Loss of EIF4G2 mediates aggressiveness in distinct human endometrial cancer subpopulations with poor survival outcome in patients.

The non-canonical translation initiation factor EIF4G2 plays essential roles in cellular stress responses via translation of selective mRNA cohorts. Currently there is limited and conflicting information regarding its involvement in cancer development and progression. Here we assessed its role in endometrial cancer (EC), in a cohort of 280 EC patients across different types, grades, and stages, and found that low EIF4G2 expression highly correlated with poor overall- and recurrence-free survival in Grade 2 EC patients, monitored over a period of up to 12 years. To establish a causative connection between low EIF4G2 expression and cancer progression, we stably knocked-down EIF4G2 in two human EC cell lines in parallel. EIF4G2 depletion resulted in increased resistance to conventional therapies and increased the prevalence of molecular markers for aggressive cell subsets, altering their transcriptional and proteomic landscapes. Prominent among the proteins with decreased abundance were Kinesin-1 motor proteins, KIF5B and KLC1, 2, 3. Multiplexed imaging of the EC patient tumor cohort showed a correlation between decreased expression of the kinesin proteins, and poor survival in patients with tumors of certain grades and stages. These findings reveal potential novel biomarkers for Grade 2 EC with ramifications for patient stratification and therapeutic interventions.

Loss-of-function cancer-linked mutations in the EIF4G2 non-canonical translation initiation factor.

Tumor cells often exploit the protein translation machinery, resulting in enhanced protein expression essential for tumor growth. Since canonical translation initiation is often suppressed because of cell stress in the tumor microenvironment, non-canonical translation initiation mechanisms become particularly important for shaping the tumor proteome. EIF4G2 is a non-canonical translation initiation factor that mediates internal ribosome entry site (IRES)- and uORF-dependent initiation mechanisms, which can be used to modulate protein expression in cancer. Here, we explored the contribution of EIF4G2 to cancer by screening the COSMIC database for EIF4G2 somatic mutations in cancer patients. Functional examination of missense mutations revealed deleterious effects on EIF4G2 protein-protein interactions and, importantly, on its ability to mediate non-canonical translation initiation. Specifically, one mutation, R178Q, led to reductions in protein expression and near-complete loss of function. Two other mutations within the MIF4G domain specifically affected EIF4G2's ability to mediate IRES-dependent translation initiation but not that of target mRNAs with uORFs. These results shed light on both the structure-function of EIF4G2 and its potential tumor suppressor effects.

Redifferentiated cardiomyocytes retain residual dedifferentiation signatures and are protected against ischemic injury.

Cardiomyocyte proliferation and dedifferentiation have fueled the field of regenerative cardiology in recent years, whereas the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterized by the restoration of function lost during dedifferentiation. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: transient dedifferentiation and redifferentiation. Here we survey the temporal transcriptomic and proteomic landscape of dedifferentiation-redifferentiation in adult mouse hearts and reveal that well-characterized dedifferentiation features largely return to normal, although elements of residual dedifferentiation remain, even after the contractile function is restored. These hearts appear rejuvenated and show robust resistance to ischemic injury, even 5 months after redifferentiation initiation. Cardiomyocyte redifferentiation is driven by negative feedback signaling and requires LATS1/2 Hippo pathway activity. Our data reveal the importance of cardiomyocyte redifferentiation in functional restoration during regeneration but also protection against future insult, in what could lead to a potential prophylactic treatment against ischemic heart disease for at-risk patients.

BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures.

Bloom's syndrome (BLM) protein is a known nuclear helicase that is able to unwind DNA secondary structures such as G-quadruplexes (G4s). However, its role in the regulation of cytoplasmic processes that involve RNA G-quadruplexes (rG4s) has not been previously studied. Here, we demonstrate that BLM is recruited to stress granules (SGs), which are cytoplasmic biomolecular condensates composed of RNAs and RNA-binding proteins. BLM is enriched in SGs upon different stress conditions and in an rG4-dependent manner. Also, we show that BLM unwinds rG4s and acts as a negative regulator of SG formation. Altogether, our data expand the cellular activity of BLM and shed light on the function that helicases play in the dynamics of biomolecular condensates.

