Proteomic analysis of the developing mammalian brain links PCDH19 to the Wnt/ß-catenin signalling pathway.

Clustering Epilepsy (CE) is a neurological disorder caused by pathogenic variants of the Protocadherin 19 (PCDH19) gene. PCDH19 encodes a protein involved in cell adhesion and Estrogen Receptor α mediated-gene regulation. To gain further insights into the molecular role of PCDH19 in the brain, we investigated the PCDH19 interactome in the developing mouse hippocampus and cortex. Combined with a meta-analysis of all reported PCDH19 interacting proteins, our results show that PCDH19 interacts with proteins involved in actin, microtubule, and gene regulation. We report CAPZA1, αN-catenin and, importantly, ß-catenin as novel PCDH19 interacting proteins. Furthermore, we show that PCDH19 is a regulator of ß-catenin transcriptional activity, and that this pathway is disrupted in CE individuals. Overall, our results support the involvement of PCDH19 in the cytoskeletal network and point to signalling pathways where PCDH19 plays critical roles.

Proteome profiling of enriched membrane-associated proteins unraveled a novel sophorose and cello-oligosaccharide transporter in Trichoderma reesei.

BACKGROUND: Trichoderma reesei is an organism extensively used in the bioethanol industry, owing to its capability to produce enzymes capable of breaking down holocellulose into simple sugars. The uptake of carbohydrates generated from cellulose breakdown is crucial to induce the signaling cascade that triggers cellulase production. However, the sugar transporters involved in this process in T. reesei remain poorly identified and characterized. RESULTS: To address this gap, this study used temporal membrane proteomics analysis to identify five known and nine putative sugar transporters that may be involved in cellulose degradation by T. reesei. Docking analysis pointed out potential ligands for the putative sugar transporter Tr44175. Further functional validation of this transporter was carried out in Saccharomyces cerevisiae. The results showed that Tr44175 transports a variety of sugar molecules, including cellobiose, cellotriose, cellotetraose, and sophorose. CONCLUSION: This study has unveiled a transporter Tr44175 capable of transporting cellobiose, cellotriose, cellotetraose, and sophorose. Our study represents the first inventory of T. reesei sugar transportome once exposed to cellulose, offering promising potential targets for strain engineering in the context of bioethanol production.

Precision Targeting of Endogenous Epidermal Growth Factor Receptor (EGFR) by Structurally Aligned Dual-Modifier Labeling.

Covalent modification of endogenous proteins by chemical probes is used for proteome-wide profiling of cellular protein function and drug discovery. However, probe selectivity in the complex cellular environment is a challenge, and new probes with better target selectivity are continuously needed. On the basis of the success of monocovalent activity-based and reactivity-based probes, an approach of structurally aligned dual-modifier labeling (SADL) was investigated here on its potential in improving target precision. Two reactive groups, based on the acrylamide and NHS ester chemistry, were linked with structural alignment to be under the same anilinoquinazoline ligand-directive for targeting the epidermal growth factor receptor (EGFR) protein kinase as the model system for proteome-wide profiling. The SADL approach was compared with its monocovalent precursors in a label-free MaxLFQ workflow using MDA-MB-468 triple negative breast cancer cells. The dual-modifier probe consistently showed labeling of EGFR with improved precision over both monocovalent precursors under various controls. The workflow also labeled endogenous USP34 and PKMYT1 with high selectivity. Precision labeling with two covalent modifiers under a common ligand directive may broaden protein identification opportunities in the native environment to complement genetic and antibody-based approaches for elucidating biological or disease mechanisms, as well as accelerating drug target discovery.

Multiple Abiotic Stresses Applied Simultaneously Elicit Distinct Responses in Two Contrasting Rice Cultivars.

