Cryptosporidium is a protozoan parasite capable of infecting humans and animals and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms underlying invasive infection and its pathogenesis remain largely unknown. To better understand the molecular mechanism of the interaction between C. parvum and host cells, we profiled the changes of host cells membrane proteins extracted using native membrane protein extraction kit between C. parvum-infected HCT-8 cells and the control group after C. parvum infected 6 h combined with quantitative Tandem Mass Tags (TMT) liquid chromatography-dual mass spectrometry proteomic analysis. Among the 4844 quantifiable proteins identified, the expression levels of 625 were upregulated, and those of 116 were downregulated at 6 h post-infection compared with controls (1.5-fold difference in abundance, p < 0.05). Enrichment analysis of the function, protein domain and Kyoto Encyclopedia of Genes and Genomes pathway of the differentially expressed proteins revealed that the differentially expressed proteins were mainly related to biological functions related to the cytoskeleton and cytoplasmic matrix. We also found that infection with C. parvum may destroy HCT-8 intercellular space adhesion. Six proteins were further verified using quantitative real-time reverse transcription polymerase chain reaction and western blotting. Through systematic analysis of proteomics related to HCT-8 cell membranes infected by C. parvum, we found many host membrane proteins that can serve as potential receptors in C. parvum adhesion or invasion. C. parvum infection destroyed host cell barrier function and caused extensive changes in host cytoskeleton proteins, providing a deeper understanding of the molecules and their functions involved in the host-C. parvum interaction. SIGNIFICANCE: There is a lack of systematic research on the molecular mechanisms underlying the interaction of C. parvum with host cells. Changes of host cell membrane proteins after C. parvum infection may be used to examine the host cell receptors for parasite adhesion and invasion, and how the parasite interacts with these receptors. It is of great significance that host cells undergo membrane fusion to mediate invasion. Through proteomic studies on the host cell membrane after infection with HCT-8 cells by C. parvum, we observed disruption of the host cell cellular barrier function and widespread alteration of host cytoskeletal proteins caused by C. parvum infection, providing a deeper understanding of the molecules and their functions involved in host-C. parvum interaction.

Primary Sjogren's Syndrome (pSS) is a chronic autoimmune disease, with unclear pathogenies. Lysine-malonylation (Kmal) as a novel post-translational modification (PTMs) was found associated with metabolic, immune, and inflammatory processes. For purpose of investigating the proteomic profile and functions of kmal in pSS, liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based analysis and bioinformatics analysis are performed based on twenty-eight pSS patients versus twenty-seven healthy controls (HCs). A total of 331 down-regulated proteins and 289 up-regulated proteins are observed in differentially expressed proteins (DEPs) of pSS. We discover the expression of transforming growth factor beta-1 (TGFB1) and CD40 ligand downregulate which enriches in the inflammatory associated pathway. Expression of signal transducer and activator of transcription 1-alpha/beta (STAT1) show upregulation and enrich in type I interferon signaling pathway and IL-27-mediated signaling pathway. In differentially malonylated proteins (DMPs) of pSS, we identify 3 proteins are down-regulated in 7 sites and 18 proteins are up-regulated in 19 sites. Expression of malonylated integrin-linked kinase (ILK) significantly enrich in the focal adhesion pathway. Together, our data provide evidence that downregulation of TGFB1 and CD40LG play a critical role in the inflammatory process of pSS, while upregulation of STAT1 may be associated with IL-27 immunity and pSS immune dysfunction. Moreover, kmal modification at the kinase domain of ILK may destabilize ILK that thus contributing to pSS pathogenies by regulating the focal adhesion pathway. SIGNIFICANCE: Our research offered the first characterization of Kmal, a newly identified form of lysine acylation in pSS, as well as proteomic data on individuals with pSS. In this study, we found that several key DMPs were associated with focal adhesion pathway, which contributes to the development of pSS. The present results provide an informative dataset for the future exploration of Kmal in pSS.