DAP5 drives translation of specific mRNA targets with upstream ORFs in human embryonic stem cells.

Death associated protein 5 (DAP5/eIF4G2/NAT1) is a member of the eIF4G translation initiation factors that has been shown to mediate noncanonical and/or cap-independent translation. It is essential for embryonic development and for differentiation of embryonic stem cells (ESCs), specifically its ability to drive translation of specific target mRNAs. In order to expand the repertoire of DAP5 target mRNAs, we compared ribosome profiles in control and DAP5 knockdown (KD) human ESCs (hESCs) to identify mRNAs with decreased ribosomal occupancy upon DAP5 silencing. A cohort of 68 genes showed decreased translation efficiency in DAP5 KD cells. Mass spectrometry confirmed decreased protein abundance of a significant portion of these targets. Among these was KMT2D, a histone methylase previously shown to be essential for ESC differentiation and embryonic development. We found that nearly half of the cohort of DAP5 target mRNAs displaying reduced translation efficiency of their main coding sequences upon DAP5 KD contained upstream open reading frames (uORFs) that are actively translated independently of DAP5. This is consistent with previously suggested mechanisms by which DAP5 mediates leaky scanning through uORFs and/or reinitiation at the main coding sequence. Crosslinking protein-RNA immunoprecipitation experiments indicated that a significant subset of DAP5 mRNA targets bound DAP5, indicating that direct binding between DAP5 protein and its target mRNAs is a frequent but not absolute requirement for DAP5-dependent translation of the main coding sequence. Thus, we have extended DAP5's function in translation of specific mRNAs in hESCs by a mechanism allowing translation of the main coding sequence following upstream translation of short ORFs.

Utilization of diverse organophosphorus pollutants by marine bacteria.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

A new function for the serine protease HtrA2 in controlling radiation-induced senescence in cancer cells.

Radiation therapy can induce cellular senescence in cancer cells, leading to short-term tumor growth arrest but increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells. The irradiated cells exhibited a typical senescent phenotype, including upregulation of p53 and its main target, p21, followed by a sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity. Mass spectrometry-based proteomic profiling of the senescent cells indicated downregulation of proteins involved in cell cycle progression and DNA repair, and upregulation of proteins associated with malignancy. A functional siRNA screen using a cell death-related library identified mitochondrial serine protease HtrA2 as being necessary for sustained growth arrest of the senescent cells. In search of direct HtrA2 substrates following radiation, we determined that HtrA2 cleaves the intermediate filament protein vimentin, affecting its cytoplasmic organization. Ectopic expression of active cytosolic HtrA2 resulted in similar changes to vimentin filament assembly. Thus, HtrA2 is involved in the cytoskeletal reorganization that accompanies radiation-induced senescence and the continuous maintenance of proliferation arrest.

Host transcriptome signatures in human faecal-washes predict histological remission in patients with IBD.

BACKGROUND: Colonoscopy is the gold standard for evaluation of inflammation in inflammatory bowel diseases (IBDs), yet entails cumbersome preparations and risks of injury. Existing non-invasive prognostic tools are limited in their diagnostic power. Moreover, transcriptomics of colonic biopsies have been inconclusive in their association with clinical features. AIMS: To assess the utility of host transcriptomics of faecal wash samples of patients with IBD compared with controls. METHODS: In this prospective cohort study, we obtained biopsies and faecal-wash samples from patients with IBD and controls undergoing lower endoscopy. We performed RNAseq of biopsies and matching faecal-washes, and associated them with endoscopic and histological inflammation status. We also performed faecal mass-spectrometry proteomics on a subset of samples. We inferred cell compositions using computational deconvolution and used classification algorithms to identify informative genes. RESULTS: We analysed biopsies and faecal washes from 39 patients (20 IBD, 19 controls). Host faecal-transcriptome carried information that was distinct from biopsy RNAseq and faecal proteomics. Transcriptomics of faecal washes, yet not of biopsies, from patients with histological inflammation were significantly correlated to one another (p=5.3×10-12). Faecal-transcriptome had significantly higher statistical power in identifying histological inflammation compared with transctiptome of intestinal biopsies (150 genes with area under the curve >0.9 in faecal samples vs 10 genes in biopsy RNAseq). These results were replicated in a validation cohort of 22 patients (10 IBD, 12 controls). Faecal samples were enriched in inflammatory monocytes, regulatory T cells, natural killer-cells and innate lymphoid cells. CONCLUSIONS: Faecal wash host transcriptome is a statistically powerful biomarker reflecting histological inflammation. Furthermore, it opens the way to identifying important correlates and therapeutic targets that may be obscured using biopsy transcriptomics.

