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Journal of Biological Physics Jun 2023Alpha-2-macroglobulin (αM) is an essential antiproteinase that is widely distributed in human plasma. The present study was aimed at investigating the binding of a...
Alpha-2-macroglobulin (αM) is an essential antiproteinase that is widely distributed in human plasma. The present study was aimed at investigating the binding of a potential therapeutic dietary flavonol, morin, with human αM using a multi-spectroscopic and molecular docking approach. Recently, flavonoid-protein interaction has gained significant attention, because a majority of dietary bioactive components interact with proteins, thereby altering their structure and function. The results of the activity assay exhibited a 48% reduction in the antiproteolytic potential of αM upon interaction with morin. Fluorescence quenching tests unequivocally confirmed quenching in the fluorescence of αM in the presence of morin, conforming complex formation and demonstrating that the binding mechanism involves a dynamic mode of interaction. Synchronous fluorescence spectra of αM with morin showed perturbation in the microenvironment around tryptophan residues. Furthermore, structural changes were observed through CD and FT-IR, showing alterations in the secondary structure of αM induced by morin. FRET further supports the results of the dynamic mode of quenching. Moderate interaction is shown by binding constant values using Stern-Volmer's fluorescence spectroscopy. Morin binds to αM at 298 K with a binding constant of 2.7 × 10 M, indicating the strength of the association. The αM-morin system was found to have negative ΔG values, which suggests that the binding process was spontaneous. Molecular docking also reveals the different amino acid residues involved in this binding process, revealing that the binding energy is -8.1 kcal/mol.
Topics: Humans; Pregnancy; Female; Pregnancy-Associated alpha 2-Macroglobulins; Molecular Docking Simulation; Spectroscopy, Fourier Transform Infrared; Flavonoids; Protein Binding
PubMed: 36913165
DOI: 10.1007/s10867-023-09629-z -
Frontiers in Pharmacology 2023Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America, characterized by glomerular accumulation of extracellular matrix (ECM) proteins....
Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America, characterized by glomerular accumulation of extracellular matrix (ECM) proteins. High glucose (HG) induction of glomerular mesangial cell (MC) profibrotic responses plays a central role in its pathogenesis. We previously showed that the endoplasmic reticulum resident GRP78 translocates to the cell surface in response to HG, where it mediates Akt activation and downstream profibrotic responses in MC. Transforming growth factor β1 (TGFβ1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGFβ1 is stored in the ECM in a latent form, requiring release for biological activity. The matrix glycoprotein thrombospondin 1 (TSP1), known to be increased in DKD and by HG in MC, is an important factor in TGFβ1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGFβ1 activation by HG. Primary mouse MC were used. TSP1 and TGFβ1 were assessed using standard molecular biology techniques. Inhibitors of csGRP78 were: 1) vaspin, 2) the C-terminal targeting antibody C38, 3) siRNA downregulation of its transport co-chaperone MTJ-1 to prevent GRP78 translocation to the cell surface, and 4) prevention of csGRP78 activation by its ligand, active α2-macroglobulin (α2M*), with the neutralizing antibody Fα2M or an inhibitory peptide. TSP1 transcript and promoter activity were increased by HG, as were cellular and ECM TSP1, and these required PI3K/Akt activity. Inhibition of csGRP78 prevented HG-induced TSP1 upregulation and deposition into the ECM. The HG-induced increase in active TGFβ1 in the medium was also inhibited, which was associated with reduced intracellular Smad3 activation and signaling. Overexpression of csGRP78 increased TSP-1, and this was further augmented in HG. These data support an important role for csGRP78 in regulating HG-induced TSP1 transcriptional induction via PI3K/Akt signaling. Functionally, this enables TGFβ1 activation in response to HG, with consequent increase in ECM proteins. Means of inhibiting csGRP78 signaling represent a novel approach to preventing fibrosis in DKD.
PubMed: 36909183
DOI: 10.3389/fphar.2023.1098321 -
BMC Medicine Mar 2023Pre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with...
BACKGROUND
Pre-eclampsia (PE) is one of the leading causes of maternal and fetal morbidity/mortality during pregnancy, and alpha-2-macroglobulin (A2M) is associated with inflammatory signaling; however, the pathophysiological mechanism by which A2M is involved in PE development is not yet understood.
