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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.

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.

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.

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.

Cardiomyocyte renewal by dedifferentiation and proliferation has fueled the field of regenerative cardiology in recent years, while the reverse process of redifferentiation remains largely unexplored. Redifferentiation is characterised by the restoration of function that is lost during dedifferentiation and is key to the healing process following injury. Previously, we showed that ERBB2-mediated heart regeneration has these two distinct phases: dedifferentiation, followed by redifferentiation. Here, using temporal RNAseq and proteomics, we survey the landscape of the dedifferentiation-redifferentiation process in the adult mouse heart. We find well characterised dedifferentiation pathways, such as reduced oxphos, increased proliferation and increased EMT-like features, largely return to normal, though elements of residual dedifferentiation remain, even after contractile function is restored. These hearts appeared rejuvenated and showed robust resistance to ischaemic injury. We find that redifferentiation is driven by negative feedback signalling, notably through LATS1/2 Hippo pathway activity. Disabling LATS1/2 in dedifferentiated cardiomyocytes augments dedifferentiation in vitro and prevents redifferentiation in vivo. Taken together, our data reveal the non-trivial nature of redifferentiation, whereby elements of dedifferentiation linger in a surprisingly beneficial manner. This cycle of dedifferentiation-redifferentiation protects against future insult, in what could become a novel prophylactic treatment against ischemic heart disease for at-risk patients.

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.

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, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-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-β-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 β-ODAP production in vivo. The identification of BOS may pave the way toward engineering β-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Anthropogenic organophosphates (AOPs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents and pesticides. Soil bacteria bearing a phosphotriesterase (PTE) can degrade AOPs, but whether bacteria are capable of utilizing AOPs as a phosphorus source, and how widespread PTEs are in nature, remains unclear. Here, we report the utilization of diverse AOPs by four model marine bacteria and seventeen bacterial isolates from seawater samples. To unravel the details of AOP utilization, two novel PTEs from marine bacteria were isolated and characterized. When expressed in E. coli, these PTEs enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPs provides bacteria with a source of phosphorus in depleted environments and offers a new prospect for the bioremediation of a pervasive class of anthropogenic pollutants.One sentence summaryWidespread utilization of diverse organophosphate pollutants by over 20 marine bacterial strains represents a new hope for ocean bioremediation.

BackgroundColonoscopy 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.AimsTo assess the utility of host transcriptomics of faecal wash samples of patients with IBD compared with controls.MethodsIn 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.ResultsWe 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.ConclusionsFaecal 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.

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 for the global identification of redox-modified cysteine (Cys) sites and quantification of their 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 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 oxidation state and protein abundance. Using SPEAR, we were able to quantify the in-vivo 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 chloroplast-targeted proteins. SPEAR provides a simple, straightforward, and cost-effective means of studying redox proteome dynamics. The presented data provide a global quantitative view of cysteine 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.

Background Colonoscopy is the gold standard for evaluation of inflammation in inflammatory bowel diseases (IBD), 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. Our aim was to assess the utility of host transcriptomics of fecal wash samples of IBD patients compared to controls. Methods In this prospective cohort study, we obtained biopsies and fecal-wash samples from IBD patients and controls undergoing lower endoscopy. We performed RNAseq of biopsies and matching fecal-washes, and associated them with endoscopic and histological inflammation status. We also performed fecal 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 analyzed biopsies and fecal washes from 39 patients (19 IBD, 20 controls). Host fecal-transcriptome carried information that was distinct from biopsy RNAseq and fecal proteomics. Transcriptomics of fecal washes, yet not of biopsies, from patients with histological inflammation were significantly correlated to one another (p=5.3*10–12). Fecal-transcriptome was significantly more powerful in identifying histological inflammation compared to transcriptome of intestinal biopsies (150 genes with area-under-the-curve >0.9 in fecal samples versus 10 genes in biopsy RNAseq). Fecal samples were enriched in inflammatory monocytes, regulatory T cells, natural killer-cells and innate lymphoid cells. Figure 1 - Fecal-wash host transcriptome predicts histological inflammation. A) Study layout, B) Clustergram of fecal-wash host cell mRNA signatures, demonstrating that patients with histological inflammation (red) are clustered when measuring fecal wash transcriptome yet not biopsy transcriptomes. C-D) Principle Component Analysis demonstrating improved separation of inflamed patients based on fecal host transcriptomes. E, F) Expression of host genes in fecal washes has higher statistical power (Area under the Curve, AUC) in classifying histological inflammation compared to biopsies. D shows NFKBIA as an example, E shows the AUC of the 5% best classifying genes, F shows the overall AUC based on biopsies or washes. Gray areas have AUC>0.9. G) UMAP of cells obtained from scRNAseq of mouse small intestine fecal washes. Conclusion Fecal wash host transcriptome is a powerful biomarker reflecting histological inflammation. Furthermore, it opens the way to identifying important correlates and therapeutic targets that may be obscure using biopsy transctriptomics.

