Tandem Mass Tag-Labeled Quantitative Proteome Analyses Identify C1R and A2M as Novel Serum Biomarkers in Pregnant Women with Obstructive Sleep Apnea.

The aim of this study was to identify serum diagnostic biomarkers associated with the severity of obstructive sleep apnea (OSA) during pregnancy. Differentially expressed proteins (DEPs) were identified in the control (C), mild (O), and moderate (MO) OSA groups (n = 3 in each group). Bioinformatics analysis was conducted to identify the underlying functions, pathways, and networks of the proteins. Receiver operating characteristic curves were used to assess the diagnostic value of the identified DEPs. The enzyme-linked immunoassay was performed to detect serum levels of the complement C1r subcomponent (C1R) and alpha-2-macroglobulin (A2M) in 79 pregnant women with OSA (mild OSA [n = 32]; moderate OSA [n = 29], and severe OSA [n = 18]) and 65 healthy pregnant women without OSA. Pearson's correlation analysis was conducted to analyze the correlation between C1R and A2M levels and OSA clinicopathological factors. In total, 141 DEPs, 29 DEPs, and 103 DEPs were identified in the three groups (i.e., the mild OSA vs control group, the moderate OSA vs mild apnea group, and the moderate OSA vs control group, respectively). C1R and A2M were identified as continuously up-regulated proteins, and the levels of C1R and A2M were associated with OSA severity. C1R and A2M were found to be correlated with body mass index, systolic blood pressure, apnea-hypopnea index, oxygen desaturation index, time with saturation below 90%, and lowest SaO2. Adverse maternal and neonatal outcomes were observed in pregnant women with OSA. C1R and A2M have been identified as diagnostic biomarkers and are associated with the severity of OSA during pregnancy.

A2ML1 Inhibits Esophageal Squamous Cell Carcinoma Progression and Serves as a Novel Prognostic Biomarker.

Studies have established a correlation between α2-macroglobulin-like 1 (A2ML1) and the prognosis of lung, pancreatic, and breast cancers; however, research on its involvement in the pathogenesis of esophageal carcinoma remains limited. Therefore, in this study, we aimed to investigate the role of A2ML1 in the progression of esophageal squamous cell carcinoma (ESCC). Immunohistochemical staining was employed to assess the expression level of A2ML1 protein in both tumor and adjacent normal tissues of patients with ESCC. The Kaplan-Meier method, along with univariate and multivariate Cox risk ratio analyses, was used to determine survival rates and prognostic factors. Furthermore, two human ESCC cell lines, KYSE30 and KYSE150, were used to assess the effect of A2ML1 overexpression on cell proliferation and apoptosis. A human apoptosis antibody kit was also used to analyze the downstream action proteins of A2ML1, and a nude mouse xenotransplantation model was used to evaluate the effect of A2ML1 on ESCC tumorigenesis in vivo. The protein level of A2ML1 in ESCC tissues was significantly lower than that in normal esophageal tissues, and higher A2ML1 protein levels were associated with smaller ESCC tumor sizes and improved tumor-specific survival rates. Multivariate analysis established A2ML1 as a novel independent prognostic factor for ESCC. Moreover, A2ML1 overexpression significantly inhibited ESCC cell proliferation and promoted apoptosis. A2ML1 consistently inhibited tumor growth in mouse models. Furthermore, the human apoptotic antibody kit results showed increased expression of the proliferation-inhibiting protein p21 downstream of KYSE150 cells overexpressing A2ML1. Our findings demonstrate that a correlation exists between A2ML1 and ESCC prognosis and that A2ML1 plays an antitumor role in ESCC progression. This study underscores the potential of A2ML1 as a novel biomarker for predicting the prognosis of ESCC.

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.

A2M Serves as Promising Biomarker for Chronic Obstructive Pulmonary Disease.

