Insight into the molecular interaction between the anticancer drug, enzalutamide and human alpha-2-macroglobulin: Biochemical and biophysical approach.

The drug pharmacokinetics is affected upon binding with proteins, thus making drug-protein interactions crucial. This study investigated the interaction between enzalutamide and human major antiproteinase alpha-2-macroglobulin (α2M) by using multi spectroscopic and calorimetric techniques. The spectroscopic techniques such as circular dichroism (CD), intrinsic fluorescence, and UV-visible absorption were used to determine the mechanism of enzalutamide-α2M interaction. Studies on the quenching of fluorescence at three different temperatures showed that the enzalutamide-α2M complex is formed through static quenching mechanism. The change in microenvironment around tyrosine residues in protein was detected through synchronised fluorescence. The secondary structure of α2M was slightly altered by enzalutamide according to far UV-CD spectral analysis. Changes in position of amide I band in FTIR spectra further confirm the secondary structural alteration in α2M. According to thermodynamic characteristics such as fluorescence quenching and isothermal titration calorimetry (ITC), hydrogen bonds and hydrophobic interactions were involved in the interaction machanism. The ITC reiterated the exothermic and spontaneous nature of the interaction. The lower proteinase inhibitory activity of the α2M-enzalutamide conjugate as reflects the disruption of the native α2M structure upon interaction with enzalutamide.

Probing the binding of morin with alpha-2-macroglobulin using multi-spectroscopic and molecular docking approach : Interaction of morin with α2M.

Alpha-2-macroglobulin (α2M) 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 α2M 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 α2M upon interaction with morin. Fluorescence quenching tests unequivocally confirmed quenching in the fluorescence of α2M in the presence of morin, conforming complex formation and demonstrating that the binding mechanism involves a dynamic mode of interaction. Synchronous fluorescence spectra of α2M 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 α2M 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 α2M at 298 K with a binding constant of 2.7 × 104 M-1, indicating the strength of the association. The α2M-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.

Exploring the interaction of myricetin with human alpha-2-macroglobulin: biophysical and in-silico analysis.

Myricetin (MYR) is a bioactive secondary metabolite found in plants that is recognized for its nutraceutical value and is an essential constituent of various foods and beverages. It is reported to exhibit a plethora of activities, including antioxidant, antimicrobial, antidiabetic, anticancer, and anti-inflammatory. Alpha-2-macroglobulin (α2M) is a major plasma anti-proteinase that can inhibit proteinases of both human and non-human origin, regardless of their specificity and catalytic mechanism. Here, we explored the interaction of MYR-α2M using various biochemical and biophysical techniques. It was found that the interaction of MYR brings subtle change in its anti-proteolytic potential and thereby alters its structure and function, as can be seen from absorbance and fluorescence spectroscopy. UV spectroscopy of α2M in presence of MYR indicated the occurrence of hyperchromism, suggesting complex formation. Fluorescence spectroscopy reveals that MYR reduces the fluorescence intensity of native α2M with a shift in the wavelength maxima. At 318.15 K, MYR binds to α2M with a binding constant of 2.4 × 103 M-1, which indicates significant binding. The ΔG value was found to be - 7.56 kcal mol-1 at 298.15 K, suggesting the interaction to be spontaneous and thermodynamically favorable. The secondary structure of α2M does not involve any major change as was confirmed by CD analysis. The molecular docking indicates that Asp-146, Ser-172, Glu-174, and Tyr-180 were the key residues involved in α2M-MYR complex formation. This study contributes to our understanding of the function and mechanism of protein and flavonoid binding by providing a molecular basis of the interaction between MYR and α2M.

Comprehensive insight into the molecular interaction of an anticancer drug-ifosfamide with human alpha-2-macroglobulin: biophysical and in silico studies.