Rice crops are often subject to multiple abiotic stresses simultaneously in both natural and cultivated environments, resulting in yield reductions beyond those expected from single stress. We report physiological changes after a 4 day exposure to combined drought, salt and extreme temperature treatments, following a 2 day salinity pre-treatment in two rice genotypes-Nipponbare (a paddy rice) and IAC1131 (an upland landrace). Stomata closed after two days of combined stresses, causing intercellular CO2 concentrations and assimilation rates to diminish rapidly. Abscisic acid (ABA) levels increased at least five-fold but did not differ significantly between the genotypes. Tandem Mass Tag isotopic labelling quantitative proteomics revealed 6215 reproducibly identified proteins in mature leaves across the two genotypes and three time points (0, 2 and 4 days of stress). Of these, 987 were differentially expressed due to stress (cf. control plants), including 41 proteins that changed significantly in abundance in all stressed plants. Heat shock proteins, late embryogenesis abundant proteins and photosynthesis-related proteins were consistently responsive to stress in both Nipponbare and IAC1131. Remarkably, even after 2 days of stress there were almost six times fewer proteins differentially expressed in IAC1131 than Nipponbare. This contrast in the translational response to multiple stresses is consistent with the known tolerance of IAC1131 to dryland conditions.

Genome survey sequencing of wild cotton (Gossypium robinsonii) reveals insights into proteomic responses of pollen to extreme heat.

Heat stress specifically affects fertility by impairing pollen viability but cotton wild relatives successfully reproduce in hot savannas where they evolved. An Australian arid-zone cotton (Gossypium robinsonii) was exposed to heat events during pollen development then mature pollen was subjected to deep proteomic analysis using 57 023 predicted genes from a genomic database we assembled for the same species. Three stages of pollen development, including tetrads (TEs), uninucleate microspores (UNs) and binucleate microspores (BNs) were exposed to 36°C or 40°C for 5 days and the resulting mature pollen was collected at anthesis (p-TE, p-UN and p-BN, respectively). Using the sequential windowed acquisition of all theoretical mass spectra proteomic analysis, 2704 proteins were identified and quantified across all pollen samples analysed. Proteins predominantly decreased in abundance at all stages in response to heat, particularly after exposure of TEs to 40°C. Functional enrichment analyses demonstrated that extreme heat increased the abundance of proteins that contributed to increased messenger RNA splicing via spliceosome, initiation of cytoplasmic translation and protein refolding in p-TE40. However, other functional categories that contributed to intercellular transport were inhibited in p-TE40, linked potentially to Rab proteins. We ascribe the resilience of reproductive processes in G. robinsonii at temperatures up to 40°C, relative to commercial cotton, to a targeted reduction in protein transport.

The phosphoproteome of rice leaves responds to water and nitrogen supply.

The scarcity of freshwater is an increasing concern in flood-irrigated rice, whilst excessive use of nitrogen fertilizers is costly and contributes to environmental pollution. To co-ordinate growth adaptation under prolonged exposure to limited water or excess nitrogen supply, plants employ complex systems for signalling and regulation of metabolic processes. There is limited information on the involvement of one of the most important post-translational modifications (PTMs), protein phosphorylation, in plant adaptation to long-term changes in resource supply. Oryza sativa cv. Nipponbare was grown under two regimes of nitrogen from the time of germination to final harvest. Twenty-five days after germination, water was withheld from half the pots in each nitrogen treatment and low water supply continued for an additional 26 days, while the remaining pots were well watered. Leaves from all four groups of plants were harvested after 51 days in order to test whether phosphorylation of leaf proteins responded to prior abiotic stress events. The dominant impact of these resources is exerted in leaves, where PTMs have been predicted to occur. Proteins were extracted and phosphopeptides were analysed by nanoLC-MS/MS analysis, coupled with label-free quantitation. Water and nitrogen regimes triggered extensive changes in phosphorylation of proteins involved in membrane transport, such as the aquaporin OsPIP2-6, a water channel protein. Our study reveals phosphorylation of several peptides belonging to proteins involved in RNA-processing and carbohydrate metabolism, suggesting that phosphorylation events regulate the signalling cascades that are required to optimize plant response to resource supply.

Wild and Cultivated Species of Rice Have Distinctive Proteomic Responses to Drought.

