Determination of the levels and possible associations of alpha2-macroglobulin with autoantibodies in the serum of patients with various forms of autoimmune thyroiditis.

Antibodies to thyroid peroxidase (AB-TPO), antibodies to thyroglobulin (AB-TG), and the content of α2-macroglobulin (α2-MG) have been studied in serum samples of patients with autoimmune thyroiditis (AIT). All the patients were divided into 3 groups depending on age: 25-35, 36-50, 51-65 years. We found a significant change in the thyroid panel parameters in AIT, but without significant changes in the average concentration of α2-MG in the age groups of patients. This may be due to the accumulation and retention of complexes of defective forms of α2-MG in the circulation associated with their decreased ability to bind to receptors.

Effect of Short PSG Peptides on Inflammatory Markers in Allogeneic Bone Marrow Cell Transplantation in Wistar Rats.

Short linear peptide fragments of placental trophoblastic ß1-glycoprotein (PSG) (YECE, YQCE, YVCS, and YACS) were studied in the context of their immunomodulatory effects at the level of inflammatory markers. The original host-versus-graft model was used in male Wistar rats without prior conditioning of recipient bone marrow. A composition of PSG peptide fragments was injected to animals after allogeneic transplantation of bone marrow cells in a dynamic experiment, inflammatory markers α1-acid glycoprotein (AGP, orosomucoid), α2-macroglobulin (α2M) were assayed by ELISA, and biochemical parameters (total protein, glucose, creatinine, and urea) were measured. The levels of α2M and AGP increased in response to allotransplantation, whereas administration of PSG peptides normalized serum α2M levels by the end of the experiment. The decrease in α2M level coincided with the independent effect of PSG peptide administration. The levels of total protein, glucose, creatinine, and urea in rat serum after allotransplantation were reduced throughout the experiment. Administration of PSG peptides contributed to normalization of serum total protein, creatinine, and urea levels by the end of the experiment. Administration of PSG peptides after allogeneic transplantation of bone marrow suspension contributed to normalization of the levels of α2M, total protein, creatinine, and urea, which can be interpreted as an anti-inflammatory effect of these peptides.

LincRNA00612 inhibits apoptosis and inflammation in LPS-induced BEAS-2B cells via enhancing interaction between p-STAT3 and A2M promoter.

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 via recruiting STAT3 to bind to A2M. This conclusion will serve as a theoretical foundation for the treatment of COPD.

Evolution of protease activation and specificity via alpha-2-macroglobulin-mediated covalent capture.

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 A2Mcap that covalently captures protease catalysts by repurposed alpha-2-macroglobulin (A2Ms). To demonstrate the utility of A2Mcap 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 A2Mcap to overcome key challenges en route to tailor-made proteases suggests easier access to such reagents in the future.

Mesenchymal Stem Cell-Originated Exosomal Lnc A2M-AS1 Alleviates Hypoxia/Reperfusion-Induced Apoptosis and Oxidative Stress in Cardiomyocytes.

BACKGROUND: Mesenchymal stem cell (MSC)-derived exosomes play significant roles in ameliorating cardiac damage after myocardial ischemia-reperfusion (I/R) injury. Long non-coding RNA alpha-2-macroglobulin antisense RNA 1 (Lnc A2M-AS1) was found that might protect against myocardial I/R. However, whether Lnc A2M-AS1 delivery via MSC-derived exosomes could also regulate myocardial I/R injury remains unknown. METHODS: Exosomes were isolated by ultracentrifugation, and qualified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blot. Hypoxia/reoxygenation (H/R) treatment in human cardiomyocytes was used to mimic the process of myocardial I/R in vitro. The viability and apoptosis of cardiomyocytes were detected using cell counting kit-8, flow cytometry, and Western blot assays. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were evaluated using corresponding commercial kits. The quantitative real-time polymerase chain reaction and Western blot were used to determine the expression levels of Lnc A2M-AS1, microRNA (miR)-556-5p, and X-linked inhibitor of apoptosis protein (XIAP). The binding interaction between miR-556-5p and Lnc A2M-AS1 or XIAP was confirmed by the dual-luciferase reporter, RIP and pull-down assays. RESULTS: Exosomes isolated from hMSCs (hMSCs-exo) attenuated H/R-induced apoptosis and oxidative stress in cardiomyocytes. Lnc A2M-AS1 was lowly expressed in AMI patients and H/R-induced cardiomyocytes. Besides, Lnc A2M-AS1 was detectable in hMSCs-exo, exosomes derived from Lnc A2M-AS1-transfected hMSCs weakened H/R-induced apoptosis and oxidative stress, and enhanced the protective action of hMSCs-exo on H/R-induced cardiomyocytes. Further mechanism analysis showed that Lnc A2M-AS1 acted as a sponge for miR-556-5p to increase XIAP expression level. Importantly, miR-556-5p overexpression or XIAP knockdown reversed the action of exosomal Lnc A2M-AS1 on H/R-induced cardiomyocytes. CONCLUSION: Lnc A2M-AS1 delivery via MSC-derived exosomes ameliorated H/R-induced cardiomyocyte apoptosis and oxidative stress via regulating miR-556-5p/XIAP, opening a new window into the pathogenesis of myocardial I/R injury.

