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International Journal of Molecular... Jan 2024and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily...
Glyceraldehyde 3-Phosphate Dehydrogenase on the Surface of and Cells-A Moonlighting Protein That Binds Human Vitronectin and Plasminogen and Can Adsorb to Pathogenic Fungal Cells via Major Adhesins Als3 and Epa6.
and other closely related pathogenic yeast-like fungi carry on their surface numerous loosely adsorbed "moonlighting proteins"-proteins that play evolutionarily conserved intracellular functions but also appear on the cell surface and exhibit additional functions, e.g., contributing to attachment to host tissues. In the current work, we characterized this "moonlighting" role for glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) of and . GAPDH was directly visualized on the cell surface of both species and shown to play a significant part in the total capacity of fungal cells to bind two selected human host proteins-vitronectin and plasminogen. Using purified proteins, both host proteins were found to tightly interact with GAPDH, with dissociation constants in an order of 10 M, as determined by bio-layer interferometry and surface plasmon resonance measurements. It was also shown that exogenous GAPDH tightly adheres to the surface of candidal cells, suggesting that the cell surface location of this moonlighting protein may partly result from the readsorption of its soluble form, which may be present at an infection site (e.g., due to release from dying fungal cells). The major dedicated adhesins, covalently bound to the cell wall-agglutinin-like sequence protein 3 (Als3) and epithelial adhesin 6 (Epa6)-were suggested to serve as the docking platforms for GAPDH in and , respectively.
Topics: Humans; Candida albicans; Glyceraldehyde-3-Phosphate Dehydrogenases; Plasminogen; Vitronectin; Fungal Proteins
PubMed: 38256088
DOI: 10.3390/ijms25021013 -
Cardiovascular Diabetology Jan 2024Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2-4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin...
BACKGROUND
Type 2 diabetes mellitus (T2DM) increases the risk of coronary heart disease (CHD) by 2-4 fold, and is associated with endothelial dysfunction, dyslipidaemia, insulin resistance, and chronic hyperglycaemia. The aim of this investigation was to assess, by a multimarker mass spectrometry approach, the predictive role of circulating proteins as biomarkers of cardiovascular damage progression associated with diabetes mellitus.
METHODS
The study considered 34 patients with both T2DM and CHD, 31 patients with T2DM and without CHD, and 30 patients without diabetes with a diagnosis of CHD. Plasma samples of subjects were analysed through a multiplexed targeted liquid chromatography mass spectrometry (LC-MS)-based assay, namely Multiple Reaction Monitoring (MRM), allowing the simultaneous detection of peptides derived from a protein of interest. Gene Ontology (GO) Analysis was employed to identify enriched GO terms in the biological process, molecular function, or cellular component categories. Non-parametric multivariate methods were used to classify samples from patients and evaluate the relevance of the analysed proteins' panel.
RESULTS
A total of 81 proteins were successfully quantified in the human plasma samples. Gene Ontology analysis assessed terms related to blood microparticles, extracellular exosomes and collagen-containing extracellular matrix. Preliminary evaluation using analysis of variance (ANOVA) of the differences in the proteomic profile among patient groups identified 13 out of the 81 proteins as significantly different. Multivariate analysis, including cluster analysis and principal component analysis, identified relevant grouping of the 13 proteins. The first main cluster comprises apolipoprotein C-III, apolipoprotein C-II, apolipoprotein A-IV, retinol-binding protein 4, lysozyme C and cystatin-C; the second one includes, albeit with sub-grouping, alpha 2 macroglobulin, afamin, kininogen 1, vitronectin, vitamin K-dependent protein S, complement factor B and mannan-binding lectin serine protease 2. Receiver operating characteristic (ROC) curves obtained with the 13 selected proteins using a nominal logistic regression indicated a significant overall distinction (p < 0.001) among the three groups of subjects, with area under the ROC curve (AUC) ranging 0.91-0.97, and sensitivity and specificity ranging from 85 to 100%.
CONCLUSIONS
Targeted mass spectrometry approach indicated 13 multiple circulating proteins as possible biomarkers of cardiovascular damage progression associated with T2DM, with excellent classification results in terms of sensitivity and specificity.
Topics: Humans; Diabetes Mellitus, Type 2; Proteomics; Biomarkers; Peptides; Blood Proteins
PubMed: 38245742
DOI: 10.1186/s12933-024-02125-1 -
Journal of Structural Biology Mar 2024The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically...
The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.
Topics: Humans; Vitronectin; Durapatite; Magnetic Resonance Spectroscopy; Magnetic Resonance Imaging; Nuclear Magnetic Resonance, Biomolecular
PubMed: 38185342
DOI: 10.1016/j.jsb.2024.108061 -
The Journal of Veterinary Medical... Mar 2024Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to...
