Quantitative Proteomics Approach Research Articles

Integrative Proteome Analysis Revels 3-Hydroxybutyrate Exerts Neuroprotective Effect by Influencing Chromatin Bivalency.

3-hydroxybutyrate (3OHB) has been proved to act as a neuroprotective molecule in multiple neurodegenerative diseases. Here, we employed a quantitative proteomics approach to assess the changes of the global protein expression pattern of neural cells upon 3OHB administration. In combination with a disease-related, protein-protein interaction network we pinpointed a hub marker, histone lysine 27 trimethylation, which is one of the key epigenetic markers in multiple neurodegenerative diseases. Integrative analysis of transcriptomic and epigenomic datasets highlighted the involvement of bivalent transcription factors in 3OHB-mediated disease protection and its alteration of neuronal development processes. Transcriptomic profiling revealed that 3OHB impaired the fate decision process of neural precursor cells by repressing differentiation and promoting proliferation. Our study provides a new mechanism of 3OHB's neuroprotective effect, in which chromatin bivalency is sensitive to 3OHB alteration and drives its neuroprotective function both in neurodegenerative diseases and in neural development processes.

Modified N-acyl-L-homoserine lactone compounds abrogate Las-dependent quorum-sensing response in human pathogen Pseudomonas aeruginosa.

Quorum sensing (QS) is a mode of cell-cell communication that bacteria use to sense population density and orchestrate collective behaviors. The common opportunistic human pathogen Pseudomonas aeruginosa employs QS to regulate a large set of genes involved in virulence and host-pathogen interactions. The Las circuit positioned on the top of the QS hierarchy in P. aeruginosa makes use of N-acyl-L-homoserine lactones (AHLs) as signal molecules, like N-3-oxo-dodecanoyl-L-homoserine lactone (3O-C12-HSL). Disabling QS circuits by certain small-molecule compounds, known as quorum-sensing inhibitors (QSIs), has been proposed as a strategy to attenuate bacterial pathogenicity. In this study, four new AHL analogs were designed by incorporating a tert-butoxycarbonyl Boc group in amide and β-keto (3-oxo) moiety. Compounds were evaluated on a molecular and phenotypic basis as a QSI using the screening strategy linked to the assignment of the Las QS system in P. aeruginosa. Using a LasR-based bioreporter, we found that the compounds decreased LasR-controlled light activity and competed efficiently with natural 3O-C12-HSL. The compounds reduced the production of the cognate 3O-C12-HSL and certain virulence traits, like total protease activity, elastase activity, pyocyanin production, and extracellular DNA release. Furthermore, a quantitative proteomic approach was used to study the effect of the compounds on QS-regulated extracellular proteins. Among the four compounds tested, one of them showed the most significant difference in the appearance of the 3O-C12-HSL-responsive reference proteins related to QS communication and virulence, i.e., a distinct activity as a QSI. Moreover, by combining experimental data with computational chemistry, we addressed the effect of LasR protein flexibility on docking precision and assessed the advantage of using a multi-conformational docking procedure for binding mode prediction of LasR modulators. Thus, the four new AHL compounds were tested for their interaction with the AHL-binding site in LasR to identify the key interferences with the activity of LasR. Our study provides further insight into molecular features that are required for small-molecule modulation of LasR-dependent QS communication in P. aeruginosa. This should facilitate rational design of the next generation of antivirulence tools to study and manipulate QS-controlled fitness in bacteria and, thereby, handle bacterial infections in a new way.

Secretome Profile of Leukocyte-Platelet-Rich Fibrin (L-PRF) Membranes.

In the last years, platelet concentrates such as leukocyte-platelet-rich fibrin (L-PRF) have been used in different clinical scenarios as a huge source of growth factors to enhance wound healing. However, platelet concentrates release many other proteins that also participate in tissue regeneration processes. In this context, the analysis of the L-PRF secretome would provide relevant information on the different proteins and growth factors released by these platelet concentrates, how such secretion varies with the time, and how relevant this could be for the regenerative properties of these products. In the present chapter, we will provide a protocol for isolation, culture, and secretome analysis of L-PRF membranes. Qualitative and quantitative proteomic approaches will be presented, including gel-based and quantitative Sequential Window Acquisition of All Theoretical Mass Spectra (SWATH-MS)-based approaches. This protocol has been recently applied with success to define the L-PRF secretome composition, setting the stage for further research that can provide relevant information on the clinical properties of these platelet concentrates' subtype.