Radiation therapy can induce cellular senescence in cancer cells leading to short-term tumor growth arrest, yet increased long-term recurrence. To better understand the molecular mechanisms involved, we developed a model of radiation-induced senescence in cultured cancer cells, which exhibited a typical senescent phenotype, including upregulation of p53 and its target p21, followed by sustained reduction in cellular proliferation, changes in cell size and cytoskeleton organization, and senescence-associated beta-galactosidase activity. A functional siRNA screen using a cell death-related library identified the mitochondrial Ser protease HtrA2 as necessary for senescence development. 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, while irradiation with HtrA2 inhibition upregulated cell proliferation components. 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, contributes to several hallmarks of senescence and is involved in the cytoskeletal reorganization that accompanies radiation-induced senescence.SummaryHere the authors identify the Ser protease HtrA2 as a novel mediator of radiation-induced senescence, necessary for sustained proliferation arrest and reorganization of the vimentin filament network.

Argonaute 2 (AGO2), the effector protein partner of microRNAs (miRNAs) in the cytoplasmic RNA induced silencing complex, is further involved in nuclear RNA processing. However, a role for AGO2 in regulation of alternative polyadenylation was not yet demonstrated. Here, we reveal unexpected abundance of AGO2 in mouse neuronal nuclei and characterize nuclear AGO2 interactors by mass spectrometry. We discover that AGO2 broadly regulated alternative polyadenylation (APA) in neuronal cells. Specifically, we demonstrate how two isoforms of Ret mRNA, which encodes a receptor tyrosine kinase are regulated by AGO2-depenent APA, affecting downstream GDNF signaling in primary motor neurons.

Grass pea (Lathyrus sativusL.) is a grain legume commonly grown in parts of Asia and Africa for food and forage. While being a highly nutritious and robust crop, able to survive both drought and floods, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder if consumed as a main diet component. So far, the enzyme that catalyzes the formation of β-ODAP has not been identified. By combining protein purification and enzymatic assays with transcriptomic and proteomic analyses, we were able to identify the enzyme β-ODAP synthetase (BOS) from grass pea. We show that BOS is an HXXXD-type acyltransferase of the BAHD superfamily and that its crystal structure is highly similar to that of plant hydroxycinnamoyl transferases. The identification of BOS, more than 50 years after it was proposed, paves the way towards the generation of non-toxic grass pea cultivars safe for human and animal consumption.

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.

