Comparative proteomics in tall fescue to reveal underlying mechanisms for improving Photosystem II thermotolerance during heat stress memory.

BACKGROUND: The escalating impacts of global warming intensify the detrimental effects of heat stress on crop growth and yield. Among the earliest and most vulnerable sites of damage is Photosystem II (PSII). Plants exposed to recurring high temperatures develop heat stress memory, a phenomenon that enables them to retain information from previous stress events to better cope with subsequent one. Understanding the components and regulatory networks associated with heat stress memory is crucial for the development of heat-resistant crops. RESULTS: Physiological assays revealed that heat priming (HP) enabled tall fescue to possess higher Photosystem II photochemical activity when subjected to trigger stress. To investigate the underlying mechanisms of heat stress memory, we performed comparative proteomic analyses on tall fescue leaves at S0 (control), R4 (primed), and S5 (triggering), using an integrated approach of Tandem Mass Tag (TMT) labeling and Liquid Chromatography-Mass Spectrometry. A total of 3,851 proteins were detected, with quantitative information available for 3,835 proteins. Among these, we identified 1,423 differentially abundant proteins (DAPs), including 526 proteins that were classified as Heat Stress Memory Proteins (HSMPs). GO and KEGG enrichment analyses revealed that the HSMPs were primarily associated with the "autophagy" in R4 and with "PSII repair", "HSP binding", and "peptidase activity" in S5. Notably, we identified 7 chloroplast-localized HSMPs (HSP21, DJC77, EGY3, LHCA4, LQY1, PSBR and DEGP8, R4/S0 > 1.2, S5/S0 > 1.2), which were considered to be effectors linked to PSII heat stress memory, predominantly in cluster 4. Protein-protein interaction (PPI) analysis indicated that the ubiquitin-proteasome system, with key nodes at UPL3, RAD23b, and UCH3, might play a role in the selective retention of memory effectors in the R4 stage. Furthermore, we conducted RT-qPCR validation on 12 genes, and the results showed that in comparison to the S5 stage, the R4 stage exhibited reduced consistency between transcript and protein levels, providing additional evidence for post-transcriptional regulation in R4. CONCLUSIONS: These findings provide valuable insights into the establishment of heat stress memory under recurring high-temperature episodes and offer a conceptual framework for breeding thermotolerant crops with improved PSII functionality.

Comparative proteomics reveals energy and carbon metabolism changes in Scenedesmus quadricauda mutants induced by heavy-ion beam irradiation.

Microalgae's superior ability to fix carbon dioxide into biomass and high-value bioproducts remains underutilized in biotechnological applications due to a lack of comprehensive understanding of their carbon metabolism and energy conversion. In this work, the strain improvement technique heavy-ion beams (HIB) mutagenesis was employed on the environmentally adaptable microalgae Scenedesmus quadricauda. After several rounds of screening, two contrasting mutants were identified. S-#4 showed low photosynthetic activity and biomass productivity, while S-#26 exhibited adaptability to prolonged high light stress, achieving a 28.34 % increase in biomass yield compared to the wild-type strain. Integrating their photosynthetic characteristics and comparative proteomic analysis revealed that the contrasting protein regulations from central carbon metabolism mainly affects the two mutants' opposite biomass accumulation. Therefore, the divergent regulation of the tricarboxylic acid cycle following HIB mutagenesis could be potential targets for engineering microalgae with superior biomass and high-value products.

Analysis of sulfide signaling in rice highlights specific drought responses.

Hydrogen sulfide regulates essential plant processes, including adaptation responses to stress situations, and the best characterized mechanism of action of sulfide consists of the posttranslational modification of persulfidation. In this study, we reveal the first persulfidation proteome described in rice including 3443 different persulfidated proteins that participate in a broad range of biological processes and metabolic pathways. In addition, comparative proteomics revealed specific proteins involved in sulfide signaling during drought responses. Several proteins involved in the maintenance of cellular redox homeostasis, the TCA cycle and energy-related pathways, and ion transmembrane transport and cellular water homeostasis, highlighting the aquaporin family, showed the highest differential levels of persulfidation. We revealed that water transport activity is regulated by sulfide which correlates to an increasing level of persulfidation of aquaporins. Our findings emphasize the impact of persulfidation on total ATP levels, fatty acid composition, ROS levels, antioxidant enzymatic activities, and relative water content. Interestingly, the persulfidation role on aquaporin transport activity as an adaptation response in rice differs from the current knowledge in Arabidopsis, which emphasizes the distinct role of sulfide improving rice tolerance to drought.

