Search Results (149)

Thiol isomerases are a family of enzymes that participate in oxidative protein folding. They contain highly reactive vicinal thiols in a CXXC motif within their catalytic domains to mediate thiol-disulfide switching as part of their reductase, oxidase, and isomerase activity. In addition, they participate in chaperone function by binding to partially folded or misfolded proteins and preventing aggregation, thereby facilitating correct protein folding. The CXXC motif is conducive to oxidative influence based on the sulfur nucleophilicity. Redox modification of the CXXC motif may influence the enzymatic function. In this review we briefly discuss the family of thiol isomerases as it relates to thrombotic disorders. We then discuss the chemical mechanisms of making and breaking disulfides by the enzymes. Enzymatic and chemical models of oxidizing the CXXC motif are proposed. Lastly, we highlight evidence that natural galloylated polyphenols can inhibit both the coronavirus main protease Mpro and thiol isomerases, supporting a therapeutic strategy for COVID-19-associated coagulopathy and thrombosis by targeting the CXXC motif with these anti-oxidative compounds. Full article

Background: Argas persicus is a hematophagous ectoparasite of poultry and is the vector of several agents infectious to poultry. This study aims to explore the key genes affecting the ovarian development of A. persicus. Methods: RNA-seq was performed on the ovaries of A. persicus before blood-feeding, on the day of engorgement, and 6 days post-engorgement. Utilizing the threshold padj < 0.05 and|log₂(foldchange)| > 1, differentially expressed genes were identified, and hub genes were determined by constructing protein–protein interaction (PPI) networks. Results: A total of 1008 differentially expressed genes were obtained during the feeding period, including 448 up-regulated and 560 down-regulated genes. Further, 2179 differentially expressed genes were screened in the preoviposition stage, including 1957 up-regulated and 222 down-regulated genes. These genes are mainly annotated in functions such as peptidase activity (especially serine protease activity), protein folding, protein assembly, and cell component assembly, and enriched in pathways such as protein processing in endoplasmic reticulum, lysosome, glutathione metabolism, and sphingolipid metabolism. In addition, some proteins that are closely related to ovarian development, including heat shock protein 70, protein disulfide isomerase, paramyosin, troponin I, hexosaminidase, serine protease, Kunitz serine protease inhibitors, and vitellogenin, were obtained. Conclusions: These findings fill the gap in the biological data for the ovarian development of soft ticks, provide a reference database for subsequent proteomics research, and offer fundamental support for the screening and development of candidate antigens for anti-tick vaccines. Full article

Almost every cell of a multicellular organism is in contact with the extracellular matrix (ECM), which provides the shape and mechanic stability of tissue, organs and the entire body. At the molecular level, cells contact the ECM via integrins. Integrins are transmembrane cell adhesion molecules that connect the ECM to the cytoskeleton, which they bind with their extracellular and intracellular domains. Cysteine residues are abundant in both integrin subunits α and β. If pairwise oxidized into disulfide bridges, they stabilize the folding and molecular structure of the integrin. However, despite the oxidative environment of the extracellular space, not all pairs of cysteines in the extracellular integrin domains are permanently engaged in disulfide bridges. Rather, the reversible and temporary linkage of cystine bridges of these cysteine pairs by oxidation or their reductive cleavage can cause major conformational changes within the integrin, thereby changing ligand binding affinity and altering cellular functions such as adhesion and migration. During recent years, several oxidoreductases and thiol isomerases have been characterized which target such allosteric disulfide bridges. This outlines much better, albeit not comprehensively, the role that such thiol switches play in the redox regulation of integrins. The platelet integrin αIIbβ3 is the best examined example so far. Mostly referring to this integrin, this review will provide insights into the thiol switch-based redox regulation of integrins and the known effects of their allosteric disulfide bridges on conformational changes and cell functions, as well as on the machinery of redox-modifying enzymes that contribute to the redox regulation of cell contacts with the ECM. Full article