Background: Chronic obstructive pulmonary disease (COPD) often associated with cigarette smoking. However, increasing evidence suggests that non-smoking COPD is much higher than previously thought. This study aims to identify a nonsmoking COPD biomarker and examined its value in diagnosis and prediction of acute exacerbation. Methods: A total of 35 stable COPD patients, 70 acute exacerbation chronic obstructive pulmonary disease (AECOPD) patients and 35 healthy control subjects were included. Plasma α 2 macroglobulin (A2M) and matrix metalloproteinase-9 (MMP-9) levels were measured using the enzyme-linked immunosorbent assay (ELISA) method on all participants. Their association with clinical characteristics and lung function parameters were determined by regression analysis. Receiver operating characteristic (ROC) curve was used to determine the diagnostic sensitivity and specificity. Correlation coefficients were evaluated using Pearson's correlation. Results: Plasma A2M concentration was decreased and MMP-9 concentration, MMP-9/A2M ratio were elevated in stable COPD patients compared with control groups. And MMP-9 expression was significantly higher in AECOPD patients. A2M level was increased in AECOPD patients with infection compared with those without. In addition, there was no statistical difference in A2M levels between smokers and nonsmokers COPD or healthy control subjects. Furthermore, A2M, MMP-9 and MMP-9/A2M were correlated with forced expiratory volume in one second (FEV1)%, FEV1/ forced vital capacity (FVC), CAT and mMRC score in COPD patients, but had no correlation with fraction of exhaled nitric oxide (FeNO) and concentration of alveolar nitric oxide (CaNO). Conclusion: A2M is altered in peripheral blood of COPD patients and correlated with severity and infection. Moreover, there was no significant correlation between the change in A2M and smoking, FeNO and CaNO, suggesting A2M may reflect the overall rather than local inflammation in COPD patients and serve as a potential biomarker for nonsmoking COPD patients.

Alpha-2-Macroglobulin (A2M) and Dopamine Receptor D2 (DRD2) expression analysis and Influence of Separation from Parents in Childhood on the Suicide and Self-injury Behavior and Psychological Adjustment in Adolescence.

To explore the influence of separation from parents in childhood on suicide and self-injury behavior and psychological adjustment in adolescence. A total of 880 subjects were selected, including 197 students who were separated from their parents in childhood and 683 students who were not separated from their parents in childhood. The scores of psychological resilience, self-compassion, forgiveness and suicide and self-injury were investigated and analyzed. Logistic regression analysis was made on the relationship between suicide and self-injury behavior and psychological adjustment in adolescence. The scores of psychological resilience, self-compassion, forgiveness and suicide and self-injury were statistically significant between children who were separated from their parents and those who were not separated. The students who were not separated had better psychological adjustment abilities and a lower rate of suicide and self-injury (P<0.05). There was a positive correlation between separation from parents in childhood and suicide and self-injury behavior and psychological adjustment in adolescence (P<0.05). The separation from parents in childhood is closely related to psychological resilience, forgiveness, self-compassion, and suicide-related psychological behavior and self-injury behavior in adolescence. Suicide and self-injury behavior can be reduced by reducing separation from parents in childhood and improving self-psychological adjustment ability in adolescence. During the past years, genetics, heritability, and genes' contribution to depression disorders have been well established. Alpha-2-Macroglobulin (A2M) and Dopamine Receptor D2 (DRD2) genes are very effective in behavioral and mood disorders. The results of this study showed the expression of these genes in different organs, especially in connection with the cerebrospinal system, so investigating the mechanism of their effect is very effective and promising, and it is hoped that they will be used in other research.

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.

Audiologic Measures in an Indigenous Community with A2ML1- and FUT2-Related Otitis Media.

Background: Many indigenous peoples are at elevated risk for otitis media, however there is limited information on hearing loss due to OM in these communities. An Indigenous Filipino community that has previously been described with an elevated prevalence of OM that is due to rare A2ML1 variants and a common FUT2 variant underwent additional phenological testing. In this study, we describe the audiologic profiles in A2ML1- and FUT2-related otitis media and the validity of otoscopy and genotyping for A2ML1 and FUT2 variants in screening for otitis media and hearing loss. Method: We analyzed A2ML1 and FUT2 genotypes together with demographic, otologic and audiologic data from tympanometry and hearing level assessments of 109 indigenous individuals. Results: We confirmed previous findings of a spectrum of nonsyndromic otitis media as associated with A2ML1 variants. A2ML1 and FUT2 variants were associated with high-frequency hearing loss at 4000 Hz. As expected, young age was associated with flat tympanograms, and eardrum perforations due to chronic otitis media were associated with severe-to-profound hearing loss across frequencies. Adding A2ML1 or FUT2 genotypes improved the validity of otoscopy as a screening test to rule out moderate-to-profound hearing loss. Conclusion: Continued multi-disciplinary management and audiologic follow-up using tympanometry and screening audiometry are needed to document and treat otitis media and prevent permanent hearing loss in the indigenous community.

