichment and inferred regulatory interactions. Differential expression of the core proteomes of the typhoidal serovars appears mainly related to cell surface components and, for the non-typhoidal serovars, to pathogenicity.Conclusions/significanceOur comparative proteome analysis indicated differences in the expression of surface proteins between Salmonella Typhi and Paratyphi A, and in pathogenesis-related proteins between Salmonella Typhimurium and Enteritidis. Our findings may guide future development of novel diagnostics and vaccines, as well as understanding of disease progression.

Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.

Abstract Here, we identified release of extracellular vesicles (EVs) by the choroid plexus epithelium (CPE) as a new mechanism of blood–brain communication. Systemic inflammation induced an increase in EVs and associated pro‐inflammatory miRNAs, including miR‐146a and miR‐155, in the CSF. Interestingly, this was associated with an increase in amount of multivesicular bodies (MVBs) and exosomes per MVB in the CPE cells. Additionally, we could mimic this using LPS‐stimulated primary CPE cells and choroid plexus explants. These choroid plexus‐derived EVs can enter the brain parenchyma and are taken up by astrocytes and microglia, inducing miRNA target repression and inflammatory gene up‐regulation. Interestingly, this could be blocked in vivo by intracerebroventricular (icv) injection of an inhibitor of exosome production. Our data show that CPE cells sense and transmit information about the peripheral inflammatory status to the central nervous system (CNS) via the release of EVs into the CSF, which transfer this pro‐inflammatory message to recipient brain cells. Additionally, we revealed that blockage of EV secretion decreases brain inflammation, which opens up new avenues to treat systemic inflammatory diseases such as sepsis.

Although several ADAMs (A disintegrin-like and metalloproteases) have been shown to contribute to the amyloid precursor protein (APP) metabolism, the full spectrum of metalloproteases involved in this metabolism remains to be established. Transcriptomic analyses centred on metalloprotease genes unraveled a 50% decrease in ADAM30 expression that inversely correlates with amyloid load in Alzheimer's disease brains. Accordingly, in vitro down- or up-regulation of ADAM30 expression triggered an increase/decrease in Aβ peptides levels whereas expression of a biologically inactive ADAM30 (ADAM30mut) did not affect Aβ secretion. Proteomics/cell-based experiments showed that ADAM30-dependent regulation of APP metabolism required both cathepsin D (CTSD) activation and APP sorting to lysosomes. Accordingly, in Alzheimer-like transgenic mice, neuronal ADAM30 over-expression lowered Aβ42 secretion in neuron primary cultures, soluble Aβ42 and amyloid plaque load levels in the brain and concomitantly enhanced CTSD activity and finally rescued long term potentiation alterations. Our data thus indicate that lowering ADAM30 expression may favor Aβ production, thereby contributing to Alzheimer's disease development.

A large portion of the proteome carries out its cellular function as part of macromolecular complexes. Here the authors describe Virotrap, a novel lysis-free approach for the isolation and identification of biologically relevant protein-protein and small molecule-protein interactions.

Alterations of hydrogen peroxide (H2O2) levels have a profound impact on numerous signaling cascades orchestrating plant growth, development, and stress signaling, including programmed cell death. To expand the repertoire of known molecular mechanisms implicated in H2O2 signaling, we performed a forward chemical screen to identify small molecules that could alleviate the photorespiratory-induced cell death phenotype of Arabidopsisthaliana mutants lacking H2O2-scavenging capacity by peroxisomal catalase2. Here, we report the characterization of pakerine, an m-sulfamoyl benzamide from the sulfonamide family. Pakerine alleviates the cell death phenotype of cat2 mutants exposed to photorespiration-promoting conditions and delays dark-induced senescence in wild-type Arabidopsis leaves. By using a combination of transcriptomics, metabolomics, and affinity purification, we identified abnormal inflorescence meristem 1 (AIM1) as a putative protein target of pakerine. AIM1 is a 3-hydroxyacyl-CoA dehydrogenase involved in fatty acid β-oxidation that contributes to jasmonic acid (JA) and salicylic acid (SA) biosynthesis. Whereas intact JA biosynthesis was not required for pakerine bioactivity, our results point toward a role for β-oxidation-dependent SA production in the execution of H2O2-mediated cell death.

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

Homologous recombination is central to repair DNA double-strand breaks, either accidently arising in mitotic cells or in a programed manner at meiosis. Crossovers resulting from the repair of meiotic breaks are essential for proper chromosome segregation and increase genetic diversity of the progeny. However, mechanisms regulating crossover formation remain elusive. Here, we identified through genetic and protein-protein interaction screens FIDGETIN-LIKE-1 INTERACTING PROTEIN (FLIP) as a new partner of the previously characterized anti-crossover factor FIDGETIN-LIKE-1 (FIGL1) in Arabidopsis thaliana. We showed that FLIP limits meiotic crossover together with FIGL1. Further, FLIP and FIGL1 form a protein complex conserved from Arabidopsis to human. FIGL1 interacts with the recombinases RAD51 and DMC1, the enzymes that catalyze the DNA strand exchange step of homologous recombination. Arabidopsis flip mutants recapitulate the figl1 phenotype, with enhanced meiotic recombination associated with change in counts of DMC1 and RAD51 foci. Our data thus suggests that FLIP and FIGL1 form a conserved complex that regulates the crucial step of strand invasion in homologous recombination.