Proteomic and physiological signatures of altitude adaptation in a Myrsine coriacea population under common garden conditions.

Plants exhibit phenotypic plasticity in response to environmental variations, which can lead to stable genetic and physiological adaptations if exposure to specific conditions is prolonged. Myrsine coriacea demonstrates this through its ability to thrive in diverse environments. The objective of the article is to investigate potential differences in protein accumulation and physiological responses of M. coriacea by cultivating plants from seeds collected from four populations at different altitudes in a common garden experiment. Additionally, we aim to evaluate whether these differences exhibit genetic fixation. Through integrated physiological and proteomic analyses, we identified 170 differentially accumulated proteins and observed significant physiological differences among the populations. The high-altitude population (POP1) exhibited a unique proteomic profile with significant down-regulation of proteins involved in carbon fixation and energy metabolism, suggesting a potential reduction in photosynthetic efficiency. Physiological analyses showed lower leaf nitrogen content, net CO2 assimilation rate, specific leaf area, and relative growth rate in stem height for POP1, alongside higher leaf carbon isotopic composition (δ13C) and leaf carbon (C) content. These findings provide insight into the complex interplay between proteomic and physiological adaptations in M. coriacea and underscore the importance of local adaptations. SIGNIFICANCE: We investigate the adaptive responses of M. coriacea, a shrub with a broad phenotypic range, by cultivating plants from seeds collected at four different altitudes in a common garden experiment. These findings provide insight into the complex interplay between proteomic and physiological adaptations in M. coriacea and underscore the importance of local adaptations in the face of climate change. This study contributes to advancing our understanding of the influence of altitude-specific selection pressures on the molecular biology and physiology of plants in natural populations. Our findings provide valuable insights that enhance our ability to predict and comprehend how plants respond to climate change.

Human peripheral blood mononuclear cells as a valuable source of disease-related biomarkers: Evidence from comparative proteomics studies.

PURPOSE: The discovery of specific and sensitive disease-associated biomarkers for early diagnostic purposes of many diseases is still highly challenging due to various complex molecular mechanisms triggered, high variability of disease-related interactions, and an overlap of manifestations among diseases. Human peripheral blood mononuclear cells (PBMCs) contain protein signatures corresponding to essential immunological interplay. Certain diseases stimulate PBMCs and contribute towards modulation of their proteome which can be effectively identified and evaluated via the comparative proteomics approach. EXPERIMENTAL DESIGN: In this review, we made a detailed survey of the PBMCS-derived protein biomarker candidates for a variety of diseases, published in the last 15 years. Articles were preselected to include only comparative proteomics studies. RESULTS: PBMC-derived biomarkers were investigated for cancer, glomerular, neurodegenerative/neurodevelopmental, psychiatric, chronic inflammatory, autoimmune, endocrinal, infectious, and other diseases. A detailed review of these studies encompassed the proteomics platforms, proposed candidate biomarkers, their immune cell type specificity, and potential clinical application. CONCLUSIONS: Overall, PBMCs have shown a solid potential in giving early diagnostic and prognostic biomarkers for many diseases. The future of PBMC biomarker research should reveal its full potential through well-designed comparative studies and extensive testing of the most promising protein biomarkers identified so far.

A comparative proteomic analysis for non-invasive early prediction of hypoxic-ischemic injury in asphyxiated neonates.

