-
Food Chemistry Jun 2024To assess the effectiveness of carrageenan oligosaccharides (COs) in enhancing superchilling storage of crayfish, the physicochemical features of muscle and protein...
To assess the effectiveness of carrageenan oligosaccharides (COs) in enhancing superchilling storage of crayfish, the physicochemical features of muscle and protein abundance in the refrigerated sample (RS), superchilled sample (SS) and COs soaked superchilled sample (CS) were evaluated. Microstructural and SDS-PAGE analyses suggested that CS exhibited fewer pores, with a microstructure and protein subunits distribution more similar to RS. Tandem Mass Tags quantitative proteomic analysis revealed 66 up-regulated differentially abundant proteins (DAPs) in the CS vs. SS batch, including myosin light chain 2, neural cadherin, integrin beta, lectin-like protein, toll-1, reticulon-1, and moesin/ezrin/radixin homolog 1, which facilitate cells adhesion and maintain membrane/cytoskeleton integrity. Eukaryotic Clusters of Orthologous Groups results confirmed that COs treatment increased the stability of crayfish myofibrillar proteins by up-regulating DAPs, which were concentrated in functional categories such as "posttranslation modification, protein turnover, chaperones", "signal transduction mechanisms", "energy production and conversion", and "cytoskeleton".
PubMed: 38936119
DOI: 10.1016/j.foodchem.2024.140126 -
PLoS Pathogens Jun 2024The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been...
The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.
PubMed: 38935804
DOI: 10.1371/journal.ppat.1012333 -
International Journal of Cancer Jun 2024Protein function alteration and protein mislocalization are cancer hallmarks that drive oncogenesis. N-methyladenosine (mA) deposition mediated by METTL3, METTL16, and... (Review)
Review
Protein function alteration and protein mislocalization are cancer hallmarks that drive oncogenesis. N-methyladenosine (mA) deposition mediated by METTL3, METTL16, and METTL5 together with the contribution of additional subunits of the mA system, has shown a dramatic impact on cancer development. However, the cellular localization of mA proteins inside tumor cells has been little studied so far. Interestingly, recent evidence indicates that mA methyltransferases are not always confined to the nucleus, suggesting that epitranscriptomic factors may also have multiple oncogenic roles beyond mA that still represent an unexplored field. To date novel epigenetic drugs targeting mA modifiers, such as METTL3 inhibitors, are entering into clinical trials, therefore, the study of the potential onco-properties of mA effectors beyond mA is required. Here we will provide an overview of methylation-independent functions of the mA players in cancer, describing the molecular mechanisms involved and the future implications for therapeutics.
PubMed: 38935523
DOI: 10.1002/ijc.35067 -
Cell Reports Jun 2024With exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23),...
With exercise, muscle and bone produce factors with beneficial effects on brain, fat, and other organs. Exercise in mice increased fibroblast growth factor 23 (FGF23), urine phosphate, and the muscle metabolite L-β-aminoisobutyric acid (L-BAIBA), suggesting that L-BAIBA may play a role in phosphate metabolism. Here, we show that L-BAIBA increases in serum with exercise and elevates Fgf23 in osteocytes. The D enantiomer, described to be elevated with exercise in humans, can also induce Fgf23 but through a delayed, indirect process via sclerostin. The two enantiomers both signal through the same receptor, Mas-related G-protein-coupled receptor type D, but activate distinct signaling pathways; L-BAIBA increases Fgf23 through Gαs/cAMP/PKA/CBP/β-catenin and Gαq/PKC/CREB, whereas D-BAIBA increases Fgf23 indirectly through sclerostin via Gαi/NF-κB. In vivo, both enantiomers increased Fgf23 in bone in parallel with elevated urinary phosphate excretion. Thus, exercise-induced increases in BAIBA and FGF23 work together to maintain phosphate homeostasis.
PubMed: 38935499
DOI: 10.1016/j.celrep.2024.114397 -
Hepatology Communications Jul 2024The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown.
BACKGROUND
The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown.
METHODS
Here, we established an animal model of gallbladder dysfunction and assessed the role of a diseased gallbladder in cholestasis-induced hepatic fibrosis (CIHF).
RESULTS
Mice with smooth muscle-specific deletion of Mypt1, the gene encoding the main regulatory subunit of myosin light chain phosphatase (myosin phosphatase target subunit 1 [MYPT1]), had apparent dysfunction of gallbladder motility. This dysfunction was evidenced by abnormal contractile responses, namely, inhibited cholecystokinin 8-mediated contraction and nitric oxide-resistant relaxation. As a consequence, the gallbladder displayed impaired bile filling and biliary tract dilation comparable to the alterations in CIHF. Interestingly, the mutant animals also displayed CIHF features, including necrotic loci by the age of 1 month and subsequently exhibited progressive fibrosis and hyperplastic/dilated bile ducts. This pathological progression was similar to the phenotypes of the animal model with bile duct ligation and patients with CIHF. The characteristic biomarker of CIHF, serum alkaline phosphatase activity, was also elevated in the mice. Moreover, we observed that the myosin phosphatase target subunit 1 protein level was able to be regulated by several reagents, including lipopolysaccharide, exemplifying the risk factors for gallbladder dysfunction and hence CIHF.