Protein C-termini containing valuable biological information plays a vital role in various physiological processes, such as protein localization, protein recognition, and signal transduction in organisms. However, C-terminal peptide identification is still challenging due to their low abundance and similar physicochemical properties to other digested peptides. Herein, we developed a simple and mild strategy for the enrichment of C-terminal peptides that incorporates selectively 2-pyridinecarbaldehyde (2-PCA) derivatization of α-amine with negative enrichment by NHS resin. Two synthesized peptides were utilized to evaluate the efficiency of 2-PCA derivatization and optimize the coupling conditions of NHS resin. The feasibility of the method was further validated by enriching the C-terminus of the bovine serum albumin (BSA). Finally, this method was successfully applied to the C-terminus analysis of mouse brain tissue, identifying 404 protein C-termini with physicochemical properties unbiasedly. Additionally, the GO and KEGG analyses revealed that these identified proteins are crucial for proper brain function. In summary, our proposed method is effective and has the potential to facilitate comprehensive C-terminal analysis of proteins. SIGNIFICANCE: Effective enrichment methods are essential for the identification of the proteins C-terminus. In this study, a mild and simple method for negative C-terminal enrichment combined with site-specific derivatization was developed. The enrichment process was simplified and minimized sample loss simultaneously, using 2-PCA derivatization which has high α-amino specificity. Up to 346C-terminal proteins were identified in mouse brain tissue unbiasedly and reliably. This approach has the potential to facilitate comprehensive analysis of protein C-termini in a variety of biological contexts.

Magnaporthe oryzae snodprot1 homologous protein (MSP1) is known to function as a pathogen-associated molecular pattern (PAMP) and trigger PAMP-triggered immunity (PTI) in rice including induction of programmed cell death and expression of defense-related genes. The involvement of several post-translational modifications (PTMs) in the regulation of plant immune response, especially PTI, is well established, however, the information on the regulatory roles of these PTMs in response to MSP1-induced signaling is currently elusive. Here, we report the phosphoproteome, ubiquitinome, and acetylproteome to investigate the MSP1-induced PTMs alterations in MSP1 overexpressed and wild-type rice. Our analysis identified a total of 4666 PTMs-modified sites in rice leaves including 4292 phosphosites, 189 ubiquitin sites, and 185 acetylation sites. Among these, the PTM status of 437 phosphorylated, 53 ubiquitinated, and 68 acetylated peptides was significantly changed by MSP1. Functional annotation of MSP1 modulated peptides by MapMan analysis revealed that these were majorly associated with cellular immune responses including signaling, transcription factors, DNA and RNA regulation, and protein metabolism, among others. Taken together, our study provides novel insights into post-translational mediated regulation of rice proteins in response to M. oryzae secreted PAMP which help in understanding the molecular mechanism of MSP1-induced signaling in rice in greater detail. SIGNIFICANCE: The research investigates the effect of overexpression of MSP1 protein in rice leaves on the phosphoproteome, acetylome, and ubiquitinome. The study found that MSP1 is involved in rice protein phosphorylation, particularly in signaling pathways, and identified a key component, PTAC16, in MSP1-induced signaling. The analysis also revealed MSP1's role in protein degradation and modification by inducing ubiquitination of the target rice proteins. The research identified potential kinases involved in the phosphorylation of rice proteins, including casein kinase II, 14-3-3 domain binding motif, β-adrenergic receptor kinase, ERK1,2 kinase substrate motif, and casein kinase I motifs. Overall, the findings provide insights into the molecular mechanisms underlying of MSP1 induced signaling in rice which may have implications for improving crop yield and quality.