Overexpression of lncRNA A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/bone morphogenetic protein 3 axis in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is a malignant tumor that occurs in the lungs. Numerous reports have substantiated the participation of long non-coding RNAs (lncRNAs) in the tumorigenesis of LUAD. Previously, lncRNA alpha-2-macroglobulin antisense RNA 1 (A2M-AS1) was confirmed to be an important regulator in the biological processes of LUAD and dysregulation of A2M-AS1 was associated with non-small cell lung cancer (NSCLC) progression. However, the precise mechanism of A2M-AS1 in LUAD has not been elucidated. Therefore, our study was designed to investigate the detailed molecular mechanism of A2M-AS1 in LUAD. Herein, the expression of lncRNA A2M-AS1, microRNA (miRNA) miR-587, and bone morphogenetic protein 3 (BMP3) in LUAD cell lines and tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR) and western blotting. The viability, proliferation, migration and invasion of LUAD cells were tested by cell counting kit-8 (CCK-8), colony formation and Transwell assays. In vivo tumor growth was investigated by xenograft animal experiment. Interactions among A2M-AS1, miR-587 and BMP3 were measured by RNA pulldown and luciferase reporter assays. In this study, A2M-AS1 was downregulated in LUAD tissues and cells and related to poor prognosis in LUAD patients. A2M-AS1 overexpression suppressed LUAD cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistically, A2M-AS1 directly bound with miR-587 to promote BMP3 expression in LUAD cells. Low expression of BMP3 was found in LUAD tissues and cells and was closely correlated with poor prognosis in LUAD patients. BMP3 deficiency reserved the inhibitory influence of A2M-AS1 overexpression on LUAD cell behaviors. Overall, A2M-AS1 inhibits cell growth and aggressiveness via regulating the miR-587/BMP3 axis in LUAD.

LncRNA A2M-AS1 Promotes Ferroptosis in Pancreatic Cancer via Interacting With PCBP3.

Ferroptosis is a newly-discovered cell death mechanism involved in the progression of various tumors, the role of noncoding RNAs (ncRNAs) in it was relatively less explored. This study identified the low levels of a recently studied long noncoding RNA (lncRNA), A2M-AS1, in pancreatic cancer and suggested its positive correlation with the overall survival time of patients with pancreatic cancer. A2M-AS1 was mainly localized in the cytoplasm, inhibiting the cellular proliferation, migration, and invasion as well as the tumor growth of the pancreatic cancer cells. Moreover, the Erastin-induced ferroptosis increased the expression levels of A2M-AS1. The overexpression of A2M-AS1 promoted ferroptosis in the pancreatic cancer, which was inhibited by the silencing of A2M-AS1. Mechanically, A2M-AS1 could directly interact with the poly (rC) binding protein 3 (PCBP3), which plays an important role in the process of iron metabolism, thereby promoting the ferroptosis in pancreatic cancer. In addition, the A2M-AS1/PCBP3 axis could facilitate the p38 activation and inhibit the phosphorylation of the AKT-mTOR signaling pathway; all these participate in regulating ferroptosis. In conclusion, the regulation of ferroptosis by targeting the A2M-AS1/PCBP3 axis might provide a novel target for the treatment of pancreatic cancer in the future. IMPLICATIONS: A2M-AS1 might be a potential novel therapeutic target for patients with pancreatic cancer in the future.

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo­electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.