Protein Disulfide Isomerases (PDIs) are emerging targets in anticancer therapy, with several PDI inhibitors demonstrating anticancer efficacy in preclinical models. Research has largely focused on “canonical” PDIs, such as PDIA1, which contain CXXC active site motifs where C represents Cysteine. Canonical PDIs have well-studied, critical roles in forming, breaking, and exchanging/scrambling disulfide bonds during protein folding. In contrast, non-canonical PDIs, which harbor CXXS active site motifs, remain less well-studied despite their role as sensors or effectors of protein folding quality control during protein trafficking in the secretory pathway. Here, we provide a review of the literature relating to the non-canonical PDIs ERp44, AGR2, and AGR3, which have been identified as strong dependencies in specific cancer subtypes according to the DepMap database. The biological and biochemical functions of ERp44, AGR2, and AGR3 are discussed, highlighting the role of ERp44 in two mechanisms of protein folding quality control, AGR2 as a selective sensor of mucin protein misfolding, and a unique role for AGR3 in cilia. Finally, we discuss recent efforts to develop small molecule inhibitors of ERp44, AGR2, and AGR3 as tool compounds and experimental therapeutics. Full article

The expression of recombinant proteins in heterologous hosts is a common strategy to obtain larger quantities of the “protein of interest” (POI) for scientific, therapeutic or commercial purposes. However, the experimental success of such an approach critically depends on the choice of an appropriate host system to obtain biologically active forms of the POI. The correct folding of the molecule, mediated by disulfide bond formation, is one of the most critical steps in that process. Here we describe the recombinant expression of hirudin, a leech-derived anticoagulant and thrombin inhibitor, in the yeast Komagataella phaffii (formerly known and mentioned throughout this publication as Pichia pastoris) and in two different strains of Escherichia coli, one of them being especially designed for improved disulfide bond formation through expression of a protein disulfide isomerase. Cultivation of the heterologous hosts and expression of hirudin were performed at different temperatures, ranging from 22 to 42 °C for the bacterial strains and from 20 to 30 °C for the yeast strain, respectively. The thrombin-inhibitory potencies of all hirudin preparations were determined using the thrombin time coagulation assay. To our surprise, the hirudin preparations of P. pastoris were considerably less potent as thrombin inhibitors than the respective preparations of both E. coli strains, indicating that a eukaryotic background is not per se a better choice for the expression of a biologically active eukaryotic protein. The hirudin preparations of both E. coli strains exhibited comparable high thrombin-inhibitory potencies when the strains were cultivated at their respective optimal temperatures, whereas lower or higher cultivation temperatures reduced the inhibitory potencies. Full article

Silkworms synthesize and secrete silk to produce cocoons, which are excellent materials for textile and biomaterial manufacturing applications. However, the gene regulation associated with the post-translational modification of silk proteins remains unknown. In this study, we analyzed the physicochemical properties, evolutionary relationships, and expression patterns of BmPDI in silkworms. Subsequently, we knocked out BmPDI (BmPDI-KO), resulting in significant phenotypes of BmPDI-KO silkworms with smaller silk glands and cocoons, weaker silk mechanical properties, and reduced disulfide bonds in silk-associated proteins. Transcription levels of silk protein-related genes and unfolded protein response signal pathway-related genes were significantly downregulated. In contrast, genes involved in the apoptosis pathway were significantly upregulated in BmPDI-KO silkworms. Knocking out BmPDI in silkworms affected the post-translational modifications of the silk proteins, thereby accumulating misfolded silk proteins and hindering their secretion into the extracellular cells. This further increased endoplasmic reticulum stress, activated the apoptotic pathway, accelerated silk gland cell apoptosis, and significantly reduced the silk yields and mechanical properties of BmPDI-KO silkworm. This study provides a potential exploration of BmPDI in the modification of silk yields and mechanical properties of Bombyx mori. Full article