The high purity insoluble dietary fiber (IDF) from okara is a natural component with a potentially positive effect on a high-fat diet (HFD)-induced hepatic metabolic disorders, although its regulatory mechanism remains unclear. This study aims to elucidate the potential pathways and key proteins of IDF for the amelioration of hepatic lipid metabolism in mice fed with HFD. Here, we used label-free quantitative proteomics technology to quantity and identify differentially expressed proteins in the liver that are associated with IDF treatment. The differentially expressed proteins were assessed by GO annotation and KEGG pathways. Western blot and qRT-PCR analyses were conducted to validate the potential targets regulated by IDF. In total, 73 differentially expressed proteins were identified, of which 27 were up-regulated (FC > 1.5) and 46 were down-regulated (FC < 0.667). GO analysis suggested that differentially expressed proteins were mainly located in the cell and organelles, regulated biological processes, and were associated with enzyme activity and molecular binding. The KEGG pathway enrichment analysis further demonstrated glycolysis/gluconeogenesis, pyruvate metabolism, TCA cycle, arginine biosynthesis, alanine, aspartate and glutamate metabolism, and retinol metabolism were affected. The combination of proteomics, Western blot, and qRT-PCR suggested that ACS, ACLY, GOT1, GLS2, NAGS, CYP4A10, CYP3A25, and CYP2A5 in these pathways might be key proteins for IDF intervention. Taken together, our findings elucidate new mechanisms involved in how IDF affects hepatic metabolism, provide important information for the functional food industries, and improve the added value of okara. SIGNIFICANCE: Okara is evidenced as a high-quality by-product with several nutritional components, especially dietary fiber (50-60%) labeled as "The Seventh Nutrient". Previous studies have shown that IDF has a positive potential effect on a high-fat diet (HFD)-induced hepatic metabolic disorders, but its molecular mechanism remains unclear. To elucidate the therapeutic mechanism of IDF at the protein level, a label-free quantitative proteomic analysis was used to identify the dynamic changes of the liver proteome between HIDF and HFD groups in this study. These results provide a new perspective for exploring the therapeutic mechanism of IDF at the protein level and enlightenment for promoting the comprehensive utilization of okara.

Open-search methods allow unbiased, high-throughput identification of post-translational modifications in proteins at an unprecedented scale. The performance of current open-search algorithms is diminished by experimental errors in the determination of the precursor peptide mass. In this work we propose a semi-supervised open search approach, called ReCom, that minimizes this effect by taking advantage of a priori known information from a reference database, such as Unimod or a database provided by the user. We present a proof-of-concept study using Comet-ReCom, an improved version of Comet-PTM. Comet-ReCom increased identification performance of Comet-PTM by 68%. This increased performance of Comet-ReCom to score the MS/MS spectrum comes in parallel with a significantly better assignation of the monoisotopic peak of the precursor peptide in the MS spectrum, even in cases of peptide coelution. Our data demonstrate that open searches using ultra-tolerant mass windows can benefit from using a semi-supervised approach that takes advantage from previous knowledge on the nature of protein modifications. SIGNIFICANCE: The present study introduces a novel approach to ultra-tolerant database search, which employs prior knowledge of post-translational modifications (PTMs) to improve identification of modified peptides. This method addresses the limitations related to experimental errors and precursor mass assignation of previous open-search methods. Thus, it enables the study of the biological significance of a wider variety of PTMs, including unknown or unexpected modifications that may have gone unnoticed using non-supervised search methods.

To identify and evaluate differentially expressed plasma proteins in biliary atresia (BA), we performed plasma proteome profiling using liquid chromatography with tandem mass spectrometry (LC-MS/MS) in 20 patients with BA and 10 control children. Serological assays validated the most significant and highly upregulated proteins in a cohort of 45 patients and 15 controls. Bioinformatics tools were used for functional classification and protein-protein interactions of differentially expressed proteins (DEPs). Of 405 proteins detected in patients and 360 in controls, 242 proteins, each with ≥2 unique peptides (total of 3230 peptides), were common in both groups. Compared to controls, 90 proteins in patients were differentially expressed and were dysregulated. Twenty-five were significantly upregulated with polymeric immunoglobulin receptor (PIgR), galectin-3-binding protein (Gal-3BP), complement C2, the most prominent, and 15 had low expression. The bioinformatic analysis revealed functional interaction between DEPs and their role in an inflammatory immune response. Enzyme immunoassay for PIgR and Gal-3BP in patients' plasma showed their levels raised significantly (p = 0.0021 and p = 0.0369, respectively). The PIgR and Gal-3BP are novel proteins upregulated in BA and may be tested further for their utility as potential circulating disease biomarker(s). SIGNIFICANCE: The study shows that plasma PIgR and GAL-3BP levels are significantly raised in infants with BA within the first 3 months of life. If tested in a larger cohort, these proteins may be found to have their diagnostic potential and utility as disease biomarkers. The study also provides valuable information on the involvement of several DEPs in innate immune response, chronic inflammation, and fibrosis. This strengthens the hypothesis that the immune-mediated inflammatory processes are responsible for the progressive nature of BA.