Alpha-2-macroglobulin in hemostasis and thrombosis: An underestimated old double-edged sword.

Antiproteinases such as alpha-2-macroglobulin (A2M) play a role in hemostasis. A2M is highly conserved throughout evolution and is a high molecular weight homo-tetrameric glycoprotein. A2M proteinase inhibitor activity is possible via a unique cage structure leading to proteinase entrapment without direct enzymatic activity inhibition. Following this entrapment, proteinase clearance is possible through A2M binding to the low-density lipoprotein receptor-related protein 1. A2M synthesis is regulated by pro-inflammatory cytokines and increases during several chronic or acute inflammatory diseases and varies with age. For instance, A2M plasma levels are known to be increased in patients with diabetes mellitus, nephrotic syndrome, or sepsis. Concerning hemostasis, A2M can trap many proteinases involved in coagulation and fibrinolysis. Because of its pleiotropic effects A2M can be seen as both anti- and pro-hemostatic. A2M can inhibit thrombin, factor Xa, activated protein C, plasmin, tissue-plasminogen activator, and urokinase. Through its many different functions A2M is generally put apart in the balanced regulation of hemostasis. In addition, the fact that A2M plasma levels are differently regulated during inflammatory-related diseases and that A2M can neutralize cytokines that also modify hemostasis could explain why it is difficult to link common proteins and parameters of hemostasis with the mechanisms of thrombosis in such diseases. Thus, we propose in the present review to summarize known functions of A2M, give a brief overview about diseases, and then to focus on the roles of this antiproteinase in hemostasis and thrombosis.

Effects of ethanolic extracts of Quercus, Cirsium vulgare, and Falcaria vulgaris on gastric ulcer, antioxidant and inflammatory indices, and gene expression in rats.

INTRODUCTION: Gastric ulcer is a multifaceted process and is usually caused by mucosal damage. Herbal medicines have received much attention considering the side effects of chemical drugs. Nowadays, the use of herbal medicines has received much attention considering the side effects of chemical drugs. Quercus brantii Lindl, Cirsium vulgare (Savi) Ten, and Falcaria vulgaris Bernh are plants used as traditional phytomedicine for gastric ulcer diseases. AIM OF THE STUDY: This study was aimed to investigate the protective effects of hydroalcoholic extracts of these herbs on ethanol-induced gastric ulceration, in addition, to investigate the antioxidant, anti-inflammatory, and gene expression. MATERIALS AND METHODS: Thirty Sprague Dawley rats, (200-250 g), were divided into six groups: Control: intact animals; sham: gavaged with distilled water (14 days); negative control: gavaged with 20 mg/kg of omeprazole (14 days); experimental groups I, II, and III: gavaged with 500 mg/kg of the extract of Falcaria vulgaris, Quercus brantii, and Cirsium vulgare, respectively, (14 days). The number of ulcers and pathological parameters were assessed. The serum superoxide dismutase, catalase, glutathione peroxidase, malondialdehyde, total antioxidant capacity, albumin, total protein, haptoglobin, alpha-1-acid glycoprotein, total globulin, alpha-2-macroglobulin, C-fos, C-myc, and Caspase-9 were measured by ELISA and RT-PCR. RESULTS: The extracts significantly reduced gastric ulcer (52.33%). The results showed that the Quercus brantii extract was more effective. There were significant differences between the serum levels of alpha-1-acid glycoprotein and those of alpha-2-macroglobulin. Also, there was a significant difference in the serum level of antioxidant parameters. Changes in the expression of the genes also confirmed the results suggested by other parameters. The expression levels of C-fos, C-myc, and caspase-9 were decreased, but the Bcl-2 expression increased. CONCLUSION: The hydro-alcoholic extracts revealed various protection and noticeable change in the expression of caspase-9, C-myc, C-fos, and Bcl-2 genes in rats.

Indomethacin Disrupts the Formation of ß-Amyloid Plaques via an α2-Macroglobulin-Activating lrp1-Dependent Mechanism.