CONCLUSIONS
We propose that gallbladder dysfunction caused by myosin phosphatase target subunit 1 ablation is sufficient to induce CIHF in mice, resulting in impairment of the bile transport system.
Topics: Animals; Myosin-Light-Chain Phosphatase; Mice; Disease Models, Animal; Liver Cirrhosis; Cholestasis; Gallbladder Diseases; Gallbladder; Male; Mice, Knockout
PubMed: 38934703
DOI: 10.1097/HC9.0000000000000473 -
MSystems Jun 2024Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our...
Airway microbiota are known to contribute to lung diseases, such as cystic fibrosis (CF), but their contributions to pathogenesis are still unclear. To improve our understanding of host-microbe interactions, we have developed an integrated analytical and bioinformatic mass spectrometry (MS)-based metaproteomics workflow to analyze clinical bronchoalveolar lavage (BAL) samples from people with airway disease. Proteins from BAL cellular pellets were processed and pooled together in groups categorized by disease status (CF vs. non-CF) and bacterial diversity, based on previously performed small subunit rRNA sequencing data. Proteins from each pooled sample group were digested and subjected to liquid chromatography tandem mass spectrometry (MS/MS). MS/MS spectra were matched to human and bacterial peptide sequences leveraging a bioinformatic workflow using a metagenomics-guided protein sequence database and rigorous evaluation. Label-free quantification revealed differentially abundant human peptides from proteins with known roles in CF, like neutrophil elastase and collagenase, and proteins with lesser-known roles in CF, including apolipoproteins. Differentially abundant bacterial peptides were identified from known CF pathogens (e.g., ), as well as other taxa with potentially novel roles in CF. We used this host-microbe peptide panel for targeted parallel-reaction monitoring validation, demonstrating for the first time an MS-based assay effective for quantifying host-microbe protein dynamics within BAL cells from individual CF patients. Our integrated bioinformatic and analytical workflow combining discovery, verification, and validation should prove useful for diverse studies to characterize microbial contributors in airway diseases. Furthermore, we describe a promising preliminary panel of differentially abundant microbe and host peptide sequences for further study as potential markers of host-microbe relationships in CF disease pathogenesis.IMPORTANCEIdentifying microbial pathogenic contributors and dysregulated human responses in airway disease, such as CF, is critical to understanding disease progression and developing more effective treatments. To this end, characterizing the proteins expressed from bacterial microbes and human host cells during disease progression can provide valuable new insights. We describe here a new method to confidently detect and monitor abundance changes of both microbe and host proteins from challenging BAL samples commonly collected from CF patients. Our method uses both state-of-the art mass spectrometry-based instrumentation to detect proteins present in these samples and customized bioinformatic software tools to analyze the data and characterize detected proteins and their association with CF. We demonstrate the use of this method to characterize microbe and host proteins from individual BAL samples, paving the way for a new approach to understand molecular contributors to CF and other diseases of the airway.
PubMed: 38934598
DOI: 10.1128/msystems.00929-23 -
Organic & Biomolecular Chemistry Jun 2024Sialyl Lewis (sLe), also known as cancer antigen 19-9, is a tumor-associated carbohydrate antigen. In this article, chemical and chemoenzymatic syntheses of a...
Sialyl Lewis (sLe), also known as cancer antigen 19-9, is a tumor-associated carbohydrate antigen. In this article, chemical and chemoenzymatic syntheses of a tetrasaccharide glycan 1 structurally derived from sLe are reported. Challenges involved in the chemical synthesis include the highly stereoselective construction of 1,2--α-L-fucoside and α-D-sialoside, as well as the assembly of the 3,4-disubstituted -acetylglucosamine subunit. Perbenzylated thiofucoside and -acetyl-5-,4--oxazolidinone protected sialic acid thioglycoside were employed as glycosyl donors, respectively, for the efficient preparation of the desired α-fucoside and α-sialoside. The 3,4-branched glucosamine backbone was established through a 3- and then 4- glycosylation sequence in which the 3-hydroxyl group of the glucosamine moiety was glycosylated first and then the 4-hydroxyl. A facile chemoenzymatic approach was also exploited to synthesize the target molecule. The chemically obtained free disaccharide 30 was sequentially sialylated and fucosylated in an enzyme-catalyzed regio- and stereospecific manner to form 1 in high yields. The linker appended 1 can be covalently attached to a carrier protein for further immunological studies.