Aspergillus flavus (A. flavus) infects the peanut seeds during pre-and post-harvest stages, causing seed quality destruction for humans and livestock consumption. Even though many resistant varieties were developed, the molecular mechanism of defense interactions of peanut against A. flavus still needs further investigation. Hence, an interologous host-pathogen protein interaction (HPPI) network was constructed to understand the subcellular level interaction mechanism between peanut and A. flavus. Out of the top 10 hub proteins of both organisms, protein phosphatase 2C and cyclic nucleotide-binding/kinase domain-containing protein and different ribosomal proteins were identified as candidate proteins involved in defense. Functional annotation and subcellular localization based characterization of HPPI identified protein SGT1 homolog, calmodulin and Rac-like GTP-binding proteins to be involved in defense response against fungus. The relevance of HPPI in infectious conditions was assessed using two transcriptome data which identified the interplay of host kinase class R proteins, bHLH TFs and cell wall related proteins to impart resistance against pathogen infection. Further, the pathogenicity analysis identified glycogen phosphorylase and molecular chaperone and allergen Mod-E/Hsp90/Hsp1 as potential pathogen targets to enhance the host defense mechanism. Hence, the computationally predicted host-pathogen PPI network could provide valuable support for molecular biology experiments to understand the host-pathogen interaction. SIGNIFICANCE: Protein-protein interactions execute significant cellular interactions in an organism and are influenced majorly by stress conditions. Here we reported the host-pathogen protein-protein interaction between peanut and A. flavus, and a detailed network analysis based on function, subcellular localization, gene co-expression, and pathogenicity was performed. The network analysis identified key proteins such as host kinase class R proteins, calmodulin, SGT1 homolog, Rac-like GTP-binding proteins bHLH TFs and cell wall related to impart resistance against pathogen infection. We observed the interplay of defense related proteins and cell wall related proteins predominantly, which could be subjected to further studies. The network analysis described in this study could be applied to understand other host-pathogen systems generally.

Epidermolytic palmoplantar keratoderma (EPPK), a highly penetrant autosomal dominant genodermatosis, is characterized by diffuse keratoses on palmplantar epidermis. The keratin 9 gene (KRT9) is responsible for EPPK. To date, phenotypic therapy is the primary treatment for EPPK. Because KRT9 pairs with a type II keratin-binding partner to function in epidermis, identifying the interaction partner is an essential first step in revealing EPPK pathogenesis and its fundamental treatment. In this study, we proved that keratin 6C (KRT6C) is a probable hereterodimer partner for KRT9. In silico model for KRT6C/KRT9 shows a typical coiled-coil structure in their 2B domains. Proteomics analysis shows that KRT6C/KRT9 pair is in a densely connected protein-protein interaction network, where proteins participate jointly in regulating cytoskeleton organization and keratinization. This study shows that co-immunoprecipitation coupled with mass spectroscopy and proteomics analysis provide a sensitive approach, which compensates for inevitable inadequacies of anti-keratin 6C antibody and helps discover the probable hereterodimer partner KRT6C for KRT9. The acknowledgement of KRT6C/KRT9 pairwise relationship may help re-classify EPPK and PC-K6c (a milder form of pachyonychia congenita, caused by KRT6C) as a group of hereditary defects at a molecular-based level, and lay foundation for deciphering the keratin network contributing to EPPK and PC-K6c. SIGNIFICANCE OF THE STUDY: What is already known about this topic? KRT9 and KRT6C are disease-causing factors for epidermolytic palmoplantar keratoderma (EPPK) and a milder form of pachyonychia congenita (PC-K6c), respectively. EPPK and PC-K6c have some symptom similarities. Keratins are the major structural proteins in epithelial cells. Each of the type I keratin is matched by a particular type II keratin to assemble a coiled-coil heterodimer. The hereterodimer partner for KRT9 is unknown. What does this study add? We discovered and proved that KRT6C is a probable hereterodimer partner for KRT9 in palmplantar epidermis in a native endogenous environment by using co-immunoprecipitation coupled with mass spectroscopy and proteomics analysis, etc. The proteomics analysis shows that KRT6C/KRT9 keratin pair is in a densely connected protein-protein interaction network, where proteins participate jointly in regulating intermediate filament-based cytoskeleton organization and keratinization processes. What are the implications of this work? The new understanding of probable KRT6C/KRT9 pairwise correlation may help re-classify the genetic cutaneous disorders EPPK and PC-K6c as a group of hereditary defects at a molecular-based level, and lay foundation for pathogenic mechanism research in EPPK and PC-K6c. The densely related network components derived from the proteomic data using Metascape in the study and pairwise regulation fashion of specific keratin pairs should attract more attention in the further explorations when investigators concern the physiological functions of keratins and the pathogenesis of related skin diseases.