Proteomic Alterations in Human Dermal Fibroblasts under Photo-Induced Pollution Caused by Excessive Solar Irradiations such as Infra-Red, Blue Light, UVA and UVB

Background: The skin serves as the first line of defense for the human body. Direct sunlight contains damaging radiations that can speed up the ageing process of the skin, resulting in wrinkles, leathery skin, dark patches, and solar elastosis. Objectives: To evaluate the effect of multiple solar irradiation related factors at the protein level in human dermal fibroblast (HDF). The overall effect of individual solar irradiations such as Infrared A (IRA), blue light (BL), UVA, and UVB on HDF cells and the extent of molecular level aberrations to be assessed and compared against each. Methods: Label-free quantitative proteomics (MS/MS) approach has been adopted in this study to observe the protein level changes induced in the HDF cells through various exposures of full light sources. Following that, downstream insilico analysis has been carried out. Results: In this study, it is demonstrated all the four different solar irradiations significantly contribute to the molecular degeneration of skin cells through various mechanisms. This study confirms that BL down-regulates DNA repair proteins and the skin cells-HDF stimulate the histone proteins as a response mechanism to maintain the chromosomal integrity. Conclusions: The proteomics experiment carried out in the current study intends to support the future sun care products based on full light protection technology that can be custom designed to provide complete protection from the solar radiation. Similar technology could enhance the further investigations for deeper understanding of induction, mode of action, and prevention of skin damage from extensive solar irradiation.

LIF Inhibits Proliferation of Esophageal Squamous Carcinoma Cells by Radiation Mediated Through JAK-STAT Signaling Pathway.

Background: Esophagus cancer is a malignant tumor with a high incidence rate, and radiation is an important modality for esophageal cancer therapy. However, therapeutic failure in the treatment of ESCC is often attributed to an inherent radio-resistance of the tumor cells. This study discusses effect and mechanism of carbon ion exerts tumor-inhibiting proliferation via down-regulation of LIF in esophageal squamous cell carcinoma. Methods: Colony formation, CCK8 and EdU assays were used to detect cell survival and proliferation after 0 and 2Gy carbon ion irradiation of ECA109 cells. Proteomics changes were probed in response to carbon ion irradiation using quantitative proteomics approach incorporating TMT isotope tags. Then, candidate genes were identified via bioinformatics analysis methods and microarray results were verified by real-time qPCR. Paired ESCC tumor tissues and adjacent non-tumor samples from 17 patients were collected and used for detecting expression by immunohistochemistry. Furthermore, small interfering RNA (siRNA) was transfected into ECA109 and KYSE150 cells and cell proliferation was analyzed by EdU assay. Flow cytometry and Western blot were performed to measure the and apoptosis and JAK-STAT3 protein expression level of ECA109 and KYSE150 cells combined drugs after siLIF transfection. Results: When compared with the control (0Gy), Inhibition of ECA109 cell proliferation and clonogenic survival by 2 Gy carbon ions, radiation group screened 360 differentially expressed proteins, 156 of which were up-regulated and 144 were down-regulated. Downregulation of LIF expression by siRNA enhances apoptotic in the ECA109 and KYSE150 cells, significantly inhibited esophageal squamous cell carcinoma cells proliferation. In ESCC cells, the JAK/STAT3 signaling pathway is inhibited in a LIF-dependent manner, resulting in the expression of STAT3 downstream target genes. Carbon ions combined with siLIF inhibited cell proliferation more significantly. The inhibitory cell proliferation effect was more pronounced by the combined intervention of carbon ion irradiation with siLIF. LIF expression was 18.51±9.84 and 5.82±4.50 in 17 paired ESCC tissues and adjacent non-cancerous tissues, respectively. LIF protein expression was lower in ESCC than in the adjacent normal tissue. Conclusion: The findings of this study reveal that Carbon ion knockdown was shown to downregulate LIF in ESCC cells. LIF is involved in ESCC proliferation and inhibited the ESCC cell proliferation by activating the STAT3 signaling pathways.

Quantitative proteomic analysis of heifer serum at the peri-implantation period using two complementary methods.