AIM: Hypoxic Ischemic Encephalopathy (HIE) is one of the principal causes of neonatal mortality and long-term morbidity worldwide. The neonatal signs of mild cerebral injury are subtle, making an early precise diagnosis difficult. Delayed detection, poor prognosis, and lack of specific biomarkers for the disease are increasing mortality rates. In this study, we intended to identify specific biomarkers using comparative proteomic analysis to predict the severity of perinatal asphyxia so that its outcome can also be prevented. EXPERIMENTAL DESIGN: A case-control study was conducted on 38 neonates, and urine samples were collected within 24 and 72 h of life. A tandem mass spectrometry-based quantitative proteomics approach, followed by validation via sandwich ELISA, was performed. RESULTS: The LC-MS/MS-based proteomics analysis resulted in the identification of 1201 proteins in urine, with 229, 244, and 426 being differentially expressed in HIE-1, HIE-2, and HIE-3, respectively. Axon guidance, Diseases of programmed cell death, and Detoxification of reactive oxygen species pathways were significantly enriched in mild HIE versus severe HIE. Among the differentially expressed proteins in various stages of HIE, we chose to validate four proteins - APP, AGT, FABP1, and FN1 - via sandwich ELISA. Individual and cumulative ROC curves were plotted. AGT and FABP1 together showed high sensitivity, specificity, and accuracy as potential biomarkers for early diagnosis of HIE. CONCLUSION: Establishing putative urinary biomarkers will facilitate clinicians to more accurately screen neonates for brain injury and monitor the disease progression. Prompt treatment of neonates may reduce mortality and neurodevelopmental impairment.

Comparative gut proteomics study revealing adaptive physiology of Eurasian spruce bark beetle, Ips typographus (Coleoptera: Scolytinae).

The bark beetle, Ips typographus (L.), is a major pest of Norway spruce, Picea abies (L.), causing enormous economic losses globally. The adult stage of the I. typographus has a complex life cycle (callow and sclerotized); the callow beetles feed ferociously, whereas sclerotized male beetles are more aggressive and pioneers in establishing new colonies. We conducted a comparative proteomics study to understand male and female digestion and detoxification processes in callow and sclerotized beetles. Proteome profiling was performed using high-throughput liquid chromatography-mass spectrometry. A total of >3000 proteins were identified from the bark beetle gut, and among them, 539 were differentially abundant (fold change ±2, FDR <0.05) between callow and sclerotized beetles. The differentially abundant proteins (DAPs) mainly engage with binding, catalytic activity, anatomical activity, hydrolase activity, metabolic process, and carbohydrate metabolism, and hence may be crucial for growth, digestion, detoxification, and signalling. We validated selected DAPs with RT-qPCR. Gut enzymes such as NADPH-cytochrome P450 reductase (CYC), glutathione S-transferase (GST), and esterase (EST) play a crucial role in the I. typographus for detoxification and digesting of host allelochemicals. We conducted enzyme activity assays with them and observed a positive correlation of CYC and GST activities with the proteomic results, whereas EST activity was not fully correlated. Furthermore, our investigation revealed that callow beetles had an upregulation of proteins associated with juvenile hormone (JH) biosynthesis and chitin metabolism, whereas sclerotized beetles exhibited an upregulation of proteins linked to fatty acid metabolism and the TCA cycle. These distinctive patterns of protein regulation in metabolic and functional processes are specific to each developmental stage, underscoring the adaptive responses of I. typographicus in overcoming conifer defences and facilitating their survival. Taken together, it is the first gut proteomic study comparing males and females of callow and sclerotized I. typographus, shedding light on the adaptive ecology at the molecular level. Furthermore, the information about bark beetle handling of nutritionally limiting and defence-rich spruce phloem diet can be utilized to formulate RNAi-mediated beetle management.

Comparative proteomic analysis of wall-forming bodies and oocyst wall reveals the molecular basis underlying oocyst wall formation in Eimeria necatrix.

