Proteomic profiling of fatty acid biosynthetic enzymes from oil palm chromoplast

Plant fatty acid metabolism has proven to be amenable to manipulation by conventional breeding, genetic and metabolic engineering to enhance the fatty acid profile. This can be done by engineering palm fruit to synthesise more oleic acid at the expense of palmitic acid. This would produce an oil with greater perceived nutritional quality and higher market value. Although the biochemistry of fatty acid biosynthesis in plants is well described, crosstalk between transcriptional and metabolic controls in regulating fatty acid composition remains poorly understood. Our hypothesis is that phosphorylation is one of the main regulators of acetyl-CoA carboxylase, fatty acid synthase complex and stearoyl-ACP-desaturase in increasing the oleic acid level between oil palm (Elaeis guineensis Jacq. var. Tenera) low and high oleic acid varieties. This study utilised advanced proteomic techniques to isolate, detect and identify chromoplast-based phosphorylated proteins associated with the fatty acid biosynthesis pathway. Sub-organelle isolation using differential centrifugation enriched the chromoplast fraction that contained the fatty acid biosynthetic enzymes before their protein extraction. Gel-based and non-gel based mass spectrometry techniques were then employed to separate and improve the identification of key fatty acid biosynthetic enzymes. Protein expression was analysed using isobaric labelling strategy. Five key enzymes, namely the β-ketoacyl-ACP reductase (EC 1.1.1.100), β-hydroxyacyl-ACP dehydrogenase (EC 4.2.1.58 and 4.2.1.59), 3-enoyl-ACP reductase (EC 1.3.19), β-ketoacyl-ACP synthase (EC 2.3.1.41) and stearoyl-ACP desaturase (EC 1.14.99.6) were identified using GeLC-MS/MS strategy. An additional two subunits of acetyl-CoA carboxylase (EC 6.4.1.2) were identified from the 2DLC-MS/MS strategy. The expression of β-hydroxyacyl-ACP dehydrogenase and β-ketoacyl-ACP synthase was up-regulated in the high oleic acid variety. In contrast, 3-enoyl-ACP reductase was down-regulated in the high oleic acid variety. The existence of other differentially regulated metabolic enzymes associated with fatty acid biosynthesis suggested that the control of fatty acid production, particularly the synthesis of oleic acid, involves more than just the main fatty acid biosynthetic enzymes. Subsequently, the role of phosphorylation in regulating these fatty acid biosynthetic enzymes was investigated using a novel combination of neutral loss-triggered MS3 and Selected Reaction Monitoring. Acetyl-CoA carboxylase and 3-enoyl-ACP reductase were postulated to be phosphorylated in both low oleic acid and high oleic acid-producing oil palms during the fruit maturation stage of 20th week after anthesis. However, other fatty acid biosynthetic enzymes from these oil palm varieties did not show any indication of phosphorylation despite the prediction of phosphoserine-containing peptides. The location of the phosphorylated serine residues in the protein domains of acetyl-CoA carboxylase and 3-enoyl-ACP reductase suggested that phosphorylation could have regulated their enzyme activities. This study has produced a robust method to capture and identify chromoplast-based enzymes that are related to plant fatty acid biosynthesis. The differences in their protein expression levels suggested that fatty acid biosynthetic enzymes were differentially regulated and phosphorylation might be involved in this regulation, at least in the enzyme activity. The outcomes reported in this thesis have significantly improved the knowledge of the possible regulation mechanisms in plant fatty acid biosynthesis. The logical extension of this work in future efforts will be to determine the biological significance of this differential protein expression and to understand the exact role of phosphorylation in the regulation of these enzymes

Evaluation of sodium deoxycholate as solubilization buffer for oil palm proteomics analysis.

Protein solubility is a critical prerequisite to any proteomics analysis. Combination of urea/thiourea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have been routinely used to enhance protein solubilization for oil palm proteomics studies in recent years. The goals of these proteomics analysis are essentially to complement the knowledge regarding the regulation networks and mechanisms of the oil palm fatty acid biosynthesis. Through omics integration, the information is able to build a regulatory model to support efforts in improving the economic value and sustainability of palm oil in the global oil and vegetable market. Our study evaluated the utilization of sodium deoxycholate as an alternative solubilization buffer/additive to urea/thiourea and CHAPS. Efficiency of urea/thiourea/CHAPS, urea/CHAPS, urea/sodium deoxycholate and sodium deoxycholate buffers in solubilizing the oil palm (Elaeis guineensis var. Tenera) mesocarp proteins were compared. Based on the protein yields and electrophoretic profile, combination of urea/thiourea/CHAPS were shown to remain a better solubilization buffer and additive, but the differences with sodium deoxycholate buffer was insignificant. A deeper mass spectrometric and statistical analyses on the identified proteins and peptides from all the evaluated solubilization buffers revealed that sodium deoxycholate had increased the number of identified proteins from oil palm mesocarps, enriched their gene ontologies and reduced the number of carbamylated lysine residues by more than 67.0%, compared to urea/thiourea/CHAPS buffer. Although only 62.0% of the total identified proteins were shared between the urea/thiourea/CHAPS and sodium deoxycholate buffers, the importance of the remaining 38.0% proteins depends on the applications. The only observed limitations to the application of sodium deoxycholate in protein solubilization were the interference with protein quantitation and but it could be easily rectified through a 4-fold dilution. All the proteomics data are available via ProteomeXchange with identifier PXD013255. In conclusion, sodium deoxycholate is applicable in the solubilization of proteins extracted from oil palm mesocarps with higher efficiency compared to urea/thiourea/CHAPS buffer. The sodium deoxycholate buffer is more favorable for proteomics analysis due to its proven advantages over urea/thiourea/CHAPS buffer