Systemic events resulting from mother-embryo relation at the peri-implantation period may result in specific changes to the protein composition of serum and thus provide a source of biomarkers for early detection of pregnancy. We set out to use two different quantitative proteomic approaches to test this hypothesis by comparing heifer serum at the peri-implantation period to that from cycling heifers. Two-dimensional electrophoresis (2-DE) and isobaric tags were used for relative and absolute quantitation (iTRAQ) of proteins. These methods yielded complementary data indicating biomarker candidate proteins. Different proteomic methods provide different and complementary information that needs to be analysed in order to consider proteins as potential biomarkers. In order to characterise the proteome under specific conditions, the use of complementary techniques is advisable.

A Proteomic Approach Reveals That miR-423-5p Modulates Glucidic and Amino Acid Metabolism in Prostate Cancer Cells

Recently, we have demonstrated that miR-423-5p modulates the growth and metastases of prostate cancer (PCa) cells both in vitro and in vivo. Here, we have studied the effects of miR-423-5p on the proteomic profile in order to identify its intracellular targets and the affected pathways. Applying a quantitative proteomic approach, we analyzed the effects on the protein expression profile of miR-423-5p-transduced PCa cells. Moreover, a computational analysis of predicted targets of miR-423-5p was carried out by using several target prediction tools. Proteomic analysis showed that 63 proteins were differentially expressed in miR-423-5-p-transfected LNCaP cells if compared to controls. Pathway enrichment analysis revealed that stable overexpression of miR-423-5p in LNCaP PCa cells induced inhibition of glycolysis and the metabolism of several amino acids and a parallel downregulation of proteins involved in transcription and hypoxia, the immune response through Th17-derived cytokines, inflammation via amphorin signaling, and ion transport. Moreover, upregulated proteins were related to the S phase of cell cycle, chromatin modifications, apoptosis, blood coagulation, and calcium transport. We identified seven proteins commonly represented in miR-423-5p targets and differentially expressed proteins (DEPs) and analyzed their expression and influence on the survival of PCa patients from publicly accessible datasets. Overall, our findings suggest that miR-423-5p induces alterations in glucose and amino acid metabolism in PCa cells paralleled by modulation of several tumor-associated processes.

Iron Restriction Alleviates Atherosclerosis in ApoE KO Mice: An iTRAQ Proteomic Analysis.

The "iron hypothesis" of atherosclerosis has long been controversial. Several studies have shown that dietary iron restriction or low-iron diets can effectively alleviate atherosclerosis in rabbits and mice. However, the underlying molecular mechanisms of these phenomena remain to be elucidated. In this study, we further evaluated possible correlations between a low-iron diet and atherosclerosis alleviation by using a quantitative proteomic approach. For this purpose, apolipoprotein E knockout (ApoE KO) mice were divided into three groups and fed a normal diet (ND), a high-fat diet (HFD), or a high-fat +low-iron diet (HFD + LI). Our results showed that the HFD-LI improved atherosclerosis by decreasing en face lesions of the aorta and reducing the accumulation of macrophages and disordered smooth muscle cells. HFD-LI also decreased iron levels, serum hepcidin levels and the serum concentration of low-density lipoprotein cholesterol (LDL-C). The use of the isobaric tag for absolute quantification (iTRAQ) proteomic method and subsequent multi-technique molecular validation indicated that many of the proteins involved in atherosclerotic inflammation, vascular remodeling, and focal adhesion had significant changes in their expression among the diet groups. Importantly, the proteins Gal-3 and VCAM1, which are key participants of atherosclerosis pathogenesis, revealed lower expression after a low-iron diet. The present findings widely support the "iron hypothesis" of atherosclerosis. Further studies are suggested to fully understand the implications of these results.

Liver Proteome Alterations in Red Deer (Cervus elaphus) Infected by the Giant Liver Fluke Fascioloides magna.

Liver fluke infections are recognised as diseases with worldwide distribution and considerable veterinary and public health importance. The giant liver fluke, Fascioloides magna, is an important non-native parasite which has been introduced to Europe, posing a threat to the survival of local wildlife populations such as red deer (Cervus elaphus). The aim of the study was to analyse differences in liver proteomes between F. magna-infected and control red deer groups using a label-based high-throughput quantitative proteomics approach. The proteomics analysis identified 234 proteins with differential abundance between the control and infected groups. Our findings showed that F. magna infection in this definitive host is associated with changes in the metabolism of proteins and fatty acids, oxidative stress, fibrosis, and signaling pathways. The identified proteins and associated biological pathways represent a valuable contribution to the understanding of host-parasite interactions and the pathogenesis of liver fluke infection.