Spatial discordances between mRNAs and proteins in the intestinal epithelium.

The use of transcriptomes as reliable proxies for cellular proteomes is controversial. In the small intestine, enterocytes operate for 4 days as they migrate along villi, which are highly graded microenvironments. Spatial transcriptomics have demonstrated profound zonation in enterocyte gene expression, but how this variability translates to protein content is unclear. Here we show that enterocyte proteins and messenger RNAs along the villus axis are zonated, yet often spatially discordant. Using spatial sorting with zonated surface markers, together with a Bayesian approach to infer protein translation and degradation rates from the combined spatial profiles, we find that, while many genes exhibit proteins zonated toward the villus tip, mRNA is zonated toward the villus bottom. Finally, we demonstrate that space-independent protein synthesis delays can explain many of the mRNA-protein discordances. Our work provides a proteomic spatial blueprint of the intestinal epithelium, highlighting the importance of protein measurements for inferring cell states in tissues that operate outside of steady state.

SPEAR: A proteomics approach for simultaneous protein expression and redox analysis.

Oxidation and reduction of protein cysteinyl thiols serve as molecular switches, which is considered the most central mechanism for redox regulation of biological processes, altering protein structure, biochemical activity, subcellular localization, and binding affinity. Redox proteomics allows global identification of redox-modified cysteine (Cys) sites and quantification of their reversible oxidation/reduction responses, serving as a hypothesis-generating platform to stimulate redox biology mechanistic research. Here, we developed Simultaneous Protein Expression and Redox (SPEAR) analysis, a new redox-proteomics approach based on differential labeling of reversibly oxidized and reduced cysteines with light and heavy isotopic forms of commercially available isotopically-labeled N-ethylmaleimide (NEM). The presented method does not require enrichment for labeled peptides, thus enabling simultaneous quantification of Cys reversible oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo reversible oxidation state of thousands of cysteines across the Arabidopsis proteome under steady-state and oxidative stress conditions. Functional assignment of the identified redox-sensitive proteins demonstrated the widespread effect of oxidative conditions on various cellular functions and highlighted the enrichment of chloroplastic proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of the reversible oxidation of well-known redox-regulated active sites and many novel redox-sensitive sites whose role in plant acclimation to stress conditions remains to be further explored.

A tecpr2 knockout mouse exhibits age-dependent neuroaxonal dystrophy associated with autophagosome accumulation.

Mutations in the coding sequence of human TECPR2 were recently linked to spastic paraplegia type 49 (SPG49), a hereditary neurodegenerative disorder involving intellectual disability, autonomic-sensory neuropathy, chronic respiratory disease and decreased pain sensitivity. Here, we report the generation of a novel CRISPR-Cas9 tecpr2 knockout (tecpr2-/-) mouse that exhibits behavioral pathologies observed in SPG49 patients. tecpr2-/- mice develop neurodegenerative patterns in an age-dependent manner, manifested predominantly as neuroaxonal dystrophy in the gracile (GrN) and cuneate nuclei (CuN) of the medulla oblongata in the brainstem and dorsal white matter column of the spinal cord. Age-dependent correlation with accumulation of autophagosomes suggests compromised targeting to lysosome. Taken together, our findings establish the tecpr2 knockout mouse as a potential model for SPG49 and ascribe a new role to TECPR2 in macroautophagy/autophagy-related neurodegenerative disorders.

ERBB2 drives YAP activation and EMT-like processes during cardiac regeneration.

