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Cell Communication and Signaling : CCS Apr 2023Due to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of...
BACKGROUND
Due to the unique nature of spermatozoa, which are transcriptionally and translationally silent, the regulation of capacitation is based on the formation of posttranslational modifications of proteins (PTMs). However, the interactions between different types of PTMs during the capacitation remain unclear. Therefore, we aimed to unravel the PTM-based regulation of sperm capacitation by considering the relationship between tyrosine phosphorylation and reversible oxidative PTMs (oxPTMs), i.e., S-nitrosylation and S-glutathionylation. Since reversible oxPTMs may be closely related to peroxyredoxin (PRDX) activity, the second aim was to verify the role of PRDXs in the PTM-based regulation of capacitation.
METHODS
Cryopreserved bull sperm were capacitated in vitro with or without PRDX inhibitor. Qualitative parameters of sperm and symptoms characteristic of capacitation were analyzed. Posttranslational protein modifications (S-nitrosylation, S-glutathionylation, tyrosine phosphorylation) were investigated at the cellular level (flow cytometry, fluorescence microscopy) and at the proteomic level (fluorescent gel-based proteomic approach).
RESULTS
Zona-pellucida binding proteins (ACRBP, SPAM1, ZAN, ZPBP1 and IZUMO4) were particularly rich in reversible oxPTMs. Moreover, numerous flagellar proteins were associated with all analyzed types of PTMs, which indicates that the direction of posttranslational modifications was integrated. Inhibition of PRDX activity during capacitation caused an increase in S-nitrosylation and S-glutathionylation and a decrease in tyrosine phosphorylation. Inhibition of PRDXs caused GAPDHS to undergo S-glutathionylation and the GSTO2 and SOD2 enzymes to undergo denitrosylation. Moreover, PRDX inhibition caused the AKAP proteins to be dephosphorylated.
CONCLUSIONS
Our research provides evidence that crosstalk occurs between tyrosine phosphorylation and reversible oxPTMs during bull sperm capacitation. This study demonstrates that capacitation triggers S-nitrosylation and S-glutathionylation (and reverse reactions) of zona-pellucida binding proteins, which may be a new important mechanism that determines the interaction between sperms and oocytes. Moreover, TCA-related and flagellar proteins, which are particularly rich in PTMs, may play a key role in sperm capacitation. We propose that the deglutathionylation of ODFs and IZUMO4 proteins is a new hallmark of bull sperm capacitation. The obtained results indicate a relationship between PRDX activity and protein phosphorylation, S-glutathionylation and S-nitrosylation. The activity of PRDXs may be crucial for maintaining redox balance and for providing proper PKA-mediated protein phosphorylation during capacitation. Video Abstract.
Topics: Male; Animals; Cattle; Sperm Capacitation; Proteomics; Semen; Proteins; Protein Processing, Post-Translational; Phosphorylation; Tyrosine
PubMed: 37046330
DOI: 10.1186/s12964-023-01080-w -
Journal of Dairy Science Aug 2023Gastric digestion of 2 commercial ultrafiltered milks and milk enriched with skim milk powder (to simulate concentration by reverse osmosis) was investigated and...
Gastric digestion of 2 commercial ultrafiltered milks and milk enriched with skim milk powder (to simulate concentration by reverse osmosis) was investigated and compared with the digestion of nonconcentrated milk. Curd formation and proteolysis of high-protein milks in simulated gastric conditions were studied using oscillatory rheology, extrusion testing, and gel electrophoresis. The presence of pepsin in the gastric fluid triggered coagulation at pH >6 and the elastic modulus of gels from high-protein milks was ~5 times larger than the gel from reference milk. Despite similar protein concentrations, the coagulum from milk enriched with skim milk powder showed higher resistance to shear deformation than the coagula from ultrafiltered milks. The gel structure was also more heterogeneous. During digestion, the degradation of coagula from high-protein milks was slowed down compared with the coagulum from reference milk, and intact milk proteins were still detected after 120 min. Differences in the digestion patterns of coagula from high-protein milks were observed and were associated with the proportion of minerals bound to caseins and the denaturation rate of whey proteins.
Topics: Animals; Proteolysis; Powders; Milk; Milk Proteins; Whey Proteins; Caseins; Rheology; Hydrogen-Ion Concentration
PubMed: 37291035
DOI: 10.3168/jds.2022-22714 -
Nutrients Feb 2021The dietary isothiocyanate L-sulforaphane (LSF), derived from cruciferous vegetables, is reported to have several beneficial biological properties, including...