Ifosfamide is an active alkylating chemotherapeutic drug chemically related to nitrogen mustard. The pharmacokinetics of drugs is affected upon binding with protein, making the studies on drug-protein interaction promising. The present study investigates the interaction between ifosfamide and human antiproteinase-alpha-2-macroglobulin (α2M) by using multi-spectroscopic and in silico techniques. The UV-visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods were employed to unveil the mode and mechanism of ifosfamide-α2M interaction. Fluorescence quenching studies performed at three different temperatures indicated that ifosfamide-α2M complex formation involves static quenching. Far UV-CD spectra revealed a minor alteration in the secondary structure of α2M instigated by ifosfamide. The thermodynamic parameters determined by fluorescence quenching experiment and isothermal titration calorimetry (ITC) suggested that the complex between ifosfamide and α2M involves hydrogen bonding and hydrophobic interactions. Molecular docking illustrates that ifosfamide binds with moderate affinity to Lys1240, Asn173, Ser957, Leu955, Asp953, Lys1216 and Thr1236 residues during the interaction. Molecular dynamic (MD) simulation suggested that the ifosfamide forms a stable complex with α2M. Communicated by Ramaswamy H. Sarma.

Characterization of the binding between anti-tumor drug 5-fluorouracil and human alpha-2-macroglobulin: spectroscopic and molecular docking analyses.

5-Fluorouracil (5-FU) is a well-recognized anticancer drug used in the treatment of tumors of head, neck and breast. Drug pharmacokinetics is affected upon binding with protein, thus, making drug-protein interactions imperative to study. Present work investigates the interaction between 5-FU and human major antiproteinase-alpha-2-macroglobulin (α2M) by multi-spectroscopic, calorimetric and molecular docking techniques. UV/Visible absorption, intrinsic fluorescence and circular dichroism (CD) spectroscopic methods have been employed to unveil the mode and mechanism of 5-FU-α2M interaction. Synchronous fluorescence showed alteration in the microenvironment of tryptophan and tyrosine residues of protein. Far UV-CD spectra suggest slight alterations in the secondary structure of α2M by 5-FU. Thermodynamic parameters determined by fluorescence quenching experiments and isothermal titration calorimetry (ITC) suggested the involvement of hydrogen bonds and hydrophobic interactions. Moreover, ITC corroborate the spontaneous and exothermic nature of the interaction process. Molecular docking illustrates that 5-FU binds with moderate affinity and Asp953, Tyr1264, Lys1236, Thr1232, Tyr1323 and Leu951 were the main residues involved. Molecular dynamics simulation studies suggested that 5-FU was stabilizing the α2M structure and forming a stable complex. It was concluded that 5-FU lower the antiproteolytic activity of α2M significantly and causes disruption in the native structure and conformation of α2M.Communicated by Ramaswamy H. Sarma.

A mass spectrometric approach to study the interaction of amyloid ß peptides with human α-2-macroglobulin.

We used MALDI-MS to study the interaction of amyloid ß (Aß) peptides with alpha-2-macroglobulin (α2M). The binding of amyloid beta (Aß) peptides to alpha-2-macroglobulin (α2M) was found to inhibit the ability of trypsin to cleave out the peptide α2M 705-715 (Pep-α2M) from α2M. This was observed with both purified α2M and α2M in human serum. We found that Aß 1-38, Aß1-40, and Aß 1-42, all inhibit the interaction of α2M with trypsin, with inhibition rate independent of the length of the Aß peptide. Further, we show that for complete inhibition, two peptide molecules must be attached to one α2M molecule; one for each of its two subunits. A region was revealed within the Aß sequence, in which proteolytic cleavage (Lys-28) and oxidation (Met-35) lead to a loss of their ability to inhibit the interaction of trypsin with α2M. Furthermore, we show that after the formation of a trypsin complex with α2M and cleavage of α2M to produce the α2M 705-715, Aß peptides continue to bind to the protein in the same proportions. However, Aß peptides treated with DMSO lost their ability to bind to α2M and thereby to inhibit the interaction of trypsin with α2M. While maintaining their primary structure, such an effect can be explained only by conformational changes in the peptides, suggesting the possibility to use our analytical approach to distinguish between conformational isomers of Aß peptides.

Induced forms of α2-macroglobulin neutralize heparin and direct oral anticoagulant effects.