Cardiomyocyte loss after injury results in adverse remodelling and fibrosis, inevitably leading to heart failure. The ERBB2-Neuregulin and Hippo-YAP signalling pathways are key mediators of heart regeneration, yet the crosstalk between them is unclear. We demonstrate that transient overexpression of activated ERBB2 in cardiomyocytes (OE CMs) promotes cardiac regeneration in a heart failure model. OE CMs present an epithelial-mesenchymal transition (EMT)-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration and extracellular matrix turnover. We identified YAP as a critical mediator of ERBB2 signalling. In OE CMs, YAP interacts with nuclear-envelope and cytoskeletal components, reflecting an altered mechanical state elicited by ERBB2. We identified two YAP-activating phosphorylations on S352 and S274 in OE CMs, which peak during metaphase, that are ERK dependent and Hippo independent. Viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Together, we reveal a potent ERBB2-mediated YAP mechanotransduction signalling, involving EMT-like characteristics, resulting in robust heart regeneration.

Loss of the Periplasmic Chaperone Skp and Mutations in the Efflux Pump AcrAB-TolC Play a Role in Acquired Resistance to Antimicrobial Peptides in Salmonella typhimurium.

Bacterial resistance to antibiotics is a major concern worldwide, leading to an extensive search for alternative drugs. Promising candidates are antimicrobial peptides (AMPs), innate immunity molecules, shown to be highly efficient against multidrug resistant bacteria. Therefore, it is essential to study bacterial resistance mechanisms against them. For that purpose, we used experimental evolution, and isolated a Salmonella enterica serovar typhimurium-resistant line to the AMP 4DK5L7. This AMP displayed promising features including widespread activity against Gram-negative bacteria and protection from proteolytic degradation. However, the resistance that evolved in the isolated strain was particularly high. Whole genome sequencing revealed that five spontaneous mutations had evolved. Of these, three are novel in the context of acquired AMP resistance. Two mutations are related to the AcrAB-TolC multidrug efflux pump. One occurred in AcrB, the substrate-binding domain of the system, and the second in RamR, a transcriptional regulator of the system. Together, the mutations increased the minimal inhibitory concentration (MIC) by twofold toward this AMP. Moreover, the mutation in AcrB induced hypersusceptibility toward ampicillin and colistin. The last mutation occurred in Skp, a periplasmic chaperone that participates in the biogenesis of outer membrane proteins (OMPs). This mutation increased the MIC by twofold to 4DK5L7 and by fourfold to another AMP, seg5D. Proteomic analysis revealed that the mutation abolished Skp expression, reduced OMP abundance, and increased DegP levels. DegP, a protease that was reported to have an additional chaperone activity, escorts OMPs through the periplasm along with Skp, but is also associated with AMP resistance. In conclusion, our data demonstrate that both loss of Skp and manipulation of the AcrAB-TolC system are alternative strategies of AMP acquired resistance in Salmonella typhimurium and might represent a common mechanism in other Gram-negative bacteria.

Ribonucleotide reductase from Fusarium oxysporum does not Respond to DNA replication stress.

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in dNTP biosynthesis and is tightly regulated at the transcription and activity levels. One of the best characterized responses of yeast to DNA damage is up-regulation of RNR transcription and activity and consequently, elevation of the dNTP pools. Hydroxyurea is a universal inhibitor of RNR that causes S phase arrest. It is used in the clinic to treat certain types of cancers. Here we studied the response of the fungal plant pathogen Fusarium oxysporum to hydroxyurea in order to generate hypotheses that can be used in the future in development of a new class of pesticides. F. oxysporum causes severe damage to more than 100 agricultural crops and specifically threatens banana cultivation world-wide. Although the recovery of F. oxysporum from transient hydroxyurea exposure was similar to the one of Saccharomyces cerevisiae, colony formation was strongly inhibited in F. oxysporum in comparison with S. cerevisiae. As expected, genomic and phosphoproteomic analyses of F. oxysporum conidia (spores) exposed to hydroxyurea showed hallmarks of DNA replication perturbation and activation of recombination. Unexpectedly and strikingly, RNR was not induced by either hydroxyurea or the DNA-damaging agent methyl methanesulfonate as determined at the RNA and protein levels. Consequently, dNTP concentrations were significantly reduced, even in response to a low dose of hydroxyurea. Methyl methanesulfonate treatment did not induce dNTP pools in F. oxysporum, in contrast to the response of RNR and dNTP pools to DNA damage and hydroxyurea in several tested organisms. Our results are important because the lack of a feedback mechanism to increase RNR expression in F. oxysporum is expected to sensitize the pathogen to a fungal-specific ribonucleotide inhibitor. The potential impact of our observations on F. oxysporum genome stability and genome evolution is discussed.