Canine induced pluripotent stem cells (ciPSCs) can provide useful insights into novel therapies in both veterinary and medical fields. However, limited accessibility to the present culture medium and requirement of considerable time, effort, and cost for routine ciPSC maintenance restrict advancement in ciPSC research. In addition, it is unknown whether ciPSC culture conditions influence differentiation propensity. We investigated the availability of the common human pluripotent stem cells (hPSCs) culture systems for ciPSC maintenance and the differentiation propensities of the ciPSCs maintained in these culture systems. StemFlex and mTeSR Plus supported PSC-like colony formation and pluripotency markers expression in ciPSCs even after five passages. Additionally, ciPSCs were maintained under weekend-free culture conditions with a stable growth rate, pluripotency marker expression, and differentiation abilities using vitronectin (VTN-N) and Geltrex. Following maintenance of spontaneously differentiated ciPSCs under various conditions by embryoid body formation, there were few differences in the differentiation propensities of ciPSCs among the tested culture conditions. Thus, ciPSCs were successfully cultured under weekend-free conditions for ciPSC maintenance using StemFlex or mTeSR Plus with VTN-N or Geltrex. The present study offers simpler and more effort-, time-, and cost-saving options for ciPSC culture systems, which may lead to further development in research using ciPSCs.
Topics: Animals; Dogs; Humans; Induced Pluripotent Stem Cells; Pluripotent Stem Cells; Cell Differentiation; Embryoid Bodies
PubMed: 38171744
DOI: 10.1292/jvms.23-0422 -
Terapevticheskii Arkhiv Aug 2023Focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy characterized by primary podocyte detection and high proteinuria. The search for biomarkers and...
BACKGROUND
Focal segmental glomerulosclerosis (FSGS) is a primary podocytopathy characterized by primary podocyte detection and high proteinuria. The search for biomarkers and factors associated with the progression of this disease is an important task nowdays.
AIM
To assess the proteomic profile of urine in patients with FSGS and to isolate urinary biomarkers of podocytopathies.
MATERIALS AND METHODS
The study included 41 patients diagnosed with chronic glomerulonephritis, 27 men and 14 women. According to the morphological study, 28 patients were diagnosed with FSGS, 9 with steroid-sensitive nephrotic syndrome and 14 with steroid-resistant nephrotic syndrome. The comparison group included 13 patients with membranous nephropathy. The study of the urinary proteome was carried out by targeted liquid chromatography-mass spectrometry using multiple reaction monitoring with synthetic stable isotope labelled peptide standards.
RESULTS
The main differences in the protein profile of urine were found in the subgroups of steroid-sensitive (SS) and steroid-resistant (SR) FSGS. In the FSGS SR group, at the onset of the disease, there was a high concentration of proteins reflecting damage to the glomerular filter (apo-lipoprotein A-IV, orosomucoid, cadherin, hemopexin, vitronectin), as well as proteins associated with tubulo-interstitial inflammation and accumulation of extracellular matrix (retinol- and vitamin D-binding proteins, kininogen-1, lumican and neurophilin-2). Compared with the membranous nephropathy group, FSGS patients had significantly higher urinary concentrations of carnosinase, orosomucoid, cadherin-13, tenascin X, osteopontin, and zinc-alpha-2-glycoprotein.
CONCLUSION
Thus, in patients with SR FSGS, the proteomic profile of urine includes more proteins at elevated concentrations, which reflects severe damage to various parts of the nephron compared with patients with SS FSGS and membranous nephropathy.
Topics: Male; Humans; Female; Glomerulosclerosis, Focal Segmental; Glomerulonephritis, Membranous; Proteomics; Orosomucoid; Nephrotic Syndrome; Biomarkers; Steroids; Cadherins
PubMed: 38158963
DOI: 10.26442/00403660.2023.06.202266 -
Remodeling of the Cardiac Extracellular Matrix Proteome During Chronological and Pathological Aging.Molecular & Cellular Proteomics : MCP Jan 2024Impaired extracellular matrix (ECM) remodeling is a hallmark of many chronic inflammatory disorders that can lead to cellular dysfunction, aging, and disease...
Impaired extracellular matrix (ECM) remodeling is a hallmark of many chronic inflammatory disorders that can lead to cellular dysfunction, aging, and disease progression. The ECM of the aged heart and its effects on cardiac cells during chronological and pathological aging are poorly understood across species. For this purpose, we first used mass spectrometry-based proteomics to quantitatively characterize age-related remodeling of the left ventricle (LV) of mice and humans during chronological and pathological (Hutchinson-Gilford progeria syndrome (HGPS)) aging. Of the approximately 300 ECM and ECM-associated proteins quantified (named as Matrisome), we identified 13 proteins that were increased during aging, including lactadherin (MFGE8), collagen VI α6 (COL6A6), vitronectin (VTN) and immunoglobulin heavy constant mu (IGHM), whereas fibulin-5 (FBLN5) was decreased in most of the data sets analyzed. We show that lactadherin accumulates with age in large cardiac blood vessels and when immobilized, triggers phosphorylation of several phosphosites of GSK3B, MAPK isoforms 1, 3, and 14, and MTOR kinases in aortic endothelial cells (ECs). In addition, immobilized lactadherin increased the expression of pro-inflammatory markers associated with an aging phenotype. These results extend our knowledge of the LV proteome remodeling induced by chronological and pathological aging in different species (mouse and human). The lactadherin-triggered changes in the proteome and phosphoproteome of ECs suggest a straight link between ECM component remodeling and the aging process of ECs, which may provide an additional layer to prevent cardiac aging.