Decline of stress resilience in aging rats: Focus on choroid plexus-cerebrospinal fluid-hippocampus

Objectives: This study was designed to examine the mechanisms underlying decline of stress resilience in aged rats from the perspective of CP-CSF-hippocampus. Methods: Male Wistar rats (7–8 weeks old or 20 months old) were subjected to chronic unpredictable mild stress (CUMS) for 6 weeks. The behavioral tests were conducted to assess anxiety, depression and cognitive function. Hippocampal neurogenesis, apoptosis and synaptic plasticity were detected by western blot (WB) and/or immunofluorescence (IF) assay. Differential expression of growth factors (GFs) and axon guidance proteins (AGPs) in CSF was analyzed using the quantitative proteomics approach. IF and WB were performed to detect expression of occludin-1, Ki-67/Transthyretin, and folate transporters in choroid plexus (CP). Results: Decreased proliferation, impaired structure and transport function of CP were correlated with CSF composition alterations in stressed aging rats, including reduced 5-Methyltetrahydrofolate, growth factors and axon growth factors. Nutritional support of CSF upon hippocampus was attenuated, therefore affecting hippocampal plasticity. It has led to depression-like behaviors and cognitive deficits in stressful aged rats. Conclusions: Keeping normal structure and function of CP-CSF system may be a practical strategy for neuropsychological disorders in the elderly. This work provides evidential basis for CP transplant and CSF replacement therapy in future studies.

Comprehensive Proteomic Analysis of Brucella melitensis ATCC23457 Strain Reveals Metabolic Adaptations in Response to Nutrient Stress.

In the present study, a comprehensive proteomic analysis of Brucella melitensis (B. melitensis) strain ATCC23457 was carried out to investigate proteome alterations in response to in vitro-induced nutrient stress. Our analysis resulted in the identification of 2440 proteins, including 365 hypothetical proteins and 850 potentially secretory proteins representing ~77.8% of the B. melitensis proteome. Utilizing a proteogenomics approach, we provide translational evidence for eight novel putative protein-coding genes and confirmed the coding potential of 31 putatively annotated pseudogenes, thus refining the existing genome annotation. Further, using a label-free quantitative proteomic approach, new insights into the cellular processes governed by nutrient stress, including enrichment of amino acid metabolism (E), transcription (K), energy production and conversion (C), and biogenesis (J) processes were obtained. Pathway analysis revealed the enrichment of survival and homeostasis maintenance pathways, including type IV secretion system, nitrogen metabolism, and urease pathways in response to nutrient limitation. To conclude, our analysis demonstrates the utility of in-depth proteomic analysis in enabling improved annotation of the B. melitensis genome. Further, our results indicate that B. melitensis undergoes metabolic adaptations during nutrient stress similar to other Brucella. sp, and adapts itself for long-term persistence and survival.

Serum proteomics analysis of biomarkers for evaluating clinical response to MTX/IGU therapy in early rheumatoid arthritis

Methotrexate (MTX) and iguratimod (IGU) are conventional synthetic disease modifying antirheumatic drugs widely used in the treatment of Rheumatoid arthritis (RA) in China. Although MTX combined with IGU can significantly inhibit the progression of RA, some patients do not respond to the treatment. The purpose of this study is to explore the difference of serum protein expression between RA patients with good and poor response to the combined therapy by label-free quantitative proteomic approach. From the proteomics data, a total of 782 proteins in the serum of RA patients were detected, and of which 9 were upregulated and 18 were downregulated in the good response group compared to poor response group. Among them, four significantly differentially expressed proteins (RELN, LDHA, MRC1 and TKT) were further validated by multiple reaction monitoring (MRM)-based quantification approach, and three of them (RELN, LDHA and MRC1) were confirmed to be correlated with the response to MTX/IGU therapy. Logistic regression and ROC analysis indicated that the combination of RELN, LDHA and MRC1 had good performance in evaluating the response. This result proved the different serum proteins signature fingerprint between response group and non-response group; and highlighted the potential of the label-free and mass spectrometry-based quantitative proteomic approach in screening biomarkers for evaluating clinical response to MTX/IGU therapy in RA.