METHODS
Human placenta samples, serum, and corresponding clinical data of the participants were collected to study the pathophysiologic mechanism underlying PE. Pregnant Sprague-Dawley rats were intravenously injected with an adenovirus vector carrying A2M via the tail vein on gestational day (GD) 8.5. Human umbilical artery smooth muscle cells (HUASMCs), human umbilical vein endothelial cells (HUVECs), and HTR-8/SVneo cells were transfected with A2M-expressing adenovirus vectors.
RESULTS
In this study, we demonstrated that A2M levels were significantly increased in PE patient serum, uterine spiral arteries, and feto-placental vasculature. The A2M-overexpression rat model closely mimicked the characteristics of PE (i.e., hypertension in mid-to-late gestation, histological and ultrastructural signs of renal damage, proteinuria, and fetal growth restriction). Compared to the normal group, A2M overexpression significantly enhanced uterine artery vascular resistance and impaired uterine spiral artery remodeling in both pregnant women with early-onset PE and in pregnant rats. We found that A2M overexpression was positively associated with HUASMC proliferation and negatively correlated with cell apoptosis. In addition, the results demonstrated that transforming growth factor beta 1 (TGFβ1) signaling regulated the effects of A2M on vascular muscle cell proliferation described above. Meanwhile, A2M overexpression regressed rat placental vascularization and reduced the expression of angiogenesis-related genes. In addition, A2M overexpression reduced HUVEC migration, filopodia number/length, and tube formation. Furthermore, HIF-1α expression was positively related to A2M, and the secretion of sFLT-1 and PIGF of placental origin was closely related to PE during pregnancy or A2M overexpression in rats.
CONCLUSIONS
Our data showed that gestational A2M overexpression can be considered a contributing factor leading to PE, causing detective uterine spiral artery remodeling and aberrant placental vascularization.
Topics: Animals; Female; Humans; Pregnancy; Rats; Endothelial Cells; Macroglobulins; Placenta; Placenta Growth Factor; Pre-Eclampsia; Rats, Sprague-Dawley; Uterine Artery
PubMed: 36894970
DOI: 10.1186/s12916-023-02807-9 -
PeerJ 2023Long non-coding RNAs (lncRNAs) have been reported as key regulators of chronic obstructive pulmonary disease (COPD). This study aimed to figure out the regulatory...
Long non-coding RNAs (lncRNAs) have been reported as key regulators of chronic obstructive pulmonary disease (COPD). This study aimed to figure out the regulatory mechanism as well as the effects of lncRNA00612 (LINC00612) in lipopolysaccharide (LPS)-induced inflammation and apoptosis in BEAS-2B cells. LINC00612 and its co-expressed gene alpha-2-macroglobulin (A2M) were strikingly downregulated in the peripheral venous blood of COPD patients. Overexpressed LINC00612 enhances BEAS-2B cells against apoptosis and inflammatory reactions mediated by LPS, however, an A2M knockdown can attenuate the degree of the enhancement. Bioinformatics analysis revealed putative binding sites between LINC00612, signal transducer and activator of transcription 3 (STAT3) and the A2M promoter, while RNA antisense purification and Chromatin immunoprecipitation were performed to confirm the prediction. Knockdown of LINC00612 impaired the binding of p-STAT3 to the promoter of A2M, which meant that LINC00612 was critical for the binding of STAT3 with the A2M promoter. Therefore, it can be concluded that LINC00612 ameliorates LPS-induced cell apoptosis and inflammation recruiting STAT3 to bind to A2M. This conclusion will serve as a theoretical foundation for the treatment of COPD.
Topics: Humans; alpha-Macroglobulins; Apoptosis; Inflammation; Lipopolysaccharides; Pulmonary Disease, Chronic Obstructive; RNA, Long Noncoding; STAT3 Transcription Factor; Cell Line
PubMed: 36883061
DOI: 10.7717/peerj.14986 -
Journal of Thrombosis and Haemostasis :... Mar 2023
Topics: Humans; Pregnancy; Female; Pregnancy-Associated alpha 2-Macroglobulins; Thromboinflammation; COVID-19; Inflammation; Thrombosis; Enzyme Inhibitors; Transcription Factors
PubMed: 36858795
DOI: 10.1016/j.jtha.2023.01.005 -
Micromachines Feb 2023The need for Alpha2-Macroglobulin (α2-M) detection has increased because it plays an important role in the diagnosis of diabetic nephropathy (DN). However, few sensors...