Phytohormones tightly regulate plant growth by integrating changing environmental and developmental cues. Although the key players have been identified in many plant hormonal pathways, the molecular mechanisms and mode of action of perception and signaling remain incompletely resolved. Characterization of protein partners of known signaling components provides insight into the formed protein complexes, but, unless quantification is involved, does not deliver much, if any, information about the dynamics of the induced or disrupted protein complexes. Therefore, in proteomics research, the discovery of what actually triggers, regulates or interrupts the composition of protein complexes is gaining importance. Here, tandem affinity purification coupled to mass spectrometry (TAP-MS) is combined with label-free quantification (LFQ) to a highly valuable tool to detect physiologically relevant, dynamic protein–protein interactions in Arabidopsis thaliana cell cultures. To demonstrate its potential, we focus on the signaling pathway of one of the most recently discovered phytohormones, strigolactones.

Abstract To understand the impact of alternative translation initiation on a proteome, we performed a proteome‐wide study on protein turnover using positional proteomics and ribosome profiling to distinguish between N‐terminal proteoforms of individual genes. By combining pulsed SILAC with N‐terminal COFRADIC, we monitored the stability of 1,941 human N‐terminal proteoforms, including 147 N‐terminal proteoform pairs that originate from alternative translation initiation, alternative splicing or incomplete processing of the initiator methionine. N‐terminally truncated proteoforms were less abundant than canonical proteoforms and often displayed altered stabilities, likely attributed to individual protein characteristics, including intrinsic disorder, but independent of N‐terminal amino acid identity or truncation length. We discovered that the removal of initiator methionine by methionine aminopeptidases reduced the stability of processed proteoforms, while susceptibility for N‐terminal acetylation did not seem to influence protein turnover rates. Taken together, our findings reveal differences in protein stability between N‐terminal proteoforms and point to a role for alternative translation initiation and co‐translational initiator methionine removal, next to alternative splicing, in the overall regulation of proteome homeostasis.

N-terminal acetylation is a major and vital protein modification catalyzed by N-terminal acetyltransferases (NATs). NatF, or Nα-acetyltransferase 60 (Naa60), was recently identified as a NAT in multicellular eukaryotes. Here, we find that Naa60 differs from all other known NATs by its Golgi localization. A new membrane topology assay named PROMPT and a selective membrane permeabilization assay established that Naa60 faces the cytosolic side of intracellular membranes. An Nt-acetylome analysis of NAA60-knockdown cells revealed that Naa60, as opposed to other NATs, specifically acetylates transmembrane proteins and has a preference for N termini facing the cytosol. Moreover, NAA60 knockdown causes Golgi fragmentation, indicating an important role in the maintenance of the Golgi’s structural integrity. This work identifies a NAT associated with membranous compartments and establishes N-terminal acetylation as a common modification among transmembrane proteins, a thus-far poorly characterized part of the N-terminal acetylome.

Synthetic glucocorticoids (GC) are the mainstay therapy for treatment of acute and chronic inflammatory disorders. Due to the high adverse effects associated with long-term use, GC pharmacology has focused since the nineties on more selective GC ligand-binding strategies, classified as selective glucocorticoid receptor (GR) agonists (SEGRAs) or selective glucocorticoid receptor modulators (SEGRMs). In the current study, GSK866 analogs with electrophilic covalent-binding warheads were developed with potential SEGRA properties to improve their clinical safety profile for long-lasting topical skin disease applications. Since the off-rate of a covalently binding drug is negligible compared to that of a non-covalent drug, its therapeutic effects can be prolonged and typically, smaller doses of the drug are necessary to reach the same level of therapeutic efficacy, thereby potentially reducing systemic side effects. Different analogs of SEGRA GSK866 coupled to cysteine reactive warheads were characterized for GR potency and selectivity in various biochemical and cellular assays. GR- and NFκB-dependent reporter gene studies show favorable anti-inflammatory properties with reduced GR transactivation of two non-steroidal GSK866 analogs UAMC-1217 and UAMC-1218, whereas UAMC-1158 and UAMC-1159 compounds failed to modulate cellular GR activity. These results were further supported by GR immuno-localization and S211 phospho-GR western analysis, illustrating significant GR phosphoactivation and nuclear translocation upon treatment of GSK866, UAMC-1217, or UAMC-1218, but not in case of UAMC-1158 or UAMC-1159. Furthermore, mass spectrometry analysis of tryptic peptides of recombinant GR ligand-binding domain (LBD) bound to UAMC-1217 or UAMC-1218 confirmed covalent cysteine-dependent GR binding. Finally, molecular dynamics simulations, as well as glucocorticoid receptor ligand-binding domain (GR-LBD) coregulator interaction profiling of the GR-LBD bound to GSK866 or its covalently binding analogs UAMC-1217 or UAMC-1218 revealed subtle conformational differences that might underlie their SEGRA properties. Altogether, GSK866 analogs UAMC-1217 and UAMC-1218 hold promise as a novel class of covalent-binding SEGRA ligands for the treatment of topical inflammatory skin disorders.