Differential proteomic analysis of frozen tilapia (Oreochromis niloticus) fillets with quality characteristics by a tandem mass tag (TMT)-based strategy

A tandem mass tag technology (TMT), based on the quantitative proteomics approach, was applied to investigate the associations between tilapia (Oreochromis niloticus) fillet quality characteristics (pH, color, texture, ice crystal morphology and protein denaturation) and proteome profiles during a 12-month frozen storage period. When compared with the control group (0 day), a total of 175 differentially abundant proteins (DAPs) were found in tilapia muscle from two frozen storage temperatures (−20 °C and −40 °C). Correlation analysis between the DAPs and the quality traits showed that 80 DAPs were correlated closely with the quality traits. The bioinformatics analysis revealed that 49, 9 and 2 DAPs were involved in binding proteins, metabolic enzymes and protein turnover, respectively, which were mainly closely associated with color traits and protein denaturation. These results provide a further understanding of the proteome changes and mechanisms of the quality decline of tilapia during frozen storage.

Serotype-dependent adhesion of Shiga toxin-producing Escherichia coli to bovine milk fat globule membrane proteins.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms for humans. Raw milk products are often incriminated as vehicule for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: (i) evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs), (ii) highlight STEC surface proteins associated with adhesion and (iii) evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the ζ-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The ζ-potential values were between -3.7 and - 2.9 mV for the strains and between -12.2 ± 0.14 mV for MFGs. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21,765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21,765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human gastrointestinal tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.

286 Oxidative Stress Modulation by Carnosine in Scaffold Free Human Dermis Spheroids Model: A Proteomic Study

Carnosine is an endogenous dipeptide with antioxidant and scavenger properties, shown to prevent aging and photoaging process by in vivo and in vitro models on different human tissues. Here an advanced quantitative proteomic approach (high-resolution mass spectrometry and network analyses) was applied to a 3D scaffold-free dermal spheroids model to investigate the biological responses induced by carnosine in human dermis. Primary human dermal fibroblasts from a 50-years-old donor were seeded in hanging drops to develop dermis spheroids.

Re-Sensitization of Azacytidine-Resistant MDS/AML Cells Via Modulation of KEAP1-NRF2 Redox Pathway

Introduction: The hypomethylation therapy by the cytidine analogue pro-drug 5-azacytidine (AZA) significantly extends the life expectancy of patients with myeloid malignancies, as shown in randomized trials in patients with myelodysplastic syndromes (MDS). Although AZA induces more durable hematologic responses, the rate and duration of remissions are insufficient, and most patients eventually progress on therapy, highlighting the need to better understand the mechanisms of drug resistance. Our and others’ previous data suggest that AZA resistance is the result of the interplay between multiple molecular mechanisms [Minařík L. et al. 2022]. Recent evidence highlights the important role of oxidative/reductive (redox) homeostasis in the development of resistance [Pollyea, et al. 2018, Jones CL. et al. 2019, Gu, X. et al. 2021]. Redox homeostasis is controlled by redox sensor Kelch like associated protein 1 (KEAP1), whose oxidation of cysteine under elevated oxidative stress stabilizes the transcription factor Nuclear factor erythroid 2-related factor 2 (NRF2), leading to cytoprotection against oxidative stress. In this work we aimed to investigate the role of the KEAP1-NRF2 pathway in the AZA efficacy. Methods: We derived AZA-resistant clones (AZA-R) from the human OCI-M2 MDS/acute myeloid leukemia (AML) cell line. The IC50 of AZA-R estimated by WST assay ranged from 4-16 µM. We applied CM2-H2DCFDA and ThiolTracker fluorescent probes to determine the oxidative state and glutathione levels (GSH) of AZA-sensitive (AZA-S) and AZA-R cells. To evaluate functional impact of altered redox homeostasis in AZA-R, we applied a quantitative mass spectrometry-based proteomic approach to identify protein targets of oxidative modifications in AZA-S and AZA-R after AZA treatment. The consequence of altered redox homeostasis was determined by RNA sequencing of AZA-S and AZA-R cells after 24h treatment with 1 µM AZA. The findings were functionally validated. Results: We showed that the redox state of AZA-S was increased by AZA, while the basal oxidative levels of AZA-R cells were 2-fold higher than those of AZA-S cells and were not further altered by AZA. Similarly, AZA induced GSH levels in AZA-S but not in AZA-R cells. Treatment of AZA-S with N-acetylcysteine (NAC) ameliorated oxidative stress and alleviated AZA toxicity, suggesting that AZA-induced oxidative stress is a key factor in its cytotoxic effect. Conversely, a reduction in GSH levels in these cells led to an increase in oxidative state, which was accompanied by enhanced sensitivity to AZA. Proteomic analysis showed that AZA treatment of AZA-S cells resulted in altered cysteine oxidation of 20% of cysteine proteins (578 out of 2853). Resistance to AZA was associated with oxidation state of 14% of cysteine proteins, among which the key redox sensor KEAP1 was significantly differently oxidized between AZA-R and AZA-S. Transcriptomic profiling revealed that 75 out of 478 NRF2 target genes were differentially expressed between AZA-S and AZA-R. NRF2 protein levels were significantly lower in the nucleus of AZA-R MDS/AML myeloblasts by Western blotting. These data indicate a loss of the dynamic response of AZA-R cells to oxidative stress by the KEAP1-NRF2 system. Therefore, we aimed to activate NRF2 in AZA-R by treatment with KEAP1 inhibitors (iKEAP1). We found that NRF2 activation in these cells modifies the redox balance and restores the sensitivity of AZA-R cells to AZA, presumably by restoring the antioxidant properties of the cells. Finally, we transplanted AZA-S and AZA-R cells into immunodeficient mice in order to validate our data in vivo. Generated Cell line-Derived Xenograft (CDX) AZA-R models were treated with iKEAP and indeed iKEAP1 markedly prolonged event-free survival and overall survival of MDS/AML mice upon AZA treatment. Conclusions: We demonstrate that the mechanism of AZA resistance involves the cellular adaptation to oxidative stress and that modulation of the KEAP1-NRF2 cellular antioxidant response pathway can efficiently re-sensitize AZA-resistant cells in vitro. We also demonstrated our findings of enhanced AZA efficacy by KEAP1 inhibition in vivo in an experimental CDX mouse model. This preliminary evidence supports the initiation of a randomized clinical trial to block KEAP1-NRF2 pathway in patients with high-risk-MDS progressing on AZA therapy.