The dietary isothiocyanate L-sulforaphane (LSF), derived from cruciferous vegetables, is reported to have several beneficial biological properties, including anti-inflammatory and immunomodulatory effects. However, there is limited data on how LSF modulates these effects in human immune cells. The present study was designed to investigate the immunomodulatory effects of LSF (10 µM and 50 µM) on peripheral blood mononuclear cell (PBMC) populations and cytokine secretion in healthy adult volunteers ( = 14), in the presence or absence of bacterial (lipopolysaccharide) and viral (imiquimod) toll-like receptor (TLRs) stimulations. Here, we found that LSF reduced pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and chemokines monocyte chemoattractant protein (MCP)-1 irrespective of TLR stimulations. This result was associated with LSF significantly reducing the proportion of natural killer (NK) cells and monocytes while increasing the proportions of dendritic cells (DCs), T cells and B cells. We found a novel effect of LSF in relation to reducing cluster of differentiation (CD) 14 monocytes while simultaneously increasing monocyte-derived DCs (moDCs: lineage-Human Leukocyte Antigen-DR isotype (HLA-DR)CD11b CD11c). LSF was also shown to induce a 3.9-fold increase in the antioxidant response element (ARE) activity in a human monocyte cell line (THP-1). Our results provide important insights into the immunomodulatory effects of LSF, showing in human PBMCs an ability to drive differentiation of monocytes towards an immature monocyte-derived dendritic cell phenotype with potentially important biological functions. These findings provide insights into the potential role of LSF as a novel immunomodulatory drug candidate and supports the need for further preclinical and phase I clinical studies.
Topics: Adult; Bodily Secretions; Cell Differentiation; Cell Line; Cytokines; Dendritic Cells; Female; Healthy Volunteers; Humans; Immunologic Factors; Immunomodulation; Isothiocyanates; Killer Cells, Natural; Leukocytes, Mononuclear; Male; Sulfoxides
PubMed: 33673203
DOI: 10.3390/nu13020602 -
Proceedings of the National Academy of... Aug 2023Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting -secreted protein functions are limited....
Malaria parasites uniquely depend on protein secretion for their obligate intracellular lifestyle but approaches for dissecting -secreted protein functions are limited. We report knockER, a unique DiCre-mediated knock-sideways approach to sequester secreted proteins in the ER by inducible fusion with a KDEL ER-retrieval sequence. We show conditional ER sequestration of diverse proteins is not generally toxic, enabling loss-of-function studies. We employed knockER in multiple species to interrogate the trafficking, topology, and function of an assortment of proteins that traverse the secretory pathway to diverse compartments including the apicoplast (ClpB1), rhoptries (RON6), dense granules, and parasitophorous vacuole (EXP2, PTEX150, HSP101). Taking advantage of the unique ability to redistribute secreted proteins from their terminal destination to the ER, we reveal that vacuolar levels of the PTEX translocon component HSP101 but not PTEX150 are maintained in excess of what is required to sustain effector protein export into the erythrocyte. Intriguingly, vacuole depletion of HSP101 hypersensitized parasites to a destabilization tag that inhibits HSP101-PTEX complex formation but not to translational knockdown of the entire HSP101 pool, illustrating how redistribution of a target protein by knockER can be used to query function in a compartment-specific manner. Collectively, our results establish knockER as a unique tool for dissecting secreted protein function with subcompartmental resolution that should be widely amenable to genetically tractable eukaryotes.
Topics: Plasmodium falciparum; Protozoan Proteins; Plasmodium; Protein Transport; Biological Transport; Erythrocytes
PubMed: 37552754
DOI: 10.1073/pnas.2308676120 -
Microbiological Research Sep 2021Plant pathogenic Gram-negative bacteria evade the host plant immune system by secreting Type III (T3E) and Type IV effector (T4E) proteins into the plant cytoplasm.... (Review)
Review
Plant pathogenic Gram-negative bacteria evade the host plant immune system by secreting Type III (T3E) and Type IV effector (T4E) proteins into the plant cytoplasm. Mostly T3Es are secreted into the plant cells to establish pathogenicity by affecting the vital plant process viz. metabolic pathways, signal transduction and hormonal regulation. Ubiquitin-26S proteasome system (UPS) exists as one of the important pathways in plants to control plant immunity and various cellular processes by employing several enzymes and enzyme components. Pathogenic and non-pathogenic bacteria are found to secrete effectors into plants with structural and/or functional similarity to UPS pathway components like ubiquitin E3 ligases, F-box domains, cysteine proteases, inhibitor of host UPS or its components, etc. The bacterial effectors mimic UPS components and target plant resistance proteins for degradation by proteasomes, thereby taking control over the host cellular activities as a strategy to exert virulence. Thus, the bacterial effectors circumvent plant cellular pathways leading to infection and disease development. This review highlights known bacterial T3E and T4E proteins that function and interfere with the ubiquitination pathway to regulate the immune system of plants.