Marine heatwaves (MHWs) have recently become more frequent, intense, and prolonged, posing significant threats to marine life and fisheries. In this study, transcriptomic analysis was employed to investigate the genes and pathways in Seriola dumerili that respond to MHW-induced stress at 28 °C (T28) and 32 °C (T32), using 24 °C (T24) as the control. Transcriptome sequencing revealed that 17 differentially expressed genes (DEGs) belonging to the heat shock protein (HSP) families—HSP30, HSP40, HSP70, and HSP90—were significantly upregulated under short-lasting MHW stress in the T24-4d vs. T32-4d comparison. Additionally, genes related to oxidative stress (e.g., protein disulfide isomerase family A member 6 [pdia6]), immune responses (e.g., interferon regulatory factor 5 [irf5]), and energy metabolism (e.g., hexokinase-1 [hk1]) were also identified. Enrichment analysis of DEGs in the T24-4d vs. T32-4d group revealed that S. dumerili exhibited adaptive responses to MHWs through the upregulation of HSPs and the activation of antioxidant, energy metabolism, and immune response pathways. However, in the T24-13d vs. T32-13d group, DEGs associated with these pathways were either not significantly expressed or were downregulated. These findings indicate that S. dumerili is unable to sustain its adaptive responses under repeated, intense MHW exposure, resulting in the disorder of its antioxidant defense system, immune suppression, and metabolic dysfunction. This study provides valuable insights into the molecular responses of S. dumerili to MHWs and supports the selection for thermal resistance in this species. Full article

Background/Objectives: Desiccation profoundly influences the distribution and abundance of intertidal seaweeds, necessitating robust molecular adaptations. Sargassum muticum is a brown seaweed inhabiting intertidal rocky substrates. During low tides, this species undergoes periodic aerial exposure. Such environmental conditions necessitate robust physiological mechanisms to mitigate desiccation stress. Yet, the molecular basis of this adaptation remains poorly understood. Methods: To investigate desiccation-responsive genes and elucidate the underlying mechanisms of adaptation, we exposed S. muticum to 6 h of controlled desiccation stress in sterilized ceramic trays, simulating natural tidal conditions, and performed comparative transcriptome analysis using RNA-seq on the Illumina NovaSeq 6000 platform. Results: High-quality sequencing identified 66,192 unigenes, with 1990 differentially expressed genes (1399 upregulated and 591 downregulated). These differentially expressed genes (DEGs) were categorized into regulatory genes—including mitogen-activated protein kinase (MAPK), calmodulin, elongation factor, and serine/threonine-protein kinase—and functional genes, such as heat shock protein family members (HSP20, HSP40, and HSP70), tubulin (TUBA and TUBB), and endoplasmic reticulum homeostasis-related genes (protein disulfide-isomerase A6, calreticulin, and calnexin). Gene Ontology (GO) enrichment highlighted upregulated DEGs in metabolic processes like glutathione metabolism, critical for oxidative stress mitigation, while downregulated genes were linked to transport functions, such as ammonium transport, suggesting reduced nutrient uptake during dehydration. KEGG pathway analysis revealed significant enrichment in “protein processing in endoplasmic reticulum” and “MAPK signaling pathway-plant”, implicating endoplasmic reticulum stress response and conserved signaling cascades in desiccation adaptation. Validation via qRT-PCR confirmed consistent expression trends for key genes, reinforcing the reliability of transcriptomic data. Conclusions: These findings suggest that S. muticum undergoes extensive biological adjustments to mitigate desiccation stress, highlighting candidate pathways for future investigations into recovery and tolerance mechanisms. Full article