Epidemiological studies have implied that the nonsteroidal anti-inflammatory drug (NSAID) indomethacin slows the development and progression of Alzheimer's disease (AD). However, the underlying mechanisms are notably understudied. Using a chimeric mouse/human amyloid precursor protein (Mo/HuAPP695swe) and a mutant human presenilin 1 (PS1-dE9) (APP/PS1) expressing transgenic (Tg) mice and neuroblastoma (N) 2a cells as in vivo and in vitro models, we revealed the mechanisms of indomethacin in ameliorating the cognitive decline of AD. By screening AD-associated genes, we observed that a marked increase in the expression of α2-macroglobulin (A2M) was markedly induced after treatment with indomethacin. Mechanistically, upregulation of A2M was caused by the inhibition of cyclooxygenase-2 (COX-2) and lipocalin-type prostaglandin D synthase (L-PGDS), which are responsible for the synthesis of prostaglandin (PG)H2 and PGD2, respectively. The reduction in PGD2 levels induced by indomethacin alleviated the suppression of A2M expression through a PGD2 receptor 2 (CRTH2)-dependent mechanism. Highly activated A2M not only disrupted the production and aggregation of ß-amyloid protein (Aß) but also induced Aß efflux from the brain. More interestingly, indomethacin decreased the degradation of the A2M receptor, low-density lipoprotein receptor-related protein 1 (LRP1), which facilitated the brain efflux of Aß. Through the aforementioned mechanisms, indomethacin ameliorated cognitive decline in APP/PS1 Tg mice by decreasing Aß production and clearing Aß from the brains of AD mice.

Antipsychotic clozapine binding to alpha-2-macroglobulin protects interacting partners against oxidation and preserves the anti-proteinase activity of the protein.

In this study, the interaction between clozapine, an atypical antipsychotic drug, and alpha-2-macroglobulin (α2M), a multipurpose anti-proteinase, was investigated under simulated (patho) physiological conditions using multiple spectroscopic techniques and molecular modeling. It was found that α2M binds clozapine with a moderate affinity (the binding constant of 0.9 × 105 M-1 at 37 °C). The preferable binding site for both clozapine's atropisomers was revealed to be a large pocket at the interface of C and D monomer subunits of the protein. Hydrogen bonds and the hydrophobic effect were proposed as dominant forces in complex formation. The binding of clozapine did not induce significant conformational change of the protein, as confirmed by virtually unaltered α2M secondary structure and anti-proteinase activity. However, both clozapine and α2M shielded each other from the deleterious influence of strong oxidants: sodium hypochlorite and 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH). Moreover, clozapine in a concentration range that is usually targeted in the plasma during patients' treatment effectively protected the anti-proteinase activity of α2M under AAPH-induced free radical overproduction. Our results suggest that the cooperation between α2M and clozapine may be a path by which these two molecules synergistically protect neural tissue against injury caused by disturbed proteostasis or oxidative stress.

Shedding of proangiogenic microvesicles from hypertrophic scar myofibroblasts.

Hypertrophic scars are a common complication of burn injuries and represent a major challenge in terms of prevention and treatment. These scars are characterized by a supraphysiological vascular density and by the presence of pathological myofibroblasts (Hmyos) displaying a low apoptosis propensity. However, the nature of the association between these two hallmarks of hypertrophic scarring remains largely unexplored. Here, we show that Hmyos produce signalling entities known as microvesicles that significantly increase the three cellular processes underlying blood vessel formation: endothelial cell proliferation, migration and assembly into capillary-like structures. The release of microvesicles from Hmyos was dose-dependently induced by the serum protein α-2-macroglobulin. Using flow cytometry, we revealed the presence of the α-2-macroglobulin receptor-low-density lipoprotein receptor-related protein 1-on the surface of Hmyos. The inhibition of the binding of α-2-macroglobulin to its receptor abolished the shedding of proangiogenic microvesicles from Hmyos. These findings suggest that the production of microvesicles by Hmyos contributes to the excessive vascularization of hypertrophic scars. α-2-Macroglobulin modulates the release of these microvesicles through interaction with low-density lipoprotein receptor-related protein 1.

Acute-phase protein α-1-acid glycoprotein is negatively associated with feed intake in postpartum dairy cows.