A2M inhibits inflammatory mediators of chondrocytes by blocking IL-1ß/NF-κB pathway.

A hallmark of osteoarthritis (OA) is cartilage degeneration, which has been previously correlated with dramatic increases in inflammatory enzymes. Specifically, interleukin-1ß (IL-1ß) and subsequent upregulation of nuclear factor kappa B (NF-κB) is implicated as an important player in the development of posttraumatic osteoarthritis (PTOA). Alpha 2-macroglobulin (A2M) can inhibit this inflammatory pathway, making it a promising therapy for PTOA. Herein, we demonstrate that A2M binds and neutralizes IL-1ß, blocking downstream NF-κB-induced catabolism seen in in vitro. Human chondrocytes (cell line C28) were incubated with A2M protein and then treated with IL-1ß. A2M was labeled with VivoTag™ 680 to localize the protein postincubation. The degree of binding between A2M and IL-1ß was evaluated through immunoprecipitation (IP). Catabolic proteins, including IL-1ß and NF-kB, were detected by Western blot. Pro-inflammatory and chondrocyte-related gene expression was examined by qRT-PCR. VivoTag™ 680-labeled A2M was observed in the cytoplasm of C28 human chondrocytes by fluorescence microscopy. IP experiments demonstrated that A2M could bind IL-1ß. Additionally, western blot analysis revealed that A2M neutralized IL-1ß and NF-κB in a dose-dependent manner. Moreover, A2M decreased levels of MMPs and TNF-α and increased the expression of cartilage protective genes Col2, Type2, Smad4, and aggrecan. Mostly importantly, A2M was shown to directly neutralize IL-1ß to downregulate the pro-inflammatory responses mediated by the NF-kB pathway. These results demonstrate a mechanism by which A2M reduces inflammatory catabolic activity and protects cartilage after joint injury. Further in vivo studies are needed to fully understand the potential of A2M as a novel PTOA therapy.

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.

The conformational change of the protease inhibitor α2-macroglobulin is triggered by the retraction of the cleaved bait region from a central channel.

The protease inhibitor α2-macroglobulin (A2M) is a member of the ancient α2-macroglobulin superfamily (A2MF), which also includes structurally related proteins, such as complement factor C3. A2M and other A2MF proteins undergo an extensive conformational change upon cleavage of their bait region by proteases. However, the mechanism whereby cleavage triggers the change has not yet been determined. We have previously shown that A2M remains functional after completely replacing its bait region with glycine and serine residues. Here, we use this tabula rasa bait region to investigate several hypotheses for the triggering mechanism. When tabula rasa bait regions containing disulfide loops were elongated by reducing the disulfides, we found that A2M remained in its native conformation. In addition, cleavage within a disulfide loop did not trigger the conformational change until after the disulfide was reduced, indicating that the introduction of discontinuity into the bait region is essential to the trigger. Previously, A2MF structures have shown that the C-terminal end of the bait region (a.k.a. the N-terminal region of the truncated α chain) threads through a central channel in native A2MF proteins. Bait region cleavage abolishes this plug-in-channel arrangement, as the bait region retracts from the channel and the channel itself collapses. We found that mutagenesis of conserved plug-in-channel residues disrupted the formation of native A2M. These results provide experimental evidence for a structural hypothesis in which retraction of the bait region from this channel following cleavage and the channel's subsequent collapse triggers the conformational change of A2M and other A2MF proteins.

Cryo-EM structures reveal the dynamic transformation of human alpha-2-macroglobulin working as a protease inhibitor.