Topics: Bacteria; Bacterial Proteins; Host-Pathogen Interactions; Plant Immunity; Plant Proteins; Plants; Proteasome Endopeptidase Complex; Ubiquitination
PubMed: 34246833
DOI: 10.1016/j.micres.2021.126810 -
Mucosal Immunology Nov 2021Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are... (Review)
Review
Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1β (IRE1β), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1β arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues.
Topics: Animals; Biological Evolution; Biomarkers; Endoplasmic Reticulum Stress; Endoribonucleases; Enzyme Activation; Epithelial Cells; Epithelium; Gene Expression Regulation; Homeostasis; Humans; Membrane Proteins; Mucous Membrane; Mucus; Phylogeny; Protein Serine-Threonine Kinases; Signal Transduction; Unfolded Protein Response
PubMed: 34075183
DOI: 10.1038/s41385-021-00412-8 -
Animal : An International Journal of... Jul 2023How the efficiency of utilization of essential amino acids (EffU) can be applied in dairy cow nutrition is presented in this review. The concept of EffU proposed by the... (Review)
Review
How the efficiency of utilization of essential amino acids (EffU) can be applied in dairy cow nutrition is presented in this review. The concept of EffU proposed by the National Academies of Sciences, Engineering and Medicine (NASEM, 2021) is first detailed. It represents the proportion of the metabolisable essential amino acids (mEAA) supply used to support protein secretions and accretions (scurf, metabolic fecal, milk and growth). For these processes, the efficiency of each individual EAA is variable, and considered to vary similarly for all the protein secretions and accretions. The anabolic process of gestation is ascribed to a constant efficiency (33%), whereas the efficiency of endogenous urinary loss (EndoUri) is set at 100%. Therefore, the NASEM model EffU was calculated as the sum of EAA in the true protein of secretions and accretions divided by the available EAA (mEAA - EndoUri - gestation net true protein/0.33). In this paper, the reliability of this mathematical calculation was tested through an example where the experimental efficiency of His was calculated assuming that liver removal represents catabolism. The NASEM model and experimental efficiencies were in the same range and varied in similar manner. Assuming that the NASEM model EffU reflects EAA metabolism in the dairy cow, its different applications were examined. In NASEM, target efficiencies were determined for each EAA: 75, 71, 73, 72, 73, 60, 64, 86 and 74% for His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val, respectively. From these, recommendations for mEAA supply can be calculated as: [(secretions + accretions)/(target EffU × 0.01) + EndoUri + gestation/0.33], assuming energy supply is adequate. In addition to NASEM propositions, equations to predict EffU with precision and accuracy are detailed, using the ratio of (mEAA-EndoUri) to digestible energy intake, in a quadratic model that includes days in milk. Moreover, milk true protein yield predictions from predicted EffU or efficiency of utilization of metabolisable protein are better than those from the multivariate equation of NASEM (2021) and superior to those predicted with a fixed efficiency. Finally, either the NASEM model or the predicted EffU can be used to assess the responsiveness of a ration to supplementation with a single EAA. If the EffU of the EAA to supplement is higher than the target EffU, while the EffU of the other EAA are lower than the target value, this suggests a potential improvement in milk true protein yield to supplementation with this EAA.
Topics: Female; Cattle; Animals; Amino Acids, Essential; Lactation; Reproducibility of Results; Diet; Milk; Milk Proteins
PubMed: 37268529
DOI: 10.1016/j.animal.2023.100833 -
Diabetes Jun 2022Double C2 domain Β (DOC2b) protein is required for glucose-stimulated insulin secretion (GSIS) in β-cells, the underlying mechanism of which remains unresolved. Our...