Comprehensive proteome and lysine acetylome analysis after artificial aging reveals the key acetylated proteins involved in wheat seed oxidative stress response and energy production.

Lysine acetylation is a common post-translational modification of proteins within all organisms. However, quantitative acetylome characterization in wheat seed during aging in storage has not been reported. This study reports the first large-scale acetylome analysis of wheat seeds after artificial aging treatment, using the quantitative proteomic approach. In total, 11,002 acetylation sites, corresponding to 4262 acetylated proteins were identified, of which 1207 acetylated sites, representing 783 acetylated proteins, were significantly more or less acetylated after artificial aging. Functional analysis demonstrated that the majority of the acetylated proteins are closely involved with cellular and metabolic functions. In particular, key enzymes in the oxidative stress response and energy metabolism were significantly differentially acetylated and appear to be heavily involved in wheat seed aging. The acetylome analysis was verified by quantitative real-time PCR and enzyme activity determination. Lysine-acetylation results in a weaker oxidative stress response and lower energy production efficiency, resulting in the apoptosis of wheat seed cells, insufficient energy supply at the germination stage, and consequently, marked loss of seed vigor. PRACTICAL APPLICATIONS: It is known that the loss of protein function is an important reason for the decrease of seed vigor. Therefore, the change of protein function in the process of wheat seed aging was studied by proteome and lysine acetylome analysis technology. The results showed that the oxidation-reduction imbalance and the decrease of energy production efficiency of seeds were the important reasons for the decrease of their vigor. This will provide a new idea for green and safe storage of grain.

Increased levels of cell wall degrading enzymes and peptidases are associated with aggressiveness in a virulent isolate of Pyrenophora teres f. maculata

Pyrenophora teres f. maculata (Ptm) is a fungal pathogen that causes the spot form of net blotch on barley and leads to economic losses in many of the world's barley-growing regions. Isolates of Ptm exhibit varying levels of aggressiveness that result in quantifiable changes in the severity of the disease. Previous research on plant-pathogen interactions has shown that such divergence is reflected in the proteome and secretome of the pathogen, with certain classes of proteins more prominent in aggressive isolates. Here we have made a detailed comparative analysis of the secretomes of two Ptm isolates, GPS79 and E35 (highly and mildly aggressive, respectively) using a proteomics-based approach. The secretomes were obtained in vitro using media amended with barley leaf sections. Secreted proteins therein were harvested, digested with trypsin, and fractionated offline by HPLC prior to LC-MS in a high-resolution instrument to obtain deep coverage of the proteome. The subsequent analysis used a label-free quantitative proteomics approach with relative quantification of proteins based on precursor ion intensities. A total of 1175 proteins were identified, 931 from Ptm and 244 from barley. Further analysis revealed 160 differentially abundant proteins with at least a two-fold abundance difference between the isolates, with the most enriched in the aggressive GPS79 secretome. These proteins were mainly cell-wall (carbohydrate) degrading enzymes and peptidases, with some oxidoreductases and other pathogenesis-related proteins also identified, suggesting that aggressiveness is associated with an improved ability of GPS79 to overcome cell wall barriers and neutralize host defense responses.