PubMed: 38934561
DOI: 10.1039/d4ob00809j -
FASEB Journal : Official Publication of... Jul 2024N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the...
N-glycosylation is the most common protein modification in the eukaryotic secretory pathway. It involves the attachment a high mannose glycan to Asn residues in the context of Asn-X-Ser/Thr/Cys, a motif known as N-glycosylation sequon. This process is mediated by STT3A and STT3B, the catalytic subunits of the oligosaccharyltransferase complexes. STT3A forms part of complexes associated with the SEC61 translocon and functions co-translationally. Vacant sequons have another opportunity for glycosylation by complexes carrying STT3B. Local sequence information plays an important role in determining N-glycosylation efficiency, but non-local factors can also have a significant impact. For instance, certain proteins associated with human genetic diseases exhibit abnormal N-glycosylation levels despite having wild-type acceptor sites. Here, we investigated the effect of protein stability on this process. To this end, we generated a family of 40 N-glycan acceptors based on superfolder GFP, and we measured their efficiency in HEK293 cells and in two derived cell lines lacking STT3B or STT3A. Sequon occupancy was highly dependent on protein stability, improving as the thermodynamic stability of the acceptor proteins decreases. This effect is mainly due to the activity of the STT3B-based OST complex. These findings can be integrated into a simple kinetic model that distinguishes local information within sequons from global information of the acceptor proteins.
Topics: Humans; Glycosylation; HEK293 Cells; Protein Processing, Post-Translational; Hexosyltransferases; Membrane Proteins; Protein Stability; Polysaccharides
PubMed: 38934375
DOI: 10.1096/fj.202302267R -
Heliyon Jun 2024This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased...
This study sheds light on a ground-breaking biochemical mechanotransduction pathway and reveals how Piezo1 channels orchestrate cell migration. We observed an increased cell migration rate in HEK293T (HEK) cells treated with Yoda1, a Piezo1 agonist, or in HEK cells overexpressing Piezo1 (HEK + P). Conversely, a significant reduction in cell motility was observed in HEK cells treated with GsMTx4 (a channel inhibitor) or upon silencing Piezo1 (HEK-P). Our findings establish a direct correlation between alterations in cell motility, Piezo1 expression, abnormal F-actin microfilament dynamics, and the regulation of Cofilin1, a protein involved in severing F-actin microfilaments. Here, the conversion of inactive pCofilin1 to active Cofilin1, mediated by the serine/threonine-protein phosphatase 2A catalytic subunit C (PP2AC), resulted in increased severing of F-actin microfilaments and enhanced cell migration in HEK + P cells compared to HEK controls. However, this effect was negligible in HEK-P and HEK cells transfected with hsa-miR-133b, which post-transcriptionally inhibited PP2AC mRNA expression. In summary, our study suggests that Piezo1 regulates cell migration through a biochemical mechanotransduction pathway involving PP2AC-mediated Cofilin1 dephosphorylation, leading to changes in F-actin microfilament dynamics.
PubMed: 38933959
DOI: 10.1016/j.heliyon.2024.e32458 -
Frontiers in Veterinary Science 2024Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic...
Nucleotide metabolism-related host proteins RNA polymerase II subunit and uridine phosphorylase 1 interacting with porcine epidemic diarrhea virus N proteins affect viral replication.
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic losses to the pig industry. PEDV N protein is the protein that constitutes the core of PEDV virus particles, and most of it is expressed in the cytoplasm, and a small part can also be expressed in the nucleus. However, the role of related proteins in host nucleotide metabolic pathways in regulating PEDV replication have not been fully elucidated. In this study, PEDV-N-labeled antibodies were co-immunoprecipitated and combined with LC-MS to screen for host proteins that interact with N proteins. Bioinformatics analyses showed that the selected host proteins were mainly enriched in metabolic pathways. Moreover, co-immunoprecipitation and confocal microscopy confirmed that the second-largest subunit of RNA polymerase II (RPB2) and uridine phosphorylase 1 (UPP1) interacted with the N protein. RPB2 is the main subunit of RNA polymerase II and plays an important role in eukaryotic transcription. UPP1 is an enzyme that catalyzes reversible phosphorylation of uridine to uracil and ribo-1-phosphate to promote catabolism and bio anabolism. RPB2 overexpression significantly promoted viral replication, whereas UPP1 overexpression significantly inhibited viral replication. Studies on interactions between the PEDV N and host proteins are helpful in elucidating the pathogenesis and immune escape mechanism of PEDV.
PubMed: 38933700
DOI: 10.3389/fvets.2024.1417348