Human alpha-2-macroglobulin is a well-known inhibitor of a broad spectrum of proteases and plays important roles in immunity, inflammation, and infections. Here, we report the cryo-EM structures of human alpha-2-macroglobulin in its native state, induced state transformed by its authentic substrate, human trypsin, and serial intermediate states between the native and fully induced states. These structures exhibit distinct conformations, which reveal the dynamic transformation of alpha-2-macro-globulin that acts as a protease inhibitor. The results shed light on the molecular mechanism of alpha-2-macroglobulin in entrapping substrates.

Characterization and expression profiling of serine protease inhibitors in the yellow mealworm Tenebrio molitor.

Serine protease inhibitors (SPIs) act in diverse biological processes in insects such as immunity, development, and digestion by preventing the unwanted proteolysis. So far, the repertoire of genes encoding SPIs has been identified from few insect species. In this study, 62 SPI genes were identified from the genome of the yellow mealworm, Tenebrio molitor. According to their modes of action, they were classified into three families, serpin (26), canonical SPI (31), and α-macroglobulins (A2M) (5). These SPIs feature eight domains including serpin, Kazal, TIL, Kunitz, WAP, Antistasin, pacifastin, and A2M. In total, 39 SPIs contain a single SPI domain, while the others encode at least two inhibitor units. Based on the amino acids in the cleaved reactive sites, the abilities of these SPIs to inhibit trypsin, chymotrypsin, or elastase-like enzymes are predicted. The expression profiling based on the RNA-seq data showed that these genes displayed stage-specific expression patterns during development, suggesting to us their significance in development. Some of the SPI genes were exclusively expressed in particular tissues such as hemocyte, fat body, gut, ovary, and testis, which may be involved in biological processes specific to the indicated tissues. These findings provide necessary information for further investigation of insect SPIs.

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.

Cryo-EM structures of human A2ML1 elucidate the protease-inhibitory mechanism of the A2M family.

A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory mechanism of human A2ML1 and determine the structures of its native and protease-cleaved conformations. The functional inhibitory unit of A2ML1 is a monomer that depends on covalent binding of the protease (mediated by A2ML1's thioester) to achieve inhibition. In contrast to the A2M tetramer which traps proteases in two internal chambers formed by four subunits, in protease-cleaved monomeric A2ML1 disordered regions surround the trapped protease and may prevent substrate access. In native A2ML1, the bait region is threaded through a hydrophobic channel, suggesting that disruption of this arrangement by bait region cleavage triggers the extensive conformational changes that result in protease inhibition. Structural comparisons with complement C3/C4 suggest that the A2M superfamily of proteins share this mechanism for the triggering of conformational change occurring upon proteolytic activation.

A2M is a potential core gene in intrahepatic cholangiocarcinoma.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is a type of malignant tumor ranking the second in the incidence of primary liver cancer following hepatocellular carcinoma. Both the morbidity and mortality have been increasing in recent years. Small duct type of ICC has potential therapeutic targets. But overall, the prognosis of patients with ICC is usually very poor. METHODS: To search latent therapeutic targets for ICC, we programmatically selected the five most suitable microarray datasets. Then, we made an analysis of these microarray datasets (GSE26566, GSE31370, GSE32958, GSE45001 and GSE76311) collected from the Gene Expression Omnibus (GEO) database. The GEO2R tool was effective to find out differentially expressed genes (DEGs) between ICC and normal tissue. Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were executed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v 6.8. The Search Tool for the Retrieval of Interacting Genes (STRING) database was used to analyze protein-protein interaction of these DEGs and protein-protein interaction of these DEGs was modified by Cytoscape3.8.2. Survival analysis was performed using Gene Expression Profiling Interactive Analysis (GEPIA) online analysis tool. RESULTS: A total of 28 upregulated DEGs and 118 downregulated DEGs were screened out. Then twenty hub genes were selected according to the connectivity degree. The survival analysis results showed that A2M was closely related to the pathogenesis and prognosis of ICC and was a potential therapeutic target for ICC. CONCLUSIONS: According to our study, low A2M expression in ICC compared to normal bile duct tissue was an adverse prognostic factor in ICC patients. The value of A2M in the treatment of ICC needs to be further studied.