Pequi oil (PO) is a natural feed additive rich in bioactive compounds, which can modulate antioxidant and immunological systems. Thus, the aim of this study was to evaluate the proteomic profile of laying hens supplemented with PO under heat stress conditions. Ninety-six 26-week-old Lohmann White hens were housed in a completely randomized design with a 2 × 2 factorial arrangement, with two climate chambers (cyclic heat stress and thermoneutral) and two diets (control and 0.6% PO). At 38 weeks old, liver samples were collected for protein extraction and digestion, and were submitted to liquid chromatography–tandem mass spectrometry (LC-MS/MS). A total of 996 differentially expressed proteins were identified in the liver proteome of laying hens fed with 0.6% PO under heat stress. These upregulated proteins (0.95 ≤ p ≤ 1.00) are associated with lipid metabolism (apolipoprotein B; vitellogenin-1; ovotransferrin), the antioxidant system (protein disulfide-isomerase A4; superoxide dismutase 1_ soluble; catalase), the immune system (Ig-like domain-containing protein) and chaperones (HSP 90; HSP 70). PO positively modulates a network of heat shock proteins and antioxidant enzymes, and the unique proteins identified can contribute to the discovery of new biomarkers related to heat stress reduction by phytogenic additives. Full article

Background/Objectives: Anterior Gradient-2 (AGR2/PDIA17) is a member of the protein disulfide isomerase (PDI) family of oxidoreductases. AGR2 is up-regulated in several solid tumors, including pancreatic ductal adenocarcinoma (PDAC). Given the dire need for new therapeutic options for PDAC patients, we investigated the expression and function of AGR2 in PDAC and developed a novel series of affinity-matured AGR2-specific single-chain variable fragments (scFvs) and monoclonal antibodies. Results: We found that AGR2 was expressed in approximately 90% of PDAC but not normal pancreas biopsies, and the level of AGR2 expression correlated with increasing disease stage. AGR2 expression was inversely related to SMAD4 status in PDAC and colorectal cancer cell models and was secreted from cells into their media. In normal tissues, a high density of AGR2 was detected in the epithelium of cells in the digestive tract but was lacking in most other normal tissue systems. The addition of recombinant AGR2 to cell culture and genetic overexpression of AGR2 increased the adhesion, motility, and invasiveness of both human and mouse PDAC cells. Human phage display library screening led to the discovery of multiple AGR2-specific scFv clones that were affinity-matured to produce monoclonal antibody (MAb) clones with low picomolar binding affinity (S31R/A53F/Y). These high-affinity MAbs inhibited AGR2-mediated cell adhesion, migration, and binding to LYPD3, which is a putative cell surface binding partner of AGR2. Conclusions: Our study provides novel, high-affinity, fully human, anti-AGR2 MAbs that neutralize the pro-tumor effects of extracellular AGR2 in PDAC. Full article

In this study, physicochemical and proteomic analyses were performed to investigate the effect of modified atmosphere packaging (MAP) on the quality of postharvest loose-leaf lettuce. The results showed that MAP enhanced the sensory characteristics of loose-leaf lettuce and delayed the incidence of postharvest deterioration by suppressing weight loss, electrolyte leakage, and reactive oxygen species levels. MAP-inhibited storage-induced programmed cell death may be attributed to a lower expression of protein disulfide isomerase and a higher expression of oligonucleotide/oligosaccharide binding fold nucleic acid binding site protein and reducing glutamine synthase levels. Also, we explore the potential of MAP to protect against oxidative damage in loose-leaf lettuce by potentially modulating the expression levels of NAC family proteins, which may enhance signaling and the expression of cytochrome c oxidase and membrane-bound pyrophosphate in the oxidative phosphorylation pathway. In addition, MAP potentially delayed postharvest senescence and extended the shelf life of lettuce by regulating key protein metabolic pathways that may reduce respiration rates. These include the NAC family of proteins, enzymes in the oxidative phosphorylation pathway, glutamine synthetize, and other crucial metabolic routes. These findings provide a scientific basis for enhancing the postharvest preservation of leafy vegetables, such as loose-leaf lettuce, through MAP technology. Full article