SummaryCardiomyocyte (CM) loss after injury results in adverse remodelling and fibrosis, which inevitably lead to heart failure. ERBB2-Neuregulin and Hippo-YAP signaling pathways are key mediators of CM proliferation and regeneration, yet the crosstalk between these pathways is unclear. Here, we demonstrate in adult mice that transient over-expression (OE) of activated ERBB2 in CMs promotes cardiac regeneration in a heart failure model. OE CMs present an EMT-like regenerative response manifested by cytoskeletal remodelling, junction dissolution, migration, and ECM turnover. Molecularly, we identified YAP as a critical mediator of ERBB2 signaling. In OE CMs, YAP interacts with nuclear envelope and cytoskeletal components, reflecting the altered mechanic state elicited by ERBB2. Hippo-independent activating phosphorylation on YAP at S352 and S274 were enriched in OE CMs, peaking during metaphase, and viral overexpression of YAP phospho-mutants dampened the proliferative competence of OE CMs. Taken together, we demonstrate a potent ERBB2-mediated YAP mechanosensory signaling, involving EMT-like characteristics, resulting in heart regeneration.HighlightsERBB2-driven regeneration of scarred hearts recapitulates core-EMT processesYAP is activated and required downstream to ERBB2 signaling in CMsYAP activity is mechanically driven by cytoskeleton and nuclear envelope remodelingYAP S274 and S352 phosphorylation is essential for CM mitosis

In search of Botrytis cinerea cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the BcXYG1 gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a B. cinerea strain overexpressing BcXYG1 produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens.

Under stress, metabolism is changing: specific up- or down-regulation of proteins and metabolites occurs as well as side effects. Distinguishing specific stress-signaling metabolites (alarmones) from side products (damage metabolites) is not trivial. One example is diadenosine tetraphosphate (Ap4A) - a side product of aminoacyl-tRNA synthetases found in all domains of life. The earliest observations suggested that Ap4A serves as an alarmone for heat stress in Escherichia coli. However, despite 50 years of research, the signaling mechanisms associated with Ap4A remain unknown. We defined a set of criteria for distinguishing alarmones from damage metabolites to systematically classify Ap4A. In a nutshell, no indications for a signaling cascade that is triggered by Ap4A were found; rather, we found that Ap4A is efficiently removed in a constitutive, nonregulated manner. Several fold perturbations in Ap4A concentrations have no effect, yet accumulation at very high levels is toxic due to disturbance of zinc homeostasis, and also because Ap4A's structural overlap with ATP can result in spurious binding and inactivation of ATP-binding proteins. Overall, Ap4A met all criteria for a damage metabolite. While we do not exclude any role in signaling, our results indicate that the damage metabolite option should be considered as the null hypothesis when examining Ap4A and other metabolites whose levels change upon stress.

Current innovations in mass-spectrometry-based technologies allow deep coverage of protein expression. Despite its immense value and in contrast to transcriptomics, only a handful of studies in crop plants engaged with global proteome assays. Here, we present large-scale shotgun proteomics profiling of tomato fruit across two key tissues and five developmental stages. A total of 7738 individual protein groups were identified and reliably measured at least in one of the analyzed tissues or stages. The depth of our assay enabled identification of 61 differentially expressed transcription factors, including renowned ripening-related regulators and elements of ethylene signaling. Significantly, we measured proteins involved in 83% of all predicted enzymatic reactions in the tomato metabolic network. Hence, proteins representing almost the complete set of reactions in major metabolic pathways were identified, including the cytosolic and plastidic isoprenoid and the phenylpropanoid pathways. Furthermore, the data allowed us to discern between protein isoforms according to expression patterns, which is most significant in light of the weak transcript-protein expression correspondence. Finally, visualization of changes in protein abundance associated with a particular process provided us with a unique view of skin and flesh tissues in developing fruit. This study adds a new dimension to the existing genomic, transcriptomic and metabolomic resources. It is therefore likely to promote translational and post-translational research in tomato and additional species, which is presently focused on transcription.

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.

During nonventilated storage of carrots, CO2 gradually accumulates to high levels and causes modifications in the carrot9s 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.