Comprehensive Interactome Analysis for the Sole Adenylyl Cyclase Cyr1 of Candida albicans.

Cyr1, the sole adenylyl cyclase of the fungal pathogen Candida albicans, is a central component of the cAMP/protein kinase A signaling pathway that controls the yeast-to-hypha transition. Cyr1 is a multivalent sensor and integrator of various external and internal signals. To better understand how these signals are relayed to Cyr1 to regulate its activity, we sought to establish the interactome of Cyr1 by using stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative proteomics to identify the proteins that coimmunoprecipitated with Cyr1. The method identified 36 proteins as candidates for authentic Cyr1-interacting partners, together with two known Cyr1-binding proteins, Cap1 and Act1. Fourteen identified proteins belonged to three functional groups, including actin regulation, cell wall components, and mitochondrial activities, that are known to play important roles in cell morphogenesis. To validate the proteomics data, we used biochemical and genetic methods to characterize two cell wall-related proteins, Mp65 and Sln1. First, coimmunoprecipitation confirmed their physical association with Cyr1. Second, deleting either MP65 or SLN1 resulted in severe defects in filamentation on serum plates. This study establishes the first Cyr1 interactome and uncovers a potential role for cell wall proteins in directly regulating Cyr1 activity to determine growth forms in C. albicans. IMPORTANCE A critical virulence trait of the human fungal pathogen Candida albicans is its ability to undergo the yeast-to-hypha transition in response to diverse environmental and cellular stimuli. Previous studies suggested that the sole adenylyl cyclase of C. albicans, Cyr1, is a multivalent signal sensor and integrator synthesizing cAMP to activate the downstream hypha-promoting events through the cAMP/protein kinase A pathway. To fully understand how Cyr1 senses and processes multiple stimuli to generate appropriate signal outputs, it was necessary to identify and characterize Cyr1-interacting partners. This study employed SILAC-based quantitative proteomic approaches and identified 36 Cyr1-associated proteins, many having functions associated with hyphal morphogenesis. Coimmunoprecipitation verified two cell surface proteins, Mp65 and Sln1. Furthermore, genetic and phenotypic analyses demonstrated the cAMP-dependent roles of these two proteins in determining hyphal growth. Our study establishes the first Cyr1 interactome and uncovers new Cyr1 regulators that mediate cell surface signals to influence the growth mode of C. albicans.

Identification of hub genes and construction of prognostic nomogram for patients with Wilms tumors.

In children, Wilms' tumors are the most common urological cancer with unsatisfactory prognosis, but few molecular prognostic markers have been discovered for it. With the rapid development of high-throughput quantitative proteomic and transcriptomic approaches, the molecular mechanisms of various cancers have been comprehensively explored. This study aimed to uncover the molecular mechanisms underlying Wilms tumor and build predictive models by use of microarray and RNA-seq data. Gene expression datasets were downloaded from Therapeutically Applicable Research to Generate Effective Treatments (TARGET) and Gene Expression Omnibus (GEO) databases. Bioinformatics methods wereutilized to identified hub genes, and these hub genes were validated by experiment. Nomogram predicting OS was developed using genetic risk score model and clinicopathological variables. CDC20, BUB1 and CCNB2 were highly expressed in tumor tissues and able to affect cell proliferation and the cell cycle of SK-NEP-1 cells. This may reveal molecular biology features and a new therapeutic target of Wilms tumour.7 genes were selected as prognostic genes after univariate, Lasso, and multivariate Cox regression analyses and had good accuracy, a prognostic nomogram combined gene model with clinical factors was completed with high accuracy. The current study discovered CDC20,BUB1 and CCNB2 as hub-genes associated with Wilms tumor, providing references to understand the pathogenesis and be considered a novel candidate to target therapy and construct novel nomogram, incorporating both clinical risk factors and gene model, could be appropriately applied in preoperative individualized prediction of malignancy in patients with Wilms tumor.