Double C2 domain Β (DOC2b) protein is required for glucose-stimulated insulin secretion (GSIS) in β-cells, the underlying mechanism of which remains unresolved. Our biochemical analysis using primary human islets and human and rodent clonal β-cells revealed that DOC2b is tyrosine phosphorylated within 2 min of glucose stimulation, and Src family kinase member YES is required for this process. Biochemical and functional analysis using DOC2bY301 mutants revealed the requirement of Y301 phosphorylation for the interaction of DOC2b with YES kinase and increased content of VAMP2, a protein on insulin secretory granules, at the plasma membrane (PM), concomitant with DOC2b-mediated enhancement of GSIS in β-cells. Coimmunoprecipitation studies demonstrated an increased association of DOC2b with ERM family proteins in β-cells following glucose stimulation or pervanadate treatment. Y301 phosphorylation-competent DOC2b was required to increase ERM protein activation, and ERM protein knockdown impaired DOC2b-mediated boosting of GSIS, suggesting that tyrosine-phosphorylated DOC2b regulates GSIS via ERM-mediated granule localization to the PM. Taken together, these results demonstrate the glucose-induced posttranslational modification of DOC2b in β-cells, pinpointing the kinase, site of action, and downstream signaling events and revealing a regulatory role of YES kinase at various steps in GSIS. This work will enhance the development of novel therapeutic strategies to restore glucose homeostasis in diabetes.
Topics: Calcium-Binding Proteins; Glucose; Insulin; Insulin-Secreting Cells; Nerve Tissue Proteins; Phosphorylation; Protein Processing, Post-Translational; Tyrosine
PubMed: 35377441
DOI: 10.2337/db21-0681 -
The Journal of Biological Chemistry Dec 2022Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in...
Most eukaryotic secretory proteins are cotranslationally translocated through Sec61 into the endoplasmic reticulum (ER). Because these proteins have evolved to fold in the ER, their mistargeting is associated with toxicity. Genetic experiments have implicated the ER heat shock protein 70 (Hsp70) Hspa13/STCH as involved in processing of nascent secretory proteins. Herein, we evaluate the role of Hspa13 in protein import and the maintenance of cellular proteostasis in human cells, primarily using the human embryonic kidney 293T cell line. We find that Hspa13 interacts primarily with the Sec61 translocon and its associated factors. Hspa13 overexpression inhibits translocation of the secreted protein transthyretin, leading to accumulation and aggregation of immature transthyretin in the cytosol. ATPase-inactive mutants of Hspa13 further inhibit translocation and maturation of secretory proteins. While Hspa13 overexpression inhibits cell growth and ER quality control, we demonstrate that HSPA13 knockout destabilizes proteostasis and increases sensitivity to ER disruption. Thus, we propose that Hspa13 regulates import through the translocon to maintain both ER and cytosolic protein homeostasis. The raw mass spectrometry data associated with this article have been deposited in the PRIDE archive and can be accessed at PXD033498.
Topics: Humans; Heat-Shock Proteins; Proteostasis; Prealbumin; Cytosol; Endoplasmic Reticulum; Protein Transport; SEC Translocation Channels
PubMed: 36244454
DOI: 10.1016/j.jbc.2022.102597 -
Biochemical Pharmacology Mar 2022Rho subfamily of G proteins (e.g., Rac1) have been implicated in glucose-stimulated insulin secretion from the pancreatic β-cell. Interestingly, metabolic stress (e.g.,... (Review)
Review
Rho subfamily of G proteins (e.g., Rac1) have been implicated in glucose-stimulated insulin secretion from the pancreatic β-cell. Interestingly, metabolic stress (e.g., chronic exposure to high glucose) results in sustained activation of Rac1 leading to increased oxidative stress, impaired insulin secretion and β-cell dysfunction. Activation-deactivation of Rho G proteins is mediated by three classes of regulatory proteins, namely the guanine nucleotide exchange factors (GEFs), which facilitate the conversion of inactive G proteins to their active conformations; the GTPase-activating proteins (GAPs), which convert the active G proteins to their inactive forms); and the GDP-dissociation inhibitors (GDIs), which prevent the dissociation of GDP from G proteins. Contrary to a large number of GEFs (82 members) and GAPs (69 members), only three members of RhoGDIs (RhoGDIα, RhoGDIβ and RhoGDIγ) are expressed in mammalian cells.Even though relatively smaller in number, the GDIs appear to play essential roles in G protein function (e.g., subcellular targeting) for effector activation and cell regulation. Emerging evidence also suggests that the GDIs are functionally regulated via post-translational modification (e.g., phosphorylation) and by lipid second messengers, lipid kinases and lipid phosphatases. We highlight the underappreciated regulatory roles of RhoGDI-Rho G protein signalome in islet β-cell function in health and metabolic stress. Potential knowledge gaps in the field, and directions for future research for the identification of novel therapeutic targets to loss of functional β-cell mass under the duress of metabolic stress are highlighted.
Topics: Animals; Humans; Insulin-Secreting Cells; Islets of Langerhans; Lipid Metabolism; rho GTP-Binding Proteins; rho-Specific Guanine Nucleotide Dissociation Inhibitors
PubMed: 34968495
DOI: 10.1016/j.bcp.2021.114886