Plants in natural environments have to cope with fluctuations in light conditions on different timescales, ranging from seconds to days. Toward this, plants employ both adaptive and protective mechanisms. Here, we studied the response of low-light-grown tomato plants to short-term high light (HL) exposure followed by a recovery period using large-scale proteomic profiling, photosynthetic- and biochemical measurements. We found that following a 6-h HL treatment, plants exhibited photosystem II photoinhibition, a tendency toward a reduced electron transport and induction of non-photochemical quenching (NPQ). The proteomic data showed that key enzymes of the Mg-branch of the tetrapyrrole pathway, leading to chlorophyll biosynthesis, were significantly down-regulated by HL. Nonetheless, no changes in chlorophyll content or in the levels of most photosynthetic proteins, including all identified photosystem and light-harvesting antenna proteins, were detected. Notably, we observed that following a recovery period (24 h), plants were characterized by higher electron transport and an increased NPQ capacity, indicative of an acclimation that resulted in better photosynthetic performance. Our proteomic analysis also revealed prominent up-regulation of several enzymes of the flavonoid pathway, as well as of enzymatic components of other plant antioxidant systems. In accord with these, we found significant increases in the content of flavonoids and ascorbate, and in the activity of ascorbate peroxidase following the HL and recovery. Together, the results highlight the key alterations involved in photo-protection and detoxification of reactive oxygen species which take part in the short-term response to HL.

In nature, bacteria communicate via chemical cues and establish complex communities referred to as biofilms, wherein cells are held together by an extracellular matrix. Much research is focusing on small molecules that manipulate and prevent biofilm assembly by modifying cellular signalling pathways. However, the bacterial cell envelope, presenting the interface between bacterial cells and their surroundings, is largely overlooked. In our study, we identified specific targets within the biosynthesis pathways of the different cell wall components (peptidoglycan, wall teichoic acids and teichuronic acids) hampering biofilm formation and the anchoring of the extracellular matrix with a minimal effect on planktonic growth. In addition, we provide convincing evidence that biofilm hampering by transglycosylation inhibitors and D-Leucine triggers a highly specific response without changing the overall protein levels within the biofilm cells or the overall levels of the extracellular matrix components. The presented results emphasize the central role of the Gram-positive cell wall in biofilm development, resistance and sustainment.

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.

During nonventilated storage of carrots, CO(2) 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 CO(2) 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 CO(2) 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 CO(2) 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 CO(2) requires further investigation. However, this study implies an overlooked role of CO(2) 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 CO(2) 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.

Presented is a data set for benchmarking MS1-based label-free quantitative proteomics using a quadrupole orbitrap mass spectrometer. Escherichia coli digest was spiked into a HeLa digest in four different concentrations, simulating protein expression differences in a background of an unchanged complex proteome. The data set provides a unique opportunity to evaluate the proteomic platform (instrumentation and software) in its ability to perform MS1-intensity-based label-free quantification. We show that the presented combination of informatics and instrumentation produces high precision and quantification accuracy. The data were also used to compare different quantitative protein inference methods such as iBAQ and Hi-N. The data can also be used as a resource for development and optimization of proteomics informatics tools, thus the raw data have been deposited to ProteomeXchange with identifier PXD001385.

A cell death-inducing apoplastic protein facilitates necrosis and establishment of the pathogen Botrytis cinerea but also is recognized by the plant immune system and triggers a defense response.

