Comparative Proteomic Analysis of Leptospira Interrogans Serogroup Icterohaemorrhagiae Human Vaccine Strain and Epidemic Isolate of China

Background: Leptospira interrogans serogroup Icterohaemorrhagiae is the predominant pathogen causing leptospirosis in China and is still used as the vaccine strain for the current human inactivated vaccine. Unlike the clade ST17, which is distributed worldwide, ST1 is the most prevalent in serogroup Icterohaemorrhagiae in China. Purpose and Methods: To further characterize leptospiral pathogens, isobaric tags for relative and absolute quantitation and parallel reaction monitoring were used to analyze differences at the proteomic level between serogroup Icterohaemorrhagiae vaccine strain 56001 (ST1) and circulating isolate 200502 (ST17) from different periods. Results: Two hundred and eighty-one proteins were differentially expressed between ST17 and ST1, of which 166 were upregulated (>1.2 fold change, P < 0.05) and 115 (>1.2-fold change, P < 0.05) were downregulated. Function prediction revealed that nine upregulated proteins were outer membrane proteins, including several known immunogenic and/or virulence-related proteins, such as ompL1, LipL71 and LipL41. Furthermore, important expression differences in carbohydrate, amino acid, and energy metabolism and transport proteins were identified between ST1 and ST17, suggesting that these differences may reflect metabolic diversity and the potential of the pathogens to adapt to different environments. Conclusion: In summary, our findings provide insights into better understanding the component strains of the Chinese human leptospirosis vaccine at the proteomic level. Additionally, these data facilitate evaluating the mechanisms by which pathogenic Leptospira species adapt to the host environment.

USP3 promotes gastric cancer progression and metastasis by deubiquitination-dependent COL9A3/COL6A5 stabilisation

As an important regulator of intracellular protein degradation, the mechanism of the deubiquitinating enzyme family in tumour metastasis has received increasing attention. Our previous study revealed that USP3 promotes tumour progression and is highly expressed in gastric cancer (GC). Herein, we report two critical targets, COL9A3 and COL6A5, downstream of USP3, via the isobaric tags for relative and absolute quantification technique. Mechanistically, we observed that USP3 interacted with and stabilised COL9A3 and COL6A5 via deubiquitination in GC. Importantly, we found that COL9A3 and COL6A5 were essential mediators of USP3-modulated oncogenic activity in vitro and in vivo. Examination of clinical samples confirmed that elevated expression of USP3, concomitant with increased COL9A3 and COL6A5 abundance, correlates with human GC progression. These data suggest that USP3 promotes GC progression and metastasis by deubiquitinating COL9A3 and COL6A5. These findings identify a mechanism of GC metastasis regarding USP3-mediated deubiquitinating enzyme activity and suggest potential therapeutic targets for GC management.

Gene excavation and expression analysis of CYP and UGT related to the post modifying stage of gypenoside biosynthesis in Gynostemma pentaphyllum (Thunb.) Makino by comprehensive analysis of RNA and proteome sequencing

Previous studies have revealed that gypenosides produced from Gynostemma pentaphyllum (Thunb.) Makino are mainly dammarane-type triterpenoid saponins with diverse structures and important biological activities, but the mechanism of diversity for gypenoside biosynthesis is still unclear. In this study, a combination of isobaric tags for relative and absolute quantification (iTRAQ) proteome analysis and RNA sequencing transcriptome analysis was performed to identify the proteins and genes related to gypenoside biosynthesis. A total of 3925 proteins were identified by proteomic sequencing, of which 2537 were quantified. Seventeen cytochrome P450 (CYP) and 11 uridine 5’-diphospho-glucuronosyltransferase (UDP-glucuronosyltransferase, UGT) candidate genes involved in the side chain synthesis and modification of gypenosides were found. Seven putative CYPs (CYP71B19, CYP77A3, CYP86A7, CYP86A8, CYP89A2, CYP90A1, CYP94A1) and five putative UGTs (UGT73B4, UGT76B1, UGT74F2, UGT91C1 and UGT91A1) were selected as candidate structural modifiers of triterpenoid saponins, which were cloned for gene expression analysis. Comprehensive analysis of RNA sequencing and proteome sequencing showed that some CYPs and UGTs were found at both the transcription and translation levels. In this study, an expression analysis of 7 CYPs and 5 UGTs that contributed to gypenoside biosynthesis and distribution in G. pentaphyllum was performed, providing consistent results that will inspire more future research on vital genes/proteins involved in gypenoside biosynthesis.