Drought often compromises yield in non-irrigated crops such as rainfed rice, imperiling the communities that depend upon it as a primary food source. In this study, two cultivated species (Oryza sativa cv. Nipponbare and Oryza glaberrima cv. CG14) and an endemic, perennial Australian wild species (Oryza australiensis) were grown in soil at 40% field capacity for 7 d (drought). The hypothesis was that the natural tolerance of O. australiensis to erratic water supply would be reflected in a unique proteomic profile. Leaves from droughted plants and well-watered controls were harvested for label-free quantitative shotgun proteomics. Physiological and gene ontology analysis confirmed that O. australiensis responded uniquely to drought, with superior leaf water status and enhanced levels of photosynthetic proteins. Distinctive patterns of protein accumulation in drought were observed across the O. australiensis proteome. Photosynthetic and stress-response proteins were more abundant in drought-affected O. glaberrima than O. sativa, and were further enriched in O. australiensis. In contrast, the level of accumulation of photosynthetic proteins decreased when O. sativa underwent drought, while a narrower range of stress-responsive proteins showed increased levels of accumulation. Distinctive proteomic profiles and the accumulated levels of individual proteins with specific functions in response to drought in O. australiensis indicate the importance of this species as a source of stress tolerance genes.

Amyloid-beta peptide neurotoxicity in human neuronal cells is associated with modulation of insulin-like growth factor transport, lysosomal machinery and extracellular matrix receptor interactions.

Extracellular deposits of the amyloid-beta peptide (Aß) are known as the main pathological hallmark of Alzheimer's disease. In Alzheimer's disease, neurons are injured and die throughout the brain, a process in which Aß neurotoxicity is considered to play an important role. However, the molecular mechanisms underlying Aß toxicity that lead to neurodegeneration are not clearly established. Here we have elucidated the molecular pathways and networks which are impacted by Aß in neurons using SH-SY5Y human neuroblastoma cells as a model. These cells were treated with Aß1-42 peptides to study changes in biochemical networks using tandem mass tag labeled quantitative proteomic technique followed by computational analysis of the data. The molecular impacts of Aß on cells were evident in a time- and dose-dependent manner, albeit the duration of treatment induced greater differential changes in cellular proteome compared to the effects of concentration. Aß induced early changes in proteins associated with lysosomes, collagen chain trimerization and extracellular matrix receptor interaction, complement and coagulation cascade, oxidative stress induced senescence, ribosome biogenesis, regulation of insulin-like growth factor transport and uptake by insulin-like growth factor-binding protein. These novel findings provide molecular insights on the effects of Aß on neurons, with implications for better understanding the impacts of Aß on early neurodegeneration in Alzheimer's disease pathology.

Proteomic Responses to Drought Vary Widely Among Eight Diverse Genotypes of Rice (Oryza sativa).

Rice is a critically important food source but yields worldwide are vulnerable to periods of drought. We exposed eight genotypes of upland and lowland rice (Oryza sativa L. ssp. japonica and indica) to drought stress at the late vegetative stage, and harvested leaves for label-free shotgun proteomics. Gene ontology analysis was used to identify common drought-responsive proteins in vegetative tissues, and leaf proteins that are unique to individual genotypes, suggesting diversity in the metabolic responses to drought. Eight proteins were found to be induced in response to drought stress in all eight genotypes. A total of 213 proteins were identified in a single genotype, 83 of which were increased in abundance in response to drought stress. In total, 10 of these 83 proteins were of a largely uncharacterized function, making them candidates for functional analysis and potential biomarkers for drought tolerance.

Probing the Role of the Chloroplasts in Heavy Metal Tolerance and Accumulation in Euglena gracilis.

The E. gracilis Zm-strain lacking chloroplasts, characterized in this study, was compared with the earlier assessed wild type Z-strain to explore the role of chloroplasts in heavy metal accumulation and tolerance. Comparison of the minimum inhibitory concentration (MIC) values indicated that both strains tolerated similar concentrations of mercury (Hg) and lead (Pb), but cadmium (Cd) tolerance of the Z-strain was twice that of the Zm-strain. The ability of the Zm-strain to accumulate Hg was higher compared to the Z-strain, indicating the existence of a Hg transportation and accumulation mechanism not depending on the presence of chloroplasts. Transmission electron microscopy (TEM) showed maximum accumulation of Hg in the cytosol of the Zm-strain and highest accumulation of Cd in the chloroplasts of the Z-strain indicating a difference in the ability of the two strains to deposit heavy metals in the cell. The highly abundant heavy metal transporter MTP2 in the Z-strain may have a role in Cd transportation to the chloroplasts. A multidrug resistance-associated protein highly increased in abundance in the Zm-strain could be a potential Hg transporter to either cytosol or mitochondria. Overall, the chloroplasts appear to have major role in the tolerance and accumulation of Cd in E. gracilis.