Klotho is an anti-aging protein whose deletion significantly reduces lifespan in mice, while its over-expression increases lifespan. Klotho is a type-I transmembrane protein that is N-glycosylated at eight positions within its ectodomain. Our study demonstrates that N-glycosylation or mutation at position N614, but not at N161, N285, or N346 in mouse Klotho, is critically involved in the transport of Klotho out of the endoplasmic reticulum (ER). Consequently, while wild-type Klotho-EGFP as well as the N-glycosylation mutants N161Q, N285Q, and N346Q were present at the plasma membrane (PM), only small amounts of the N614Q Klotho-EGFP were present at the PM, with most of the protein accumulating in the ER. Protein interactome analysis of Klotho-EGFP N614Q revealed increased interactions with proteasome-related proteins and proteins involved in ER protein processing, like heat shock proteins and protein disulfide isomerases, indicative of impaired protein folding. Co-immunoprecipitation experiments confirmed the interaction of Klotho-EGFP N614Q with ER chaperons. Interestingly, despite the low amounts of Klotho-EGFP N614Q at the PM, it efficiently induced FGF receptor-mediated ERK activation in the presence of FGF23, highlighting its efficacy in triggering downstream signaling, even in limited quantities at the PM. Full article

Difenoconazole (DIF) is a fungicide used to control various fungi. It is absorbed on the surface of different plants and contributes significantly to increased crop production. However, DIF is reported to exhibit toxicity to fungi and to aquatic plants, fish, and mammals, including humans, causing adverse effects. However, research on the impact of DIF on the mammary epithelial cells of herbivorous bovines is limited. DIF-induced damage and accumulation in the mammary glands can have direct and indirect effects on humans. Therefore, we investigated the effects and mechanisms of DIF toxicity in MAC-T cells. The current study revealed that DIF reduces cell viability and proliferation while triggering apoptotic cell death through the upregulation of pro-apoptotic proteins, including cleaved caspase 3 and Bcl-2-associated X protein (BAX), and the downregulation of leukemia type 2 (BCL-2). DIF also induced endoplasmic reticulum (ER) stress by increasing the expression of genes or proteins of Bip/GRP78, protein disulfide isomerase (PDI), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and endoplasmic reticulum oxidoreductase 1 Alpha (ERO1-Lα). We demonstrated that DIF induces mitochondria-mediated apoptosis in MAC-T cells by activating ER stress pathways. This cellular damage resulted in a significant increase in the expression of inflammatory response genes and proteins, including cyclooxygenase 2 (COX2), transforming growth factor beta 3 (TGFB3), CCAAT enhancer binding protein delta (CEBPD), and iNOS, in DIF-treated groups. In addition, spheroid formation by MAC-T cells was suppressed by DIF treatment. Our findings suggest that DIF exposure in dairy cows may harm mammary gland function and health and may indirectly affect human consumption of milk. Full article

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3. Full article

Plant extracellular vesicles are non-self-replicating particles released by living plant cells and delimited by a lipid bilayer. They contain a large amount of lipids, RNA, and proteins. Seed vigor plays an important role in agricultural production and preservation of germplasm resources. Extracellular vesicles with cross-species communication with bioactive molecules can resist pathogens, exhibit anti-aging properties, and perform other functions; however, its potential influence on seed vigor has not been reported. In this study, rice seeds with different germination percentages were used to extract extracellular vesicles, endogenous proteins, and RNA. Protein qualitative identification and miRNA differential analysis were performed to analyze the regulatory mechanism of extracellular vesicles on seed vigor. Results: The profiles of four miRNA families were found to be significantly different: osa-miR164, osa-miR168, osa-miR166, and osa-miR159. Protein correlation analysis predicted that extracellular vesicles might mediate the synthesis of the seed cell wall; glyoxic acid cycle and tricarboxylic acid cycle; non-specific lipid transfer; mitochondrial quality control; and other biological processes to regulate rice seed viability. In addition, cupin protein, phospholipase D, aldehyde dehydrogenase, seven heat shock proteins (especially BiP1 and BiP2), protein disulfide isomerase-like (PDI), thioredoxin, calnexin and calreticulin, glutathione transferase, and other proteins found in extracellular vesicles were closely related to seed vigor. This provides a novel direction for the study of the regulation mechanism of seed vigor. Full article