Previous data demonstrated that high retroperitoneal visceral fat content increases retroperitoneal soft-tissue sarcoma (RSTS) local recurrence and patients' mortality. Most RSTS tumors initiate and recur within visceral fat. The objective of the current study was to evaluate potential paracrine effects of visceral fat on RSTS. A xenograft model was used to evaluate in vivo effects of human visceral fat on STS growth. Tissue explants were prepared from visceral fat, and their conditioned medium (CM) was utilized for various in vitro experiments designed to evaluate growth, survival, migration, and invasion of STS and endothelial cells. Visceral fat–secreted protumorigenic factors were identified by mass spectrometry. The in vivo experiments demonstrated a significant increase in STS tumor growth rate when SK-LMS-1 leiomyosarcoma cells were colocalized with human visceral fat compared with subcutaneous injection of cancer cells only. The in vitro model demonstrated that visceral fat CM increased STS cellular growth and reduced doxorubicin-induced apoptosis. Visceral fat also enhanced STS cellular migration and invasion. In addition, visceral fat CM significantly increased endothelial cell tube formation, suggesting its role as a proangiogenic factor in the STS tumor microenvironment (TME). Using a robust proteomic approach, liquid chromatography and tandem mass spectrometry resolved various molecules within the visceral fat CM, of which a subset was associated with protumorigenic biologic processes. These results suggest that visceral fat directly interacts with STS cells by secreting specific adipokines into the TME, thus augmenting STS tumor cell proliferation and invasiveness. Fat-induced STS molecular deregulations should be studied to identify new potential prognostic and therapeutic targets. Implications: Visceral fat induces protumorigenic effects, in STS, through various secreted factors that should be investigated to improve our understanding of adipose–cancer cell interactions. Mol Cancer Res; 14(12); 1254–65. ©2016 AACR.

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.

Structural differences in the [2Fe-2S] ferredoxin, putidaredoxin (Pdx), from the camphor hydroxylation pathway of Pseudomonas putida have been investigated as a function of oxidation state of the iron cluster. Pdx is involved in biological electron transfer to cytochrome P450(cam) (CYP101). Redox-dependent differences have been observed previously for Pdx in terms of binding affinities to CYP101, NMR spectral differences, and dynamic properties. To further characterize these differences, structure refinement of both oxidized and reduced Pdx has been carried out using a hybrid approach utilizing paramagnetic distance restraints and NMR orientational restraints in the form of backbone (15)N residual dipolar couplings. Use of these new restraints has improved the structure of oxidized Pdx considerably over the earlier solution NMR structure without RDC restraints, with the new structure now much closer in overall fold to the recently published X-ray crystal structures. We now observe better defined relative orientations of the major secondary structure elements as also of the conformation of the metal binding loop region. Extension of this approach to structure calculation of reduced Pdx has identified structural differences that are primarily localized for residues in the C-terminal interaction domain consisting of the functionally important residue Trp 106 and regions near the metal binding loop in Pdx. These redox-dependent structural differences in Pdx correlate to dynamic changes observed before and may be linked to differences in binding and electron transfer properties between oxidized and reduced Pdx.

Quantitative proteomics by LC-MS/MS is a widely used approach for quantifying a significant portion of any complex proteome. Among the different techniques used for this purpose, one is by use of Data Independent Acquisition (DIA). We present a descriptive protocol for label-free quantitation of proteins by one DIA method termed LC-MS(E), which facilitates large-scale quantification of proteins without the need for isotopic labelling and with no theoretical limit to the number of samples included in an experiment.

The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) causes wilt and canker disease of tomato (Solanum lycopersicum). Mechanisms of Cmm pathogenicity and tomato response to Cmm infection are not well understood. To explore the interaction between Cmm and tomato, multidimensional protein identification technology (MudPIT) and tandem mass spectrometry were used to analyze in vitro and in planta generated samples. The results show that during infection Cmm senses the plant environment, transmits signals, induces, and then secretes multiple hydrolytic enzymes, including serine proteases of the Pat-1, Ppa, and Sbt familes, the CelA, XysA, and NagA glycosyl hydrolases, and other cell wall-degrading enzymes. Tomato induction of pathogenesis-related (PR) proteins, LOX1, and other defense-related proteins during infection indicates that the plant senses the invading bacterium and mounts a basal defense response, although partial with some suppressed components including class III peroxidases and a secreted serine peptidase. The tomato ethylene-synthesizing enzyme ACC-oxidase was induced during infection with the wild-type Cmm but not during infection with an endophytic Cmm strain, identifying Cmm-triggered host synthesis of ethylene as an important factor in disease symptom development. The proteomic data were also used to improve Cmm genome annotation, and thousands of Cmm gene models were confirmed or expanded.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum . Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.