Topics: Humans; Proteome; Endothelial Cells; Heart; Aging; Extracellular Matrix; Extracellular Matrix Proteins
PubMed: 38141925
DOI: 10.1016/j.mcpro.2023.100706 -
Nanotechnology, Science and Applications 2023Disorganisation of the extracellular matrix (ECM) is strongly connected to tumor progression. Even small-scale changes can significantly influence the adhesion and...
INTRODUCTION
Disorganisation of the extracellular matrix (ECM) is strongly connected to tumor progression. Even small-scale changes can significantly influence the adhesion and proliferation of cancer cells. Therefore, the use of biocompatible nanomaterials capable of supporting and partially replenishing degraded ECM might be essential to recover the niche after tumor resection. The objective of this study was to evaluate the influence of graphene, graphene oxide, fullerene, and diamond nanofilms on breast cancer and glioblastoma grade IV cell lines.
METHODS
Nanomaterials were characterized using SEM and TEM techniques; zeta potential analysis was also performed. Nanofilms of graphene, fullerene, and diamond nanoparticles were also characterized using AFM. The toxicity was tested on breast cancer MDA.MB.231 and glioblastoma grade IV U-87 MG cell lines, using LDH assay and by counting stained dead cells in bioprinted 3D models. The following parameters were analyzed: proliferation, adhesion to the nanofilm, and adhesion to particular ECM components covered with diamond nanoparticles.
RESULTS AND DISCUSSION
Our studies demonstrated that nanofilms of graphene and diamond nanoparticles are characterized by cell-specific toxicity. Those nanomaterials were non-toxic to MDA.MB.231 cells. After applying bioprinted 3D models, diamond nanoparticles were not toxic for both cell lines. Nanofilms made of diamond nanoparticles and graphene inhibit the proliferation of MDA.MB.231 cells after 48 and 72 hours. Increased adhesion on nanofilm made of diamond nanoparticles was only observed for MDA.MB.231 cells after 30 and 60 minutes from seeding the cells. However, analysis of adhesion to certain ECM components coated with diamond nanoparticles revealed enhanced adhesion to tenascin and vitronectin for both tested cell lines.
CONCLUSION
Our studies show that nanofilm made of diamond nanoparticles is a non-toxic and pro-adhesive nanomaterial that might stabilize and partially replenish the niche after breast tumor resection as it enhances the adhesion of breast cancer cells and inhibits their proliferation.
PubMed: 38111798
DOI: 10.2147/NSA.S439185 -
Frontiers in Cellular Neuroscience 2023Modification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of...
INTRODUCTION
Modification of the extracellular matrix (ECM) is one of the major processes in the pathology of brain damage following an ischemic stroke. However, our understanding of how age-related ECM alterations may affect stroke pathophysiology and its outcome is still very limited.
METHODS
We conducted an ECM-targeted re-analysis of our previously obtained RNA-Seq dataset of aging, ischemic stroke and their interactions in young adult (3-month-old) and aged (18-month-old) mice. The permanent middle cerebral artery occlusion (pMCAo) in rodents was used as a model of ischemic stroke. Altogether 56 genes of interest were chosen for this study.
RESULTS
We identified an increased activation of the genes encoding proteins related to ECM degradation, such as matrix metalloproteinases (MMPs), proteases of a disintegrin and metalloproteinase with the thrombospondin motifs (ADAMTS) family and molecules that regulate their activity, tissue inhibitors of metalloproteinases (TIMPs). Moreover, significant upregulation was also detected in the mRNA of other ECM molecules, such as proteoglycans, syndecans and link proteins. Notably, we identified 8 genes where this upregulation was enhanced in aged mice in comparison with the young ones. Ischemia evoked a significant downregulation in only 6 of our genes of interest, including those encoding proteins associated with the protective function of ECM molecules (e.g., brevican, Hapln4, Sparcl1); downregulation in brevican was more prominent in aged mice. The study was expanded by proteome analysis, where we observed an ischemia-induced overexpression in three proteins, which are associated with neuroinflammation (fibronectin and vitronectin) and neurodegeneration (link protein Hapln2). In fibronectin and Hapln2, this overexpression was more pronounced in aged post-ischemic animals.