Isobaric Tags for Relative and Absolute Quantitation-Based Proteomics Analysis Provides a New Perspective Into Unsynchronized Growth in Kuruma Shrimp (Marsupenaeus japonicus)

Unsynchronized growth is a common phenomenon in farmed crustaceans. The underlying molecular mechanism of unsynchronized growth of crustaceans is unclear. In this study, a comparative proteomic analysis focusing on growth differences was performed using kuruma shrimp Marsupenaeus japonicus, an economic crustacean species, as the model. The study analyzed kuruma shrimp at fast growth stage and steady growth stage from both fast growth group and slow growth group by an Isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analysis method. A total of 1,720 proteins, including 12,291 peptides, were identified. Fifty-two and 70 differentially expressed proteins (DEPs) were identified in the fast growth stage and steady growth stage, respectively. Interestingly, 10 DEPs, including 14-3-3-epsilon-like, GPI, GPD1, MHC-1a, and MHC-1b, were presented in both growth stages. In addition, all these 10 DEPs shared the same expression tendency at these two growth stages. The results indicated that these 10 DEPs are potential growth biomarkers of M. japonicus. Proteins associated with faster growth of M. japonicus may promote cell growth and inhibit cell apoptosis through the Hippo signaling pathway. The fast growth group of M. japonicus may also achieve growth superiority by activating multiple related metabolic pathways, including glycolysis, glycerophospholipid metabolism and Citrate cycle. The present study provides a new perspective to explore the molecular mechanism of unsynchronized growth in crustacean species.

Insights Into Mechanism of the Naphthalene-Enhanced Biodegradation of Phenanthrene by Pseudomonas sp. SL-6 Based on Omics Analysis

The existence of polycyclic aromatic hydrocarbons (PAHs) in contaminated environment is multifarious. At present, studies of metabolic regulation focus on the degradation process of single PAH. The global metabolic regulatory mechanisms of microorganisms facing coexisting PAHs are poorly understood, which is the major bottleneck for efficient bioremediation of PAHs pollution. Naphthalene (NAP) significantly enhanced the biodegradation of phenanthrene (PHE) by Pseudomonas sp. SL-6. To explore the underlying mechanism, isobaric tags for relative and absolute quantification (iTRAQ) labeled quantitative proteomics was used to characterize the differentially expressed proteins of SL-6 cultured with PHE or NAP + PHE as carbon source. Through joint analysis of proteome and genome, unique proteins were identified and quantified. The up-regulated proteins mainly concentrated in PAH catabolism, Transporters and Electron transfer carriers. In the process, the regulator NahR, activated by salicylate (intermediate of NAP-biodegradation), up-regulates degradation enzymes (NahABCDE and SalABCDEFGH), which enhances the biodegradation of PHE and accumulation of toxic intermediate–1-hydroxy-2-naphthoic acid (1H2Na); 1H2Na stimulates the expression of ABC transporter, which maintains intracellular physiological activity by excreting 1H2Na; the up-regulation of cytochrome C promotes the above process running smoothly. Salicylate works as a trigger that stimulates cell to respond globally. The conjecture was verified at transcriptional and metabolic levels. These new insights contribute to improving the overall understanding of PAHs-biodegradation processes under complex natural conditions, and promoting the application of microbial remediation technology for PAHs pollution.

Isobaric Tags for Relative and Absolute Quantification-Based Proteomics Reveals Candidate Proteins of Fat Deposition in Chinese Indigenous Sheep With Morphologically Different Tails

Background: Chinese indigenous sheep can be classified into two types according to their tail morphology: fat-rumped and thin-tailed sheep, of which the typical breeds are Altay sheep and Tibetan sheep, respectively.Methods: To identify the differentially expressed proteins (DEPs) underlying the phenotypic differences between tail types, we used isobaric tags for relative and absolute quantification (iTRAQ) combined with multi-dimensional liquid chromatography tandem-mass spectrometry (LC-MS/MS) technology to detect candidate proteins. We then subjected these to a database search and identified the DEPs. Finally, bioinformatics technology was used to carry out Gene Ontology (GO) functional and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.Results: A total of 3,248 proteins were identified, of which 44 were up-regulated and 40 were down-regulated DEPs. Analyzing their GO function terms and KEGG pathways revealed that the functions of these DEPs are mainly binding, catalytic activity, structural molecule activity, molecular function regulator, and transporter activity. Among the genes encoding the DEPs, APOA2, GALK1, ADIPOQ, and NDUFS4 are associated with fat formation and metabolism.Conclusion: The APOA2, GALK1, ADIPOQ, and NDUFS4 genes may be involved in the deposition of fat in the tail of sheep. This study provides a scientific basis for the breeding of thin-tailed sheep.

iTRAQ-Based Proteomics Analysis of Response to Solanum tuberosum Leaves Treated with the Plant Phytotoxin Thaxtomin A

Thaxtomin A (TA) is a phytotoxin secreted by Streptomyces scabies that causes common scab in potatoes. However, the mechanism of potato proteomic changes in response to TA is barely known. In this study, the proteomic changes in potato leaves treated with TA were determined using the Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) technique. A total of 693 proteins were considered as differentially expressed proteins (DEPs) following a comparison of leaves treated with TA and sterile water (as a control). Among the identified DEPs, 460 and 233 were upregulated and downregulated, respectively. Based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, many DEPs were found to be involved in defense and stress responses. Most DEPs were grouped in carbohydrate metabolism, amino acid metabolism, energy metabolism, and secondary metabolism including oxidation–reduction process, response to stress, plant–pathogen interaction, and plant hormone signal transduction. In this study, we analyzed the changes in proteins to elucidate the mechanism of potato response to TA, and we provided a molecular basis to further study the interaction between plant and TA. These results also offer the option for potato breeding through analysis of the resistant common scab.