BACKGROUND: The durable oocyst wall formed from the contents of wall-forming bodies (WFBs) protects Eimeria parasites from harsh conditions and enhances parasite transmission. Comprehending the contents of WFBs and proteins involved in oocyst wall formation is pivotal to understanding the mechanism of the oocyst wall formation and the search for novel targets to disrupt parasite transmission. METHODS: Total proteins extracted from WFBs and the oocyst wall of Eimeria necatrix were subjected to comparative proteomic analysis using tandem mass tag in conjunction with liquid chromatography tandem-mass spectrometry techniques. After functional clustering analysis of the identified proteins, three proteins, including E. necatrix disulfide isomerase (EnPDI), thioredoxin (EnTrx) and phosphoglycerate kinase (EnPGK), were selected for further study to confirm their potential roles in oocyst wall formation. RESULTS: A total of 3009 and 2973 proteins were identified from WFBs and the oocyst wall of E. necatrix, respectively. Among these proteins, 1102 were identified as differentially expressed proteins, of which 506 were upregulated and 596 downregulated in the oocyst wall compared to the WFBs. A total of 108 proteins, including compositional proteins of the oocyst wall, proteases, oxidoreductases, proteins involved in glycosylation, proteins involved in synthesis of the acid-fast lipid layer and proteins related to transport, were proposed to be involved in oocyst wall formation. The approximate molecular sizes of native EnPDI, EnTrx and EnPGK proteins were 55, 50 and 45 kDa, respectively. EnPDI was present in both type 1 and type 2 WFBs, EnTrx was present only in type 2 WFB2 and EnPGK was present only in type 1 WFBs, whereas all of them were localized to the outer layer of the oocyst wall, indicating that all of them participate in the formation of the oocyst wall. CONCLUSIONS: To the best of our knowledge, this is the first report on the proteomes of WFBs and the oocyst wall of E. necatrix. The data obtained from this study form a basis for deciphering the molecular mechanisms underlying oocyst wall formation of Eimeria parasites. They also provide valuable resources for future studies on the development of novel therapeutic agents and vaccines aimed at combating coccidian transmission.

Using Comparative Proteomics to Identify Protein Signatures in Clear Cell Renal Cell Carcinoma.

BACKGROUND/AIM: Renal cell carcinoma (RCC) is one of the most commonly diagnosed cancers in the world. Approximately 25-30% of patients identified with initial kidney cancer will have metastasized tumors, thus 5-year survival rates for these patients are poor. Therefore, biomarker research is required to identify and predict molecular signatures in RCC. MATERIALS AND METHODS: To address this, we used a mass spectrometry (MS)-based proteomics approach to identify proteins related to clear cell RCC (ccRCC) tissues from patients with T1G2, T1G3, T3G2, T3G3, and metastatic RCC (mRCC) stages. RESULTS: We identified and quantified 2,608 and 2,463 proteins, respectively, in ccRCC tissue and identified 1,449 differentially expressed proteins (DEPs). Bioinformatics analysis revealed that serpin family A member 3 (SERPINA3) qualified as biomarker for ccRCC progression. Using indirect enzyme-linked immunosorbent assay (ELISA), immunoblotting, and immunohistochemistry assays it was found that SERPINA3 expression levels in ccRCC tissues were much higher in stages before metastasis. CONCLUSION: Comparative proteomics analysis of ccRCC tissues provided new evidence of SERPINA3 association with ccRCC progression.

Quantitative proteomics analysis reveals the key proteins related to semen quality in Niangya yaks.

BACKGROUND: Proteins related to sperm motility and sperm morphology have an important impact on sperm function such as metabolism, motility and fertilisation etc. An understanding of the key proteins related to semen quality in Niangya yaks would help to provide support for breeding. However, the key proteins that affect semen quality in Niangya yaks remain unclear. METHODS: Herein, we applied tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC‒MS/MS) to analyze the expression levels of sperm proteins in groups of high- and low-quality semen from Niangya yaks. And fifteen differentially expressed proteins (DEPs) were randomly selected for expression level validation by parallel reaction monitoring (PRM). RESULTS: Of the 2,092 quantified proteins, 280 were identified as DEPs in the high-quality group versus the low-quality group. Gene Ontology (GO) analysis revealed that in terms of biological pathways, the DEPs were mainly involved in metabolic processes, cell transformation processes, and single organism metabolic processes. In terms of cell composition, the DEPs were mainly located in the cell membrane, organelle, molecular complex. In terms of molecular functions, the most abundant functions of the DEPs were catalytic activity, binding activity, transport activity, and enzyme regulation activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the DEPs were mainly involved in the cytokine and cytokine receptor interaction, notch signaling pathway, lysine biosynthesis, renal function-related protein and proteasome pathway. From protein-protein interaction (PPI) analysis of DEPs involved in important pathways, 6 related proteins affecting the semen quality of Niangya yaks were identified. And the results of the PRM and TMT analysis were consistent. CONCLUSIONS: The differential sperm proteomic analysis of high- and low-quality semen from Niangya yaks, revealed 6 proteins (PSMC5, PSMD8, PSMB3, HSP90AA1, UGP2 and HSPB1), were mainly concentrated in energy production and metabolism, might play important roles in semen quality, which could serve as candidates for the selection and breeding of Niangya yaks.