Alpha2-macroglobulin (α2M) is a physiological macromolecule that facilitates the clearance of many proteinases, cytokines and growth factors in human. Here, we explored the effect of induced forms of α2M on anticoagulant drugs. Gla-domainless factor Xa (GDFXa) and methylamine (MA)-induced α2M were prepared and characterized by electrophoresis, immunonephelometry, chromogenic, clot waveform and rotational thromboelastometry assays. Samples from healthy volunteers and anticoagulated patients were included. In vivo neutralization of anticoagulants was evaluated in C57Bl/6JRj mouse bleeding-model. Anticoagulant binding sites on induced α2M were depicted by computer-aided energy minimization modeling. GDFXa-induced α2M neutralized dabigatran and heparins in plasma and whole blood. In mice, a single IV dose of GDFXa-induced α2M following anticoagulant administration significantly reduced blood loss and bleeding time. Being far easier to prepare, we investigated the efficacy of MA-induced α2M. It neutralized rivaroxaban, apixaban, dabigatran and heparins in spiked samples in a concentration-dependent manner and in samples from treated patients. Molecular docking analysis evidenced the ability of MA-induced α2M to bind non-covalently these compounds via some deeply buried binding sites. Induced forms of α2M have the potential to neutralize direct oral anticoagulants and heparins, and might be developed as a universal antidote in case of major bleeding or urgent surgery.

Development of selective protease inhibitors via engineering of the bait region of human α2-macroglobulin.

Human α2-macroglobulin (A2M) is an abundant protease inhibitor in plasma, which regulates many proteolytic processes and is involved in innate immunity. A2M's unique protease-trapping mechanism of inhibition is initiated when a protease cleaves within the exposed and highly susceptible "bait region." As the wild-type bait region is permissive to cleavage by most human proteases, A2M is accordingly a broad-spectrum protease inhibitor. In this study, we extensively modified the bait region in order to identify any potential functionally important elements in the bait region sequence and to engineer A2M proteins with restrictive bait regions, which more selectively inhibit a target protease. A2M in which the bait region was entirely replaced by glycine-serine repeats remained fully functional and was not cleaved by any tested protease. Therefore, this bait region was designated as the "tabula rasa" bait region and used as the starting point for further bait region engineering. Cleavage of the tabula rasa bait region by specific proteases was conveyed by the insertion of appropriate substrate sequences, e.g., basic residues for trypsin. Screening and optimization of tabula rasa bait regions incorporating matrix metalloprotease 2 (MMP2) substrate sequences produced an A2M that was specifically cleaved by MMPs and inhibited MMP2 cleavage activity as efficiently as wild-type A2M. We propose that this approach can be used to develop A2M-based protease inhibitors, which selectively inhibit target proteases, which might be applied toward the clinical inhibition of dysregulated proteolysis as occurs in arthritis and many types of cancer.

Interaction of Human Alpha-2-Macroglobulin with Pesticide Aldicarb Using Spectroscopy and Molecular Docking.

BACKGROUND: Aldicarb is a carbamate pesticide commercially used in potato crop production. Once it enters human body, it interacts with diverse proteins and other substances. OBJECTIVE: Aldicarb is toxic to human health and it is also a cholinesterase inhibitor, which prevents the breakdown of acetylcholine in synapse. Human alpha-2-macroglobulin (α2M), is a large tetrameric glycoprotein of 720 kDa with antiproteinase activity, found abundantly in plasma. METHODS: In the present study, the interaction of aldicarb with alpha-2-macroglobulin was explored utilizing various spectroscopic techniques and molecular docking studies. RESULTS: UV-vis and fluorescence spectroscopy suggests the formation of a complex between aldicarb and α2M apparent by increased absorbance and decreased fluorescence with static quenching mode. CD spectroscopy indicates a slight change in the structure of alpha-2-macroglobulin. Docking studies confirm the interaction of aldicarb with Pro- 1391, Leu-1392, Lys-1393, Val-1396, Lys- 1397, Thr-1408, Glu-1409, Val-1410, Asp-282 and Glu-281 in the receptor binding domain at the C-terminal of the alpha 2 macroglobulin. DISCUSSION: In this work, aldicarb is shown to bind with alpha 2-macroglobulin at receptor binding domain which is the binding site for various extracellular and intracellular ligand too. Also, affecting the functional activity of the protein may lead to further physiological consequences. CONCLUSION: It is possible that aldicarb binds and compromises antiproteinase activity of α2M and binding properties by inducing changes in the secondary structure of the protein.