α-1-Acid glycoprotein (AGP) is an acute-phase protein that may suppress dry matter intake (DMI), potentially by acting on the leptin receptor in the hypothalamus. Our objectives were to characterize plasma AGP concentration and associations with DMI during the transition period, and to determine the utility of AGP to identify or predict cows with low DMI. Plasma samples (n = 2,086) from 434 Holstein cows in 6 studies were analyzed on d -21, -13 ± 2, -3, 1, 3, 7 ± 1, 14 ± 1, and 21 ± 1 relative to parturition. A commercially available ELISA kit specific for bovine AGP was validated, and 2 internal controls were analyzed on each plate with interplate variation of 15.0 and 17.3%, respectively. Bivariate analysis was used to assess the relationship between AGP and DMI. For significant associations, treatment(study) was added to the model, and quadratic associations were included in the model if significant. Plasma AGP concentration (±SEM) increased from 213 ± 37.3 µg/mL on d -3 to 445 ± 60.0 µg/mL on d 14. On d 3, AGP was associated negatively with DMI in a quadratic manner for wk 1 and wk 2 and linearly for wk 3. Day 7 AGP was associated negatively with DMI in a quadratic manner for wk 2 and linearly for wk 3. Similarly, d 14 AGP was negatively associated with DMI for wk 3 and wk 4. As d 3 AGP concentration increased over the interquartile range, a calculated 1.4 (8.5%), 0.5 (2.7%), and 0.4 (1.9%) kg/d reduction in predicted DMI was detected during wk 1, 2, and 3, respectively. Using bivariate analysis, d 3 AGP explained 10% of the variation in DMI during wk 1. We explored the clinical utility of d 3 AGP to diagnose low DMI, defined as wk 1 DMI >1 standard deviation below the mean. Receiver operating characteristic analysis identified a threshold of 480.9 µg/mL, providing 76% specificity and 48% sensitivity (area under the curve = 0.60). Limited associations occurred between AGP and blood biomarkers; however, AGP was associated with plasma haptoglobin concentration postpartum and incidence of displaced abomasum, retained placenta, and metritis. These results demonstrate a negative association between plasma AGP concentration and DMI in early-postpartum dairy cows, although its diagnostic performance was marginal. Further investigation into whether AGP directly suppresses DMI in dairy cattle is warranted.

Thromboinflammation in COVID-19: Can α2 -macroglobulin help to control the fire?

The complex COVID-19-associated coagulopathy appears to impair prognosis. Recently, we presented the hypothesis that children are to some extent protected by higher α2 -macroglobulin (α2 -M) levels from severe COVID-19. In addition to endothelial cells, thrombin, and platelets, neutrophil granulocytes also appear to play an important role. Neutrophils extrude extracellular nets, which are histone- and protease-coated web-like DNA structures; activate coagulation and platelets; and release radicals and proteases such as elastase. The unique phylogenetically ancient and "versatile" inhibitor α2 -M contributes particularly during childhood to the antithrombin activity of plasma, binds a broad spectrum of proteases, and interacts with other mediators of inflammation such as cytokines. It is suggested that the scope of basic research and clinical studies would include the potential role of α2 -M in COVID-19.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Epigenetic Modulation by Apabetalone Counters Cytokine-Driven Acute Phase Response In Vitro, in Mice and in Patients with Cardiovascular Disease.

Chronic systemic inflammation contributes to cardiovascular disease (CVD) and correlates with the abundance of acute phase response (APR) proteins in the liver and plasma. Bromodomain and extraterminal (BET) proteins are epigenetic readers that regulate inflammatory gene transcription. We show that BET inhibition by the small molecule apabetalone reduces APR gene and protein expression in human hepatocytes, mouse models, and plasma from CVD patients. Steady-state expression of serum amyloid P, plasminogen activator inhibitor 1, and ceruloplasmin, APR proteins linked to CVD risk, is reduced by apabetalone in cultured hepatocytes and in humanized mouse liver. In cytokine-stimulated hepatocytes, apabetalone reduces the expression of C-reactive protein (CRP), alpha-2-macroglobulin, and serum amyloid P. The latter two are also reduced by apabetalone in the liver of endotoxemic mice. BET knockdown in vitro also counters cytokine-mediated induction of the CRP gene. Mechanistically, apabetalone reduces the cytokine-driven increase in BRD4 BET occupancy at the CRP promoter, confirming that transcription of CRP is BET-dependent. In patients with stable coronary disease, plasma APR proteins CRP, IL-1 receptor antagonist, and fibrinogen γ decrease after apabetalone treatment versus placebo, resulting in a predicted downregulation of the APR pathway and cytokine targets. We conclude that CRP and components of the APR pathway are regulated by BET proteins and that apabetalone counters chronic cytokine signaling in patients.