Systematic compositional analysis of exosomal extracellular vesicles produced by cells undergoing apoptosis, necroptosis and ferroptosis.

Formation of extracellular vesicles (EVs) has emerged as a novel paradigm in cell-to-cell communication in health and disease. EVs are notably produced during cell death but it had remained unclear whether different modalities of regulated cell death (RCD) influence the biogenesis and composition of EVs. To this end, we performed a comparative analysis of steady-state (ssEVs) and cell death-associated EVs (cdEVs) following TNF-induced necroptosis (necEVs), anti-Fas-induced apoptosis (apoEVs), and ML162-induced ferroptosis (ferEVs) using the same cell line. For each RCD condition, we determined the biophysical and biochemical characteristics of the cell death-associated EVs (cdEVs), the protein cargo, and the presence of methylated ribosomal RNA. We found that the global protein content of all cdEVs was increased compared to steady-state EVs. Qualitatively, the isolated exosomal ssEVs and cdEVs, contained a largely overlapping protein cargo including some quantitative differences in particular proteins. All cdEVs were enriched for proteins involved in RNA splicing and nuclear export, and showed distinctive rRNA methylation patterns compared to ssEVs. Interestingly, necEVs and apoEVs, but strikingly not ferEVs, showed enrichment of proteins involved in ribosome biogenesis. Altogether, our work documents quantitative and qualitative differences between ssEVs and cdEVs.

Mechanistic insights into sulfur source-driven physiological responses and metabolic reorganization in the fuel-biodesulfurizing Rhodococcus qingshengii IGTS8.

Comparative proteomics and untargeted metabolomics were combined to study the physiological and metabolic adaptations of Rhodococcus qingshengii IGTS8 under biodesulfurization conditions. After growth in a chemically defined medium with either dibenzothiophene (DBT) or MgSO4 as the sulfur source, many differentially produced proteins and metabolites associated with several metabolic and physiological processes were detected including the metabolism of carbohydrates, amino acids, lipids, nucleotides, vitamins, protein synthesis, transcriptional regulation, cell envelope biogenesis, and cell division. Increased production of the redox cofactor mycofactocin and associated proteins was one of the most striking adaptations under biodesulfurization conditions. While most central metabolic enzymes were less abundant in the presence of DBT, a key enzyme of the glyoxylate shunt, isocitrate lyase, was up to 26-fold more abundant. Several C1 metabolism and oligotrophy-related enzymes were significantly more abundant in the biodesulfurizing culture. R. qingshengii IGTS8 exhibited oligotrophic growth in liquid and solid media under carbon starvation. Moreover, the oligotrophic growth was faster on the solid medium in the presence of DBT compared to MgSO4 cultures. In the DBT culture, the cell envelope and phospholipids were remodeled, with lower levels of phosphatidylethanolamine and unsaturated and short-chain fatty acids being the most prominent changes. Biodesulfurization increased the biosynthesis of osmoprotectants (ectoine and mannosylglycerate) as well as glutamate and induced the stringent response. Our findings reveal highly diverse and overlapping stress responses that could protect the biodesulfurizing culture not only from the associated sulfate limitation but also from chemical, oxidative, and osmotic stress, allowing efficient resource management. IMPORTANCE Despite decades of research, a commercially viable bioprocess for fuel desulfurization has not been developed yet. This is mainly due to lack of knowledge of the physiology and metabolism of fuel-biodesulfurizing bacteria. Being a stressful condition, biodesulfurization could provoke several stress responses that are not understood. This is particularly important because a thorough understanding of the microbial stress response is essential for the development of environmentally friendly and industrially efficient microbial biocatalysts. Our comparative systems biology studies provide a mechanistic understanding of the biology of biodesulfurization, which is crucial for informed developments through the rational design of recombinant biodesulfurizers and optimization of the bioprocess conditions. Our findings enhance the understanding of the physiology, metabolism, and stress response not only in biodesulfurizing bacteria but also in rhodococci, a precious group of biotechnologically important bacteria.