The need for Alpha2-Macroglobulin (α2-M) detection has increased because it plays an important role in the diagnosis of diabetic nephropathy (DN). However, few sensors can realize the high-sensitive detection for α2-M with characteristics of being fast, flexible, wearable and portable. Herein, a biosensor based on a MnFeO@chitosan/MWCNTs/PDMS composite film was developed for α2-M detection. Due to the excellent magnetoelastic effect of MnFeO nanoparticles, the stress signal of the biosensor surface induced by the specific antibody-antigen binding was transformed into the electrical and magnetic signal. Chitosan-coated MnFeO particles were used to provide biological modification sites for the α2-M antibody, which simplified the conventional biological functionalization modification process. The MnFeO@chitosan particles were successfully prepared by a chemical coprecipitation method and the property was studied by TEM, FT-IR and XRD. MWCNTs were employed to enhance electrical conductivity and the sensitivity of the biosensor. The detection limit (LOD) was reduced to 0.1299 ng·mL in the linear range from 10 ng∙mL to 100 µg·mL, which was significantly lower than the limit of health diagnostics. The biosensor is fabricated by a simple method, with advantages of being rapid and highly-sensitive, and having selective detection of α2-M, which provides a novel method for the early diagnosis of DN, and it has potential in the point of care (PoC) field.
PubMed: 36838101
DOI: 10.3390/mi14020401 -
Indian Dermatology Online Journal 2023Acute phase reactants (APRs) are a heterogeneous group of plasma proteins whose concentration either increases or decreases by at least 25% during an inflammatory... (Review)
Review
Acute phase reactants (APRs) are a heterogeneous group of plasma proteins whose concentration either increases or decreases by at least 25% during an inflammatory process. The conditions that commonly lead to acute phase response are infection, trauma, burns, tissue infarction, inflammatory conditions, and advanced malignancy. APRs are elevated in all infective conditions. In skin and soft tissue infection, the levels of erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) help to predict the severity of infection. Procalcitonin can be used to differentiate between viral and bacterial infections. During active stages of systemic lupus erythematosus (SLE), ESR is elevated, but CRP either remains normal or shows only moderate elevation. In the case of superadded bacterial infection in SLE, CRP is elevated. In SLE, ferritin levels are elevated during the active stage of the disease. Serum amyloid antigen (SAA) and CRP levels are significantly higher in patients with early and late stages of diffuse systemic sclerosis. Elevated levels of serum ferritin are seen in rheumatoid arthritis and adult-onset Still's disease. CRP, SAA, and 2-macroglobulin ( M) are elevated in active psoriasis. In severe psoriasis, the ferritin-iron ratio is elevated. In drug-induced maculopapular rash, drug-induced hyperaemic vasculitis, and severe drug-induced cutaneous adverse reactions, CRP levels are elevated during the active stages. Neoplastic diseases in general are accompanied by increased serum ferritin. Further detailed studies are required to explore the clinical significance of APRs in dermatology and the scope of their possible application as a diagnostic tool.
PubMed: 36776186
DOI: 10.4103/idoj.idoj_174_21 -
Cells Feb 2023Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic...
Inflammation and oxidative and nitrosative stress are involved in the pathogenesis of proliferative retinopathies (PR). In PR, a loss of balance between pro-angiogenic and anti-angiogenic factors favors the secretion of vascular endothelial growth factor (VEGF). This vascular change results in alterations in the blood-retinal barrier, with extravasation of plasma proteins such as α-macroglobulin (αM) and gliosis in Müller glial cells (MGCs, such as MIO-M1). It is well known that MGCs play important roles in healthy and sick retinas, including in PR. Nitro-fatty acids are electrophilic lipid mediators with anti-inflammatory and cytoprotective properties. Our aim was to investigate whether nitro-oleic acid (NO-OA) is beneficial against oxidative stress, gliosis, and the pro-angiogenic response in MGCs. Pure synthetic NO-OA increased HO-1 expression in a time- and concentration-dependent manner, which was abrogated by the Nrf2 inhibitor trigonelline. In response to phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS), NO-OA prevented the ROS increase and reduced the gliosis induced by αM. Finally, when hypoxic MGCs were incubated with NO-OA, the increase in VEGF mRNA expression was not affected, but under hypoxia and inflammation (IL-1β), NO-OA significantly reduced VEGF mRNA levels. Furthermore, NO-OA inhibited endothelial cell (BAEC) tubulogenesis. Our results highlight NO-OA's protective effect on oxidative damage, gliosis; and the exacerbated pro-angiogenic response in MGCs.