Chlorophyll catabolism precedes changes in chloroplast structure and proteome during leaf senescence.

The earliest visual changes of leaf senescence occur in the chloroplast as chlorophyll is degraded and photosynthesis declines. Yet, a comprehensive understanding of the sequence of catabolic events occurring in chloroplasts during natural leaf senescence is still missing. Here, we combined confocal and electron microscopy together with proteomics and biochemistry to follow structural and molecular changes during Arabidopsis leaf senescence. We observed that initiation of chlorophyll catabolism precedes other breakdown processes. Chloroplast size, stacking of thylakoids, and efficiency of PSII remain stable until late stages of senescence, whereas the number and size of plastoglobules increase. Unlike catabolic enzymes, whose level increase, the level of most proteins decreases during senescence, and chloroplast proteins are overrepresented among these. However, the rate of their disappearance is variable, mostly uncoordinated and independent of their inherent stability during earlier developmental stages. Unexpectedly, degradation of chlorophyll-binding proteins lags behind chlorophyll catabolism. Autophagy and vacuole proteins are retained at relatively high levels, highlighting the role of extra-plastidic degradation processes especially in late stages of senescence. The observation that chlorophyll catabolism precedes all other catabolic events may suggest that this process enables or signals further catabolic processes in chloroplasts.

The Chloroplast Envelope Protease FTSH11 - Interaction With CPN60 and Identification of Potential Substrates.

FTSH proteases are membrane-bound, ATP-dependent metalloproteases found in bacteria, mitochondria and chloroplasts. The product of one of the 12 genes encoding FTSH proteases in Arabidopsis, FTSH11, has been previously shown to be essential for acquired thermotolerance. However, the substrates of this protease, as well as the mechanism linking it to thermotolerance are largely unknown. To get insight into these, the FTSH11 knockout mutant was complemented with proteolytically active or inactive variants of this protease, tagged with HA-tag, under the control of the native promoter. Using these plants in thermotolerance assay demonstrated that the proteolytic activity, and not only the ATPase one, is essential for conferring thermotolerance. Immunoblot analyses of leaf extracts, isolated organelles and sub-fractionated chloroplast membranes localized FTSH11 mostly to chloroplast envelopes. Affinity purification followed by mass spectrometry analysis revealed interaction between FTSH11 and different components of the CPN60 chaperonin. In affinity enrichment assays, CPN60s as well as a number of envelope, stroma and thylakoid proteins were found associated with proteolytically inactive FTSH11. Comparative proteomic analysis of WT and knockout plants, grown at 20°C or exposed to 30°C for 6 h, revealed a plethora of upregulated chloroplast proteins in the knockout, some of them might be candidate substrates. Among these stood out TIC40, which was stabilized in the knockout line after recovery from heat stress, and three proteins that were found trapped in the affinity enrichment assay: the nucleotide antiporter PAPST2, the fatty acid binding protein FAP1 and the chaperone HSP70. The consistent behavior of these four proteins in different assays suggest that they are potential FTSH11 substrates.