Differential analysis of the haemolymph proteome of Carcinus maenas parasitized by Sacculina carcini (Cirripeda, Rhizocephala) reveals potential mechanisms of parasite control.

Sacculina carcini is an endoparasite of the green crab, Carcinus maenas. This parasite induces behavioural changes in its host and affects its metabolism by inhibiting moulting and reproduction. Using a proteomic approach in mass spectrometry, we studied the haemolymph proteomes of healthy and parasitized wild green crabs from Brittany, France to identify proteins that are differentially expressed as a consequence of parasitization. We also investigated specific proteins involved in reproduction, moulting, and immunity. We detected 77 proteins for females and 53 proteins for males that were differentially present between the healthy and parasitized crabs, some of which were sex-specific. Detection of these differentially expressed proteins suggests that the parasite can inhibit and promote different aspects of the immune response of the host. Sacculina appears to inhibit host melanisation for self-protection, while promoting the presence of immune factors, such as antimicrobial peptides to cope with possible bacterial superinfections. Moreover, one protein, juvenile hormone esterase-like carboxylesterase, was 17-times more abundant in parasitized crabs than in healthy crabs and may be responsible for inhibiting moulting and reproduction in parasitized crabs, thus ensuring the success of Sacculina reproduction.

Comparative proteomics reveals the antibiotic resistance and virulence of Cronobacter isolated from powdered infant formula and its processing environment.

Cronobacter species are opportunistic foodborne pathogens that can cause neonatal meningitis, sepsis, and necrotizing enterocolitis. In this genus, certain level strains have high mortality to infant (Cronobacter sakazakii and Cronobacter malonaticus) and antibiotic tolerance. Cronobacter has strong environmental tolerance (acid resistance, high temperature resistance, UV resistance, antibiotic resistance, etc.) and can survive in a variety of environments. It has been isolated in various production environments and products in several countries. However, the relationships between Cronobacter antibiotic tolerance and virulence remain unclear, especially at the molecular level. In this study, 96 strains of Cronobacter were isolated from powdered infant formula and its processing environment and screened for antibiotic tolerance, and proteomic maps of the representative strains of Cronobacter with antibiotic tolerance were generated by analyzing proteomics data using multiple techniques to identify protein that are implicated in Cronobacter virulence and antibiotic resistance. The increase in antibiotic tolerance of Cronobacter had a certain increase in the production of enterotoxin and hemolysin. Only triple tolerated Cronobacter sakazakii decreased the utilization of sialic acid. A total of 16,131 intracellular proteins were detected in eight representative strains, and different proteomes were present in strains with different antibiotic tolerance, including 56 virulence-related proteins. Multiple virulence proteins regulated by unknown genes were also found in the eight isolated representative strains.

Comparative analysis of wild-type and chloroplast MCU-deficient plants reveals multiple consequences of chloroplast calcium handling under drought stress.

Introduction: Chloroplast calcium homeostasis plays an important role in modulating the response of plants to abiotic and biotic stresses. One of the greatest challenges is to understand how chloroplast calcium-permeable pathways and sensors are regulated in a concerted manner to translate specific information into a calcium signature and to elucidate the downstream effects of specific chloroplast calcium dynamics. One of the six homologs of the mitochondrial calcium uniporter (MCU) was found to be located in chloroplasts in the leaves and to crucially contribute to drought- and oxidative stress-triggered uptake of calcium into this organelle. Methods: In the present study we integrated comparative proteomic analysis with biochemical, genetic, cellular, ionomic and hormone analysis in order to gain an insight into how chloroplast calcium channels are integrated into signaling circuits under watered condition and under drought stress. Results: Altogether, our results indicate for the first time a link between chloroplast calcium channels and hormone levels, showing an enhanced ABA level in the cmcu mutant already in well-watered condition. Furthermore, we show that the lack of cMCU results in an upregulation of the calcium sensor CAS and of enzymes of chlorophyll synthesis, which are also involved in retrograde signaling upon drought stress, in two independent KO lines generated in Col-0 and Col-4 ecotypes. Conclusions: These observations point to chloroplasts as important signaling hubs linked to their calcium dynamics. Our results obtained in the model plant Arabidopsis thaliana are discussed also in light of our limited knowledge regarding organellar calcium signaling in crops and raise the possibility of an involvement of such signaling in response to drought stress also in crops.