Topics: Humans; Nitrogen Dioxide; Vascular Endothelial Growth Factor A; Ependymoglial Cells; Gliosis; Oxidative Stress; Hypoxia; Inflammation; RNA, Messenger
PubMed: 36766836
DOI: 10.3390/cells12030494 -
Nature Communications Feb 2023Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving...
Tailoring of the activity and specificity of proteases is critical for their utility across industrial, medical and research purposes. However, engineering or evolving protease catalysts is challenging and often labour intensive. Here, we describe a generic method to accelerate this process based on yeast display. We introduce the protease selection system A2M that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2M for protease engineering we exemplify the directed activity and specificity evolution of six serine proteases. This resulted in a variant of Staphylococcus aureus serin-protease-like (Spl) protease SplB, an enzyme used for recombinant protein processing, that no longer requires activation by N-terminal signal peptide removal. SCHEMA-based domain shuffling was used to map the specificity determining regions of Spl proteases, leading to a chimeric scaffold that supports specificity switching via subdomain exchange. The ability of A2M to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.
Topics: Humans; Pregnancy-Associated alpha 2-Macroglobulins; Protein Sorting Signals; Recombinant Proteins; Serine Endopeptidases; Serine Proteases; alpha-Macroglobulins
PubMed: 36765057
DOI: 10.1038/s41467-023-36099-7 -
Journal of the Pediatric Infectious... Apr 2023To determine by multi-omic analysis changes in metabolites, lipids, and proteins as a consequence of transient viral rebound (tVR) in children with perinatally acquired...
BACKGROUND
To determine by multi-omic analysis changes in metabolites, lipids, and proteins as a consequence of transient viral rebound (tVR) in children with perinatally acquired HIV-1 (PHIV).
METHODS
Plasma samples from children with PHIV and with tVR (first episode of transient RNA-HIV viral load >20 copies/ml followed by suppression) on the time-point immediately before (pre-tVR) and after (post-tVR) the tVR were assessed. Multi-omic analyses were performed using nLC-Orbitrap, GC-qTOF-MS, and LC-qTOF-MS.
RESULTS
Comparing pre- and post-tVR time-points, HIV-1 children with tVR (n = 5) showed a trend to a decrease in ratio CD4/CD8 (p = 0.08) but no significant differences were observed in plasma metabolites, lipids, or proteins. Post-tVR condition was compared with a reference group of children with PHIV with persistent viral control (n = 9), paired by sex, age, and time under antiretroviral treatment. A total of 10 proteins, 8 metabolites, and 2 lipids showed significant differences (p < 0.05): serotransferrin, clusterin, kininogen-1, succinic acid, threonine, 2-hydroxyisovaleric acid, methionine, 2-hydroxyglutaric, triacylglyceride 50:0 (TG50:0), and diacylglyceride 34:1 (DG34:1) were upregulated while alpha-2-macroglobulin, apolipoprotein A-II, carboxylic ester hydrolase, apolipoprotein D, coagulation factor IX, peptidase inhibitor 16, SAA2-SAA4 readthrough, oleic acid, palmitoleic acid, and D-sucrose downregulated on post-tVR time-point compared to the reference group. Ratio CD4/CD8 correlated with apolipoprotein A-II, DG34:1, and methionine (p = 0.004; ρ = 0.71, p = 0.016; ρ = -0.63; and p = 0.032; ρ = -0.57, respectively). Nadir CD4+ correlated inversely with kininogen-1 (p = 0.022; ρ = -0.60) and positively with D-sucrose (p = 0.001; ρ = 0.77).
CONCLUSIONS
tVR followed by suppression implies changes in soluble proteins, lipids, and metabolites that correlate with immunological parameters, mainly ratio CD4/CD8, that decreased after tVR. These distinct soluble biomarkers could be considered potential biomarkers of immune progression.
Topics: Child; Humans; Apolipoprotein A-II; Biomarkers; CD8-Positive T-Lymphocytes; HIV Infections; HIV Seropositivity; HIV-1; Methionine; Viral Load; CD4-Positive T-Lymphocytes
PubMed: 36727571
DOI: 10.1093/jpids/piad008