Identification of New Proteins and Potential Mitochondrial F1F0-ATPase Inhibitor Factor 1-Associated Mechanisms in Arabidopsis thaliana Using iTRAQ-Based Quantitative Proteomic Analysis

The mitochondrial synthesis of ATP makes a vital contribution to the growth and development of biological organisms, in which the enzyme mitochondrial F1F0-ATP synthase plays a pivotal role, in that it can either synthesize or hydrolyze cellular ATP. The finding of our previous study revealed that mitochondrial F1F0-ATPase inhibitor factor 1 (IF1) in Arabidopsis thaliana has a conserved function as an endogenous inhibitor affecting cellular energy status and plays an important role in plant growth and reproduction, particularly in fertility. In this study, to gain an insight into IF1-related traits, we performed isobaric tags for relative and absolute quantitation labeling analysis. In total, 67 of 4778 identified proteins were identified as differentially expressed proteins (DEPs; 59 up-regulated and 8 down-regulated) between wild-type and if1 mutant Arabidopsis thaliana seedlings. Gene ontology enrichment analysis revealed that these DEPs were the most significantly enriched in pathways such as “long-day photoperiodism, flowering,” “positive regulation of protein import into chloroplast stroma,” and “pollen sperm cell differentiation,” which are closely associated with reproductive development. Moreover, Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that photosynthesis was the pathway most significantly enriched with DEPs. Collectively, our results revealed a global shift in protein abundance patterns corresponding to AtIF1 mutation, entailing changes in the abundance of multiple key proteins and metabolic processes, which will provide a valuable proteomic foundation for future studies.

Potential Muscle-Related Biomarkers in Predicting Curve Progression to the Surgical Threshold in Adolescent Idiopathic Scoliosis—A Pilot Proteomic Study Comparing Four Non-Progressive vs. Four Progressive Patients vs. A Control Cohort

Previous studies have reported abnormal muscle morphology and functions in patients with adolescent idiopathic scoliosis (AIS). To answer whether such abnormalities could be reflected in their circulation and their clinical implication for predicting curve progression to the surgical threshold, this preliminary study explored the presence of baseline muscle-related proteins and their association with curve progression. Plasma samples were collected at the first clinical visit for AIS, with patients divided into non-progressive or progressive groups (N = four and four) according to their Cobb angle in six-year follow-ups, with age- and sex-matched healthy subjects (N = 50). Then, the samples were subjected to isobaric tags for relative and absolute quantitation (iTRAQ) for global comparison of untargeted protein expression. Seventy-one differentially expressed proteins (DEPs) were found elevated in progressive AIS. Functional analysis showed that 18 of these are expressed in muscles and play an essential role in muscle activities. Among the muscle-related DEPs, α-actin had the highest fold change in progressive/non-progressive groups. This preliminary study firstly suggested higher circulating levels of muscle structural proteins in progressive AIS, indicating the likelihood of structural damage at the microscopic level and its association with progression to the surgical threshold. Further studies with larger sample sizes are warranted to validate these novel candidates for early diagnosis and predicting progression.

Quantitative proteomics by iTRAQ-PRM based reveals the new characterization for gout

Background Gout is a common and complex form of immunoreactive arthritis based on hyperuricemia, while the symptoms would turn to remission or even got worse. So, it is hard to early identify whether an asymptomatic hyperuricemia (AHU) patient will be susceptible to get acute gout attack and it is also hard to predict the process of gout remission to flare. Here, we report that the plasma proteins profile can distinguish among acute gout (AG), remission of gout (RG), AHU patients, and healthy controls. Methods We established an isobaric tags for relative and absolute quantification (iTRAQ) and parallel reaction monitoring (PRM) based method to measure the plasma proteins for AG group (n = 8), RG group (n = 7), AHU group (n = 7) and healthy controls (n = 8). Results Eleven differentially expressed proteins such as Histone H2A, Histone H2B, Thrombospondin-1 (THBS1), Myeloperoxidase (MPO), Complement C2, Complement component C8 beta chain (C8B), Alpha-1-acid glycoprotein 1 (ORM1), Inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), Carbonic anhydrase 1 (CA1), Serum albumin (ALB) and Multimerin-1 (MMRN1) were identified. Histone H2A, Histone H2B and THBS1 might be the strongest influential regulator to maintain the balance and stability of the gout process. The complement and coagulation cascades is one of the main functional pathways in the mechanism of gout process. Conclusions Histone H2A, Histone H2B and THBS1 are potential candidate genes for novel biomarkers in discriminating gout attack from AHU or RG, providing new theoretical insights for the prognosis, treatment, and management of gout process. Trial registration This study is not a clinical trial.