Unraveling and characterization of novel T3SS effectors in Edwardsiella piscicida.

Type III secretion system (T3SS) facilitates survival and replication of Edwardsiella piscicida in vivo. Identifying novel T3SS effectors and elucidating their functions are critical in understanding the pathogenesis of E. piscicida. E. piscicida T3SS effector EseG and EseJ was highly secreted when T3SS gatekeeper-containing protein complex EsaB-EsaL-EsaM was disrupted by EsaB deficiency. Based on this observation, concentrated secretomes of ΔesaB strain and ΔesaBΔesaN strain were purified by loading them into SDS-PAGE gel for a short electrophoresis to remove impurities prior to the in-the gel digestion and mass spectrometry. Four reported T3SS effectors and two novel T3SS effector candidates EseQ (ETAE_2009) and Trx2 (ETAE_0559) were unraveled by quantitative comparison of the identified peptides. EseQ and Trx2 were revealed to be secreted and translocated in a T3SS-dependent manner through CyaA-based translocation assay and immunofluorescent staining, demonstrating that EseQ and Trx2 are the novel T3SS effectors of E. piscicida. Trx2 was found to suppress macrophage apoptosis as revealed by TUNEL staining and cleaved caspase-3 of infected J774A.1 monolayers. Moreover, Trx2 has been shown to inhibit the p65 phosphorylation and p65 translocation into the nucleus, thus blocking the NF-κB pathway. Furthermore, depletion of Trx2 slightly but significantly attenuates E. piscicida virulence in a fish infection model. Taken together, an efficient method was established in unraveling T3SS effectors in E. piscicida, and Trx2, one of the novel T3SS effectors identified in this study, was demonstrated to suppress apoptosis and block NF- κB pathway during E. piscicida infection. IMPORTANCE Edwardsiella piscicida is an intracellular bacterial pathogen that causes intestinal inflammation and hemorrhagic sepsis in fish and human. Virulence depends on the Edwardsiella type III secretion system (T3SS). Identifying the bacterial effector proteins secreted by T3SS and defining their role is key to understanding Edwardsiella pathogenesis. EsaB depletion disrupts the T3SS gatekeeper-containing protein complex, resulting in increased secretion of T3SS effectors EseG and EseJ. EseQ and Trx2 were shown to be the novel T3SS effectors of E. piscicida by a secretome comparison between ∆esaB strain and ∆esaB∆esaN strain (T3SS mutant), together with CyaA-based translocation assay. In addition, Trx2 has been shown to suppress macrophage apoptosis and block the NF-κB pathway. Together, this work expands the known repertoire of T3SS effectors and sheds light on the pathogenic mechanism of E. piscicida.

Functional response of Acinetobacter guillouiae SFC 500-1A to tannery wastewater as revealed by a complementary proteomic approach.

Acinetobacter guillouiae SFC 500-1 A is a promising candidate for the bioremediation of tannery wastewater. In this study, we applied shotgun proteomic technology in conjunction with a gel-based assay (Gel-LC) to explore the strain's intracellular protein profile when grown in tannery wastewater as opposed to normal culture conditions. A total of 1775 proteins were identified, 52 of which were unique to the tannery wastewater treatment. Many of them were connected to the degradation of aromatic compounds and siderophore biosynthesis. On the other hand, 1598 proteins overlapped both conditions but were differentially expressed in each. Those that were upregulated in wastewater (109) were involved in the processes mentioned above, as well as in oxidative stress mitigation and intracellular redox state regulation. Particularly interesting were the downregulated proteins under the same treatment (318), which were diverse but mainly linked to the regulation of basic cellular functions (replication, transcription, translation, cell cycle, and wall biogenesis); metabolism (amino acids, lipids, sulphate, energetic processes); and other more complex responses (cell motility, exopolysaccharide production, biofilm formation, and quorum sensing). The findings suggest that SFC 500-1 A engages in survival and stress management strategies to cope with the toxic effects of tannery wastewater, and that such strategies may be mostly oriented at keeping metabolic processes to a minimum. Altogether, the results might be useful in the near future to improve the strain's effectiveness if it will be applied for bioremediation.