Skin Mucus Proteome Analysis Reveals Disease-Resistant Biomarker Signatures in Hybrid Grouper (<i>Epinephelus fuscoguttatus</i> ♀ × <i>Epinephelus lanceolatus</i> ♂) against <i>Vibrio alginolyticus</i>

Fish skin mucus is the first line of defense that provides physical and chemical barriers against pathogens and toxins. The mucus is produced continuously and sloughed off regularly from the skin to defend against infections through the skin. However, the molecular properties of the mucus content that prevent pathogen invasion are yet to be fully understood. In this study, a proteomic approach using liquid chromatography–mass spectrometry (LCMS) was applied to explore the changes in the mucus protein content of resistant and susceptible groupers in response to Vibrio alginolyticus. The Vibrio-resistant groupers showed no observable clinical sign of infection after the immersion challenge, while the Vibrio-susceptible groupers presented either hemorrhagic- or non-hemorrhagic ulceration of the skin. A comparative proteome analysis on the mucus samples yielded 1488 identified proteins. The immune-related proteins, namely Cystatin B, Complement Component C6, Complement factor 1, Allograft inflammatory factor 1, Deleted in malignant brain tumors protein, MHC class 1 and Annexin A1, that were significantly abundant in the resistant group responded to V. alginolyticus infection. Interestingly, there was an expression of immune-related proteins that possibly could be the non-invasive biomarkers, namely 3-hydroxybutyrate dehydrogenase type 2 and L-rhamnose-binding lectin SML

Higher resolution protein band visualisation via improvement of colloidal CBB-G staining by gel fxation

Background: Gel staining is a crucial step that allows the visualisation of proteins separated through SDS-PAGE. Col‑ loidal Coomassie Brilliant Blue-G (CBB-G) staining is among the commonly used visualisation methods due to several factors such as compatibility with mass spectrometry (MS) analysis, sensitivity, reproducibility, and simplicity of the staining process. However, the standard colloidal CBB-G staining has a drawback: the resolution of protein bands is compromised because of difusion of proteins during the washing step. Results: A modifcation to an established colloidal CBB-G staining method, which greatly increases the resolution of protein bands, is described. The addition of a fxation step, which prevents the difusion of proteins during the wash‑ ing step, is shown to increase protein band resolution. Conclusion: The fxation step is fast, fexible, and also retains all the advantages of the standard colloidal CBB-G stain‑ing methods. As there are no drawbacks, incorporating this fxation step into the standard colloidal CBB-G staining is an easy way to improve protein visualisation in SDS-PAGE

Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata

Background: Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. Methods: In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. Results: Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. Conclusion: Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host

Computationally Designed Anti-LuxP DNA Aptamer Suppressed Flagellar Assembly- and Quorum Sensing-Related Gene Expression in Vibrio parahaemolyticus

(1) Background: Quorum sensing (QS) is the chemical communication between bacteria that sense chemical signals in the bacterial population to control phenotypic changes through the regulation of gene expression. The inhibition of QS has various potential applications, particularly in the prevention of bacterial infection. QS can be inhibited by targeting the LuxP, a periplasmic receptor protein that is involved in the sensing of the QS signaling molecule known as the autoinducer 2 (AI-2). The sensing of AI-2 by LuxP transduces the chemical information through the inner membrane sensor kinase LuxQ protein and activates the QS cascade. (2) Methods: An in silico approach was applied to design DNA aptamers against LuxP in this study. A method combining molecular docking and molecular dynamics simulations was used to select the oligonucleotides that bind to LuxP, which were then further characterized using isothermal titration calorimetry. Subsequently, the bioactivity of the selected aptamer was examined through comparative transcriptome analysis. (3) Results: Two aptamer candidates were identified from the ITC, which have the lowest dissociation constants (Kd) of 0.2 and 0.5 micromolar. The aptamer with the lowest Kd demonstrated QS suppression and down-regulated the flagellar-assembly-related gene expression. (4) Conclusions: This study developed an in silico approach to design an aptamer that possesses anti-QS properties