Interaction of organophosphate pesticide chlorpyrifos with alpha-2-macroglobulin: Biophysical and molecular docking approach.

Organophosphate class of pesticides causes neurotoxicity and carcinogenicity in humans. Once inside the human body, these pesticides often interact with plasma proteins, such as alpha-2-macroglobulin (α2M) which is the key anti-proteinase. Our work focuses on the structural and functional alteration of α2M by chlorpyrifos (CPF), a member of organophosphates. We explored the binding interaction between alpha-2-macroglobulin and CPF by using UV absorption and fluorescence spectroscopy (steady state and synchronous), circular dichroism and molecular docking approach. The functional activity of α2M was analyzed by anti-proteinase trypsin inhibitory assay which showed dose-dependent decrease in alpha-2-macroglobulin antiproteolytic potential. UV absorption studies and fluorescence quenching experiments suggested the formation of a complex between α2M and CPF. The CD spectra suggested a reduction in the beta helical (ß helix) content of α2M. Analysis of thermodynamic parameters suggested the process is spontaneous and endothermic with the ΔG and ΔH values being -5.501 kJ/mol, 11.49 kJ/mol, respectively. CPF binds with Ile-1390, Pro-1391, Leu-1392, Lys-1393, Val-1396, Lys-1397, Arg-1407, Thr-1408, Glu-1409, Val-1410, Asp-282, Glu-281 of α2M as suggested by molecular docking.

Bilirubin binding affects the structure and function of alpha-2-macroglobulin.

Bilirubin is an endogenous antioxidant that is a metabolite of the heme in red blood cells (RBC). In blood, bilirubin is associated with albumin to form a water-soluble complex, known as unconjugated bilirubin. Alpha-2-macroglobulin (α2M) is a proteinase inhibitor found in the plasma of vertebrates. In the present study, we have investigated the interaction of photo-illuminated bilirubin with serum α2M using various biophysical and thermodynamic techniques. The binding of bilirubin to α2M leads to various functional and structural changes in α2M protein. The result of ultraviolet (UV) and fluorescence spectroscopy suggests that binding of bilirubin to α2M induces a conformational change in the secondary structure of protein which was corroborated by circular dichroism (CD) and Fourier-transform infrared spectroscopy (FT-IR). This binding leads to the conversion of ß-sheet into α-helical conformation and subsequently loss in protein activity. The thermodynamic parameters of bilirubin-α2M binding indicate that the binding is exothermic, and the reaction spontaneous. Our studies show that binding of bilirubin with α2M in the presence of light induces structural and functional modifications in the protein. Bilirubin possesses multiple biological activities, including immunomodulatory property which has not been extensively explored and which may be of interest for further study.

Fibrinogen-clotting enzyme, pictobin, from Bothrops pictus snake venom. Structural and functional characterization.

A thrombin-like enzyme, pictobin, was purified from Bothrops pictus snake venom. It is a 41-kDa monomeric glycoprotein as showed by mass spectrometry and contains approx. 45% carbohydrate by mass which could be removed with N-glycosidase. Pictobin coagulates plasma and fibrinogen, releasing fibrinopeptide A and induces the formation of a friable/porous fibrin network as visualized by SEM. The enzyme promoted platelet aggregation in human PRP and defibrination in mouse model and showed catalytic activity on chromogenic substrates S-2266, S-2366, S-2160 and S-2238. Pictobin interacts with the plasma inhibitor α2-macroglobulin, which blocks its interaction with fibrinogen but not with the small substrate BApNA. Heparin does not affect its enzymatic activity. Pictobin cross reacted with polyvalent bothropic antivenom, and its deglycosylated form reduced its catalytic action and antivenom reaction. In breast and lung cancer cells, pictobin inhibits the fibronectin-stimulated migration. Moreover, it produces strong NADH oxidation, mitochondrial depolarization, ATP decrease and fragmentation of mitochondrial network. These results suggest by first time that a snake venom serinprotease produces mitochondrial dysfunction by affecting mitochondrial dynamics and bioenergetics. Structural model of pictobin reveals a conserved chymotrypsin fold ß/ß hydrolase. These