Proteomics evaluation of five economical commercial abundant protein depletion kits for enrichment of diseases-specific biomarkers from blood serum.

Blood serum is arguably the most analyzed biofluid for disease prediction and diagnosis. Herein, we benchmarked five different serum abundant protein depletion (SAPD) kits with regard to the identification of disease-specific biomarkers in human serum using bottom-up proteomics. As expected, the IgG removal efficiency among the SAPD kits is highly variable, ranging from 70% to 93%. A pairwise comparison of database search results showed a 10%-19% variation in protein identification among the kits. Immunocapturing-based SAPD kits against IgG and albumin outperformed the others in the removal of these two abundant proteins. Conversely, non-antibody-based methods (i.e., kits using ion exchange resins) and kits leveraging a multi-antibody approach were proven to be less efficient in depleting IgG/albumin from samples but led to the highest number of identified peptides. Notably, our results indicate that different cancer biomarkers could be enriched up to 10% depending on the utilized SAPD kit compared with the undepleted sample. Additionally, functional analysis of the bottom-up proteomic results revealed that different SAPD kits enrich distinct disease- and pathway-specific protein sets. Overall, our study emphasizes that a careful selection of the appropriate commercial SAPD kit is crucial for the analysis of disease biomarkers in serum by shotgun proteomics.

Proteomics of wheat and barley cereals in response to environmental stresses: Current state and future challenges.

Wheat and barley genera represent a wide range of genotypes from Triticeae group grown around the globe. The broad plasticity of Triticeae phenotypes mirrors the robustness of their genomes revealing a high level of gene homeology. Publication and annotation of the reference genome sequences for spring barley Morex and Chinese Spring wheat represents an important milestone enabling the researchers to precisely identify and annotate nearly all proteins. Due to the broad range of environments used for wheat and barley cultivation and their economical importance, proteomic studies focused on their responses to environmental stresses including combined stress treatments. Most of the Triticeae stress proteomics studies are comparative ones aimed at determination of differentially abundant proteins (DAPs) between two or more genotypes with contrasting stress tolerance. Studies focused on subcellular fractions and protein posttranslational modifications (PTMs) are still relatively rare although PTMs play a crucial role in modulation of protein biological function. Functional and interactomics studies are needed although gene homeology and the resulting protein functional redundancy practically excludes the utilization of knock-out mutants. The alternatives could represent either gene overexpression in a heterologous system such as A. thaliana or transient posttranscriptional gene silencing using RNAi. Publication of complete reference genome sequences together with novel technological approaches such as pQTL mapping boost the Triticeae proteomics studies not only to provide data but also to contribute to designing novel genotypes with improved adaptations to ever changing environments.

The roles of plant proteases and protease inhibitors in drought response: a review.

Upon exposure to drought, plants undergo complex signal transduction events with concomitant changes in the expression of genes, proteins and metabolites. For example, proteomics studies continue to identify multitudes of drought-responsive proteins with diverse roles in drought adaptation. Among these are protein degradation processes that activate enzymes and signalling peptides, recycle nitrogen sources, and maintain protein turnover and homeostasis under stressful environments. Here, we review the differential expression and functional activities of plant protease and protease inhibitor proteins under drought stress, mainly focusing on comparative studies involving genotypes of contrasting drought phenotypes. We further explore studies of transgenic plants either overexpressing or repressing proteases or their inhibitors under drought conditions and discuss the potential roles of these transgenes in drought response. Overall, the review highlights the integral role of protein degradation during plant survival under water deficits, irrespective of the genotypes' level of drought resilience. However, drought-sensitive genotypes exhibit higher proteolytic activities, while drought-tolerant genotypes tend to protect proteins from degradation by expressing more protease inhibitors. In addition, transgenic plant biology studies implicate proteases and protease inhibitors in various other physiological functions under drought stress. These include the regulation of stomatal closure, maintenance of relative water content, phytohormonal signalling systems including abscisic acid (ABA) signalling, and the induction of ABA-related stress genes, all of which are essential for maintaining cellular homeostasis under water deficits. Therefore, more validation studies are required to explore the various functions of proteases and their inhibitors under water limitation and their contributions towards drought adaptation.