GALNT6 promotes breast cancer metastasis by increasing mucin-type O-glycosylation of α2M.

Breast cancer is the most lethal malignancy in women. N-acetylgalactosaminyltransferase 6 (GALNT6) is an enzyme which mediates the initial step of mucin-type O-glycosylation, and has been reported to be involved in mammary carcinogenesis. However, the molecular mechanism of GALNT6 in breast cancer metastasis has not been fully explored. In this study, based on online database analyses and tissue microarrays, the overall survival (OS) of breast cancer patients with high expression of GALNT6 was found to be shorter than those with low expression of GALNT6. Also, high GALNT6 expression was positively correlated with advanced pN stage and pTNM stage. GALNT6 was shown to be able to promote the migration and invasion of breast cancer cells, and enhance the level of mucin-type O-glycosylation of substrates in the supernatants of breast cancer cells. Qualitative mucin-type glycosylomics analysis identified α2M as a novel substrate of GALNT6. Further investigation showed that GALNT6 increased O-glycosylation of α2M, and the following activation of the downstream PI3K/Akt signaling pathway was involved in the promotion of migration and invasion of breast cancer cells. This study identified a new substrate of GALNT6 and provides novel understanding of the role of GALNT6 in promoting metastasis and poor prognosis in breast cancer.

Activated α2-macroglobulin binding to cell surface GRP78 induces trophoblastic cell fusion.

The villous cytotrophoblastic cells have the ability to fuse and differentiate, forming the syncytiotrophoblast (STB). The syncytialisation process is essential for placentation. Nevertheless, the mechanisms involved in cell fusion and differentiation are yet to be fully elucidated. It has been suggested that cell surface glucose-regulated protein 78 (GRP78) was involved in this process. In multiple cancer cells, cell membrane-located GRP78 has been reported to act as a receptor binding to the active form of α2-macroglobulin (α2M*), activating thus several cellular signalling pathways implicated in cell growth and survival. We hypothesised that GRP78 interaction with α2M* may also activate signalling pathways in trophoblastic cells, which, in turn, may promote cell fusion. Here, we observed that α2M mRNA is highly expressed in trophoblastic cells, whereas it is not expressed in the choriocarcinoma cell line BeWo. We thus took advantage of forskolin-induced syncytialisation of BeWo cells to study the effect of exogenous α2M* on syncytialisation. We first demonstrated that α2M* induced trophoblastic cell fusion. This effect is dependent on α2M*-GRP78 interaction, ERK1/2 and CREB phosphorylation, and unfolded protein response (UPR) activation. Overall, these data provide novel insights into the signalling molecules and mechanisms regulating trophoblastic cell fusion.

The Role of Alpha 2 Macroglobulin in IgG-Aggregation and Chronic Activation of the Complement System in Patients With Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults in the western world. One of the treatments offered for CLL is immunotherapy. These treatments activate various cellular and biochemical mechanisms, using the complement system. Recently it was shown that the complement system in CLL patients is persistently activated at a low level through the classical pathway (CP). The mechanism of chronic CP activation involves the formation of IgG-hexamers (IgG-aggregates). According to recent studies, formation of ordered IgG-hexamers occurs on cell surfaces via specific interactions between Fc regions of the IgG monomers, which occur after antigen binding. The present study investigated the formation of IgG-hexamers in CLL patients and normal (non-malignant) controls (NC), their ability to activate complement, their incidence as cell-free and cell-bound forms and the identity of the antigen causing their formation. Sera from 30 patients and 12 NC were used for separation of IgG- aggregates. The obtained IgG- aggregates were measured and used for assessment of CP activation. For evaluation of the presence of IgG- aggregates on blood cells, whole blood samples were stained and assessed by flow cytometry. Serum levels of IgG- aggregates were higher in CLL and they activated the complement system to a higher extent than in NC. Alpha 2 macroglobulin (A2M) was identified as the antigen causing the hexamerization/aggregation of IgG, and was found to be part of the hexamer structure by mass spectrometry, Western blot and flow cytometry analysis. The presence of A2M-IgG-hexamers on B-cells suggests that it may be formed on B cells surface and then be detached to become cell-free. Alternatively, it may form in the plasma and then attach to the cell surface. The exact time course of A2M-IgG-hexamers formation in CLL should be further studied. The results in this study may be useful for improvement of current immunotherapy regimens.