Pseudomonas aeruginosa Proteome under Hypoxic Stress Conditions Mimicking the Cystic Fibrosis Lung.

Pseudomonas aeruginosa is a ubiquitous Gram-negative pathogen known to inhabit hypoxic mucus plugs of cystic fibrosis (CF) patient lungs. Despite the high prevalence and related patient mortality, the protein machinery enabling the bacterium to adapt to low oxygen environment remains to be fully elucidated. We investigated this by performing both SWATH mass spectrometry and data-dependent SPS-MS3 of TMT-labeled peptides to profile the proteomes of two P. aeruginosa CF isolates, PASS2 and PASS3, and a laboratory reference strain, PAO1, grown under hypoxic stress (O2 < 1%) in media that mimic the nutrient components of the CF lung. Quantitated across all three strains were 3967 P. aeruginosa proteins, reflecting approximately 71% of predicted ORFs in PAO1 and representing the most comprehensive proteome of clinically relevant P. aeruginosa to date. Comparative analysis revealed 735, 640, and 364 proteins were altered by 2-fold or more when comparing low oxygen to aerobic growth in PAO1, PASS2, and PASS3, respectively. Strikingly, under hypoxic stress, all strains showed concurrent increased abundance of proteins required for both aerobic (cbb3-1 and cbb3-2 terminal oxidases) and anaerobic denitrification and arginine fermentation, with the two clinical isolates showing higher relative expression of proteins in these pathways. Additionally, functional annotation revealed that clinical strains portray a unique expression profile of replication, membrane biogenesis, and virulence proteins during hypoxia which may endow these bacteria with a survival advantage. These protein profiles illuminate the diversity of P. aeruginosa mechanisms to adapt to low oxygen and shows that CF isolates initiate a robust molecular response to persist under these conditions.

Pseudomonas aeruginosa Cell Membrane Protein Expression from Phenotypically Diverse Cystic Fibrosis Isolates Demonstrates Host-Specific Adaptations.

Pseudomonas aeruginosa is a Gram-negative, nosocomial, highly adaptable opportunistic pathogen especially prevalent in immuno-compromised cystic fibrosis (CF) patients. The bacterial cell surface proteins are important contributors to virulence, yet the membrane subproteomes of phenotypically diverse P. aeruginosa strains are poorly characterized. We carried out mass spectrometry (MS)-based proteome analysis of the membrane proteins of three novel P. aeruginosa strains isolated from the sputum of CF patients and compared protein expression to the widely used laboratory strain, PAO1. Microbes were grown in planktonic growth condition using minimal M9 media, and a defined synthetic lung nutrient mimicking medium (SCFM) limited passaging. Two-dimensional LC-MS/MS using iTRAQ labeling enabled quantitative comparisons among 3171 and 2442 proteins from the minimal M9 medium and in the SCFM, respectively. The CF isolates showed marked differences in membrane protein expression in comparison with PAO1 including up-regulation of drug resistance proteins (MexY, MexB, MexC) and down-regulation of chemotaxis and aerotaxis proteins (PA1561, PctA, PctB) and motility and adhesion proteins (FliK, FlgE, FliD, PilJ). Phenotypic analysis using adhesion, motility, and drug susceptibility assays confirmed the proteomics findings. These results provide evidence of host-specific microevolution of P. aeruginosa in the CF lung and shed light on the adaptation strategies used by CF pathogens.

Proteomics of hosts and pathogens in cystic fibrosis.

Cystic fibrosis (CF) is a congenital disease that results in great morbidity and mortality mainly in the Caucasian population. Although CF is a monogenic disease caused by mutation in the CF conductance transmembrane regulator (CFTR) gene, most of the related mortality can be attributed to infection mediated by opportunistic bacterial and fungal pathogens. Over the past decade, advancements in the field of proteomics have helped to gain insight into the repertoire of host and pathogen proteins involved in CF pathophysiology. This review provides an overview of the contributions of proteomic studies in advancing our knowledge of the biology of CF and disease progression associated with pathogen infection and host defense responses.