High Levels of CO2 Induce Spoilage by Leuconostoc mesenteroides by Upregulating Dextran Synthesis Genes.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot's microbiome toward dominance of Lactobacillales and Enterobacteriales The lactic acid bacterium Leuconostoc mesenteroides secretes a slimy exudate over the surface of the carrots. The objective of this study was to characterize the slime components and the potential cause for its secretion under high CO2 levels. A proteomic analysis of the exudate revealed bacterial glucosyltransferases as the main proteins, specifically, dextransucrase. A chemical analysis of the exudate revealed high levels of dextran and several simple sugars. The exudate volume and dextran amount were significantly higher when L. mesenteroides was incubated under high CO2 levels than when incubated in an aerated environment. The treatment of carrot medium plates with commercial dextransucrase or exudate protein extract resulted in similar sugar profiles and dextran production. Transcriptome analysis demonstrated that dextran production is related to the upregulation of the L. mesenteroides dextransucrase-encoding genes dsrD and dsrT during the first 4 to 8 h of exposure to high CO2 levels compared to aerated conditions. A phylogenetic analysis of L. mesenteroides YL48 dsrD revealed a high similarity to other dsr genes harbored by different Leuconostoc species. The ecological benefit of dextran production under elevated CO2 requires further investigation. However, this study implies an overlooked role of CO2 in the physiology and fitness of L. mesenteroides in stored carrots, and perhaps in other food items, during storage under nonventilated conditions.IMPORTANCE The bacterium Leuconostoc mesenteroides is known to cause spoilage of different types of foods by secreting a slimy fluid that damages the quality and appearance of the produce. Here, we identified a potential mechanism by which high levels of CO2 affect the spoilage caused by this bacterium by upregulating dextran synthesis genes. These results have broader implications for the study of the physiology, degradation ability, and potential biotechnological applications of Leuconostoc.

The Disordered Landscape of the 20S Proteasome Substrates Reveals Tight Association with Phase Separated Granules.

Proteasomal degradation is the main route of regulated proteostasis. The 20S proteasome is the core particle (CP) responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. It has been previously shown that the 20S CP is active in degradation of certain intrinsically disordered proteins (IDP) lacking structural constrains. Here, a comprehensive analysis of the 20S CP substrates in vitro is conducted. It is revealed that the 20S CP substrates are highly disordered. However, not all the IDPs are 20S CP substrates. The group of the IDPs that are 20S CP substrates, termed 20S-IDPome are characterized by having significantly more protein binding partners, more posttranslational modification sites, and are highly enriched for RNA binding proteins. The vast majority of them are involved in splicing, mRNA processing, and translation. Remarkably, it is found that low complexity proteins with prion-like domain (PrLD), which interact with GR or PR di-peptide repeats, are the most preferential 20S CP substrates. The finding suggests roles of the 20S CP in gene transcription and formation of phase-separated granules.

The Xanthomonas euvesicatoria type III effector XopAU is an active protein kinase that manipulates plant MAP kinase signaling.

The Gram-negative bacterium Xanthomonas euvesicatoria (Xe) is the causal agent of bacterial spot disease of pepper and tomato. Xe delivers effector proteins into host cells through the type III secretion system to promote disease. Here, we show that the Xe effector XopAU, which is conserved in numerous Xanthomonas species, is a catalytically active protein kinase and contributes to the development of disease symptoms in pepper plants. Agrobacterium-mediated expression of XopAU in host and non-host plants activated typical defense responses, including MAP kinase phosphorylation, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death, that were dependent on the kinase activity of the effector. XopAU-mediated cell death was not dependent on early signaling components of effector-triggered immunity and was also observed when the effector was delivered into pepper leaves by Xanthomonas campestris pv. campestris, but not by Xe. Protein-protein interaction studies in yeast and in planta revealed that XopAU physically interacts with components of plant immunity-associated MAP kinase cascades. Remarkably, XopAU directly phosphorylated MKK2 in vitro and enhanced its phosphorylation at multiple sites in planta. Consistent with the notion that MKK2 is a target of XopAU, silencing of the MKK2 homolog or overexpression of the catalytically inactive mutant MKK2K99R in N. benthamiana plants reduced XopAU-mediated cell death and MAPK phosphorylation. Furthermore, yeast co-expressing XopAU and MKK2 displayed reduced growth and this phenotype was dependent on the kinase activity of both proteins. Together, our results support the conclusion that XopAU contributes to Xe disease symptoms in pepper plants and manipulates host MAPK signaling through phosphorylation and activation of MKK2.