CONCLUSION
Based on these results, we can conclude that the ratio between the protecting and degrading mechanisms in the aged brain is shifted toward degradation and contributes to the aged tissues' increased sensitivity to ischemic insults. Altogether, our data provide fresh perspectives on the processes underlying ischemic injury in the aging brain and serve as a freely accessible resource for upcoming research.
PubMed: 38107409
DOI: 10.3389/fncel.2023.1296455 -
Cornea Mar 2024The purpose of this study was to extensively evaluate the efficacy of integrin αvβ3 antagonists for the treatment of experimental dry eye (EDE).
PURPOSE
The purpose of this study was to extensively evaluate the efficacy of integrin αvβ3 antagonists for the treatment of experimental dry eye (EDE).
METHODS
Vitronectin, an αvβ3 ligand, was used to induce tumor necrosis factor-α gene expression in human THP-1 macrophages. To induce EDE, C57BL/6 mice were housed in a low-humidity controlled environment chamber and injected subcutaneously with scopolamine for 7 days. Subsequently, αvβ3 antagonists, including RGDfD, c(RGDfD), c(RGDiD), c(RGDfK), ATN-161, SB273005, and cilengitide, were administered topically to EDE animals under controlled environment chamber conditions. Corneal epithelial damage in EDE was assessed by fluorescein staining. The density of conjunctival goblet cells and secretion of tears was measured by period acid-Schiff staining and phenol red-impregnated cotton threads, respectively. Inflammation markers, including tumor necrosis factor-α, interleukin (IL)-1β, IL-6, IL-17A, and metalloproteinase (MMP)-9, in the pooled cornea and conjunctiva tissues were examined by real-time polymerase chain reaction.
RESULTS
The inhibitory effects of αvβ3 antagonists on the vitronectin-induced tumor necrosis factor-α gene expression and integrin-mediated inflammatory signaling were validated in THP-1 macrophages. αvβ3 antagonists ameliorated the impairment of the corneal epithelial barrier with varying therapeutic efficacies, compared with vehicle-treated mice. c(RGDfD) and c(RGDiD) significantly protected against goblet cell loss, tear reduction, and proinflammatory gene expression in EDE.
CONCLUSIONS
Topical applications of αvβ3 antagonists yield therapeutic benefits in EDE by promoting corneal epithelial defect healing and reducing inflammation. Antagonistic targeting αvβ3 may be a novel promising strategy to treat patients with dry eye disease.
Topics: Humans; Animals; Mice; Integrin alphaVbeta3; Tumor Necrosis Factor-alpha; Vitronectin; Mice, Inbred C57BL; Dry Eye Syndromes; Tears; Conjunctiva; Cornea; Inflammation; Disease Models, Animal
PubMed: 38015979
DOI: 10.1097/ICO.0000000000003427 -
Veterinary Research Nov 2023Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines...
Mycoplasma bovis is responsible for various inflammatory diseases in cattle. The prevention and control of M. bovis are complicated by the absence of effective vaccines and the emergence of multidrug-resistant strains, resulting in substantial economic losses worldwide in the cattle industry. Lipoproteins, vital components of the Mycoplasmas cell membrane, are deemed potent antigens for eliciting immune responses in the host upon infection. However, the functions of lipoproteins in M. bovis remain underexplored due to their low sequence similarity with those of other bacteria and the scarcity of genetic manipulation tools for M. bovis. In this study, the lipoprotein LppA was identified in all examined M. bovis strains. Utilizing immunoelectron microscopy and Western blotting, it was observed that LppA localizes to the surface membrane. Recombinant LppA demonstrated dose-dependent adherence to the membrane of embryonic bovine lung (EBL) cells, and this adhesion was inhibited by anti-LppA serum. In vitro binding assays confirmed LppA's ability to associate with fibronectin, collagen IV, laminin, vitronectin, plasminogen, and tPA, thereby facilitating the conversion of plasminogen to plasmin. Moreover, LppA was found to bind and enhance the accumulation of Annexin A2 (ANXA2) on the cell membrane. Disrupting LppA in M. bovis significantly diminished the bacterium's capacity to adhere to EBL cells, underscoring LppA's function as a bacterial adhesin. In conclusion, LppA emerges as a novel adhesion protein that interacts with multiple host extracellular matrix proteins and ANXA2, playing a crucial role in M. bovis's adherence to host cells and dissemination. These insights substantially deepen our comprehension of the molecular pathogenesis of M. bovis.
Topics: Animals; Cattle; Mycoplasma bovis; Bacterial Adhesion; Plasminogen; Annexin A2; Lipoproteins; Extracellular Matrix; Mycoplasma Infections; Cattle Diseases
PubMed: 37978536
DOI: 10.1186/s13567-023-01242-1