Congenital cataracts are a threat to visual development in children, and the visual impairment persists after surgical treatment; however, the mechanisms involved remain unclear. Previous clinical studies have identified the effect of congenital cataracts on retinal morphology and function. To further understand the molecular mechanisms by which congenital cataracts affect retinal development, we analyzed retina samples from 7-week-old GJA8-knockout rabbits with congenital cataracts and controls by four-dimensional label-free quantification proteomics and untargeted metabolomics. Bioinformatics analysis of proteomic data showed that retinol metabolism, oxidative phosphorylation, and fatty acid degradation pathways were downregulated in the retinas of rabbits with congenital cataracts, indicating that their visual cycle and mitochondrial function were affected. Additional validation of differentially abundant proteins related to the visual cycle and mitochondrial function was performed using Parallel reaction monitoring and western blot experiments. Untargeted metabolome analysis showed significant upregulation of the antioxidant glutathione and ascorbic acid in the retinas of rabbits with congenital cataracts, indicating that their oxidative stress balance was not dysregulated. SIGNIFICANCE: Congenital cataracts in children can alter retinal structure and function, yet the mechanisms are uncertain. Here is the first study to use proteomics and metabolomics approaches to investigate the effects of congenital cataracts on retinal development in the early postnatal period. Our findings suggest that congenital cataracts have an impact on the retinal visual cycle and mitochondrial function. These findings give insight on the molecular pathways behind congenital cataract-induced visual function impairment in the early postnatal period.

This study investigated the effect of agro-industrial hazelnut skin by-products supplementation on lamb meat color variation and the changes in the sarcoplasmic muscle proteome during post-mortem storage (0, 4 and 7 days). Gel-based proteomics and bioinformatics approaches were applied to better understand the potential role of feeding strategies in modulating the mechanisms underpinning meat discoloration and post-mortem changes during storage. Therefore, twenty-two Valle del Belice male lambs were randomly assigned to two dietary treatments: control (C), lambs fed with maize-barley diet, and hazelnut skin (H), lambs fed hazelnut skin by-product as maize partial replacer in the concentrate diet. Hazelnut dietary treatment led to better lamb meat color stability as evidenced by the lowest decrease in redness and saturation index values. Proteomics and bioinformatics results revealed changes in the abundance of 41 proteoforms, which were mainly involved in glycolytic processes, responses to oxidative stress, and immune and endocrine system. The proteins allowed revealing interconnected pathways to be behind meat color variation as a consequence of using hazelnut skin by-products to sustainable feed lamb. The proteins can be used as potential predictors of lamb meat color variation. Accordingly, the regression equations developed in this paper revealed triosephosphate isomerase (TPI1) as a reliable candidate biomarker of color stability in lamb meat. SIGNIFICANCE: The use of agro-industrial by-products in animal feeding can be a potential sustainable strategy to reduce the environmental impacts of the food production chain and consequently improve animal welfare and product quality. The inclusion of hazelnut skin by-products in the animal's diet, due to the high concentration of polyphenols, represents an effective strategy to improve the oxidative stability of meat, with significant implications on color. The use of proteomics combined with bioinformatics on the sarcoplasmic proteome is a powerful approach to decipher the underlying mechanism. Accordingly, this approach allowed in this trial a deeper understanding of the molecular mechanisms involved in the post-mortem processes through the discovery of several biological pathways linked with lamb meat color variation. Glycolysis, followed by responses to oxidative stress, and other proteins involved in the immune and endocrine system were found as the major interconnected pathways that could act as potential predictors of lamb meat color stability. Candidate proteins biomarkers were further revealed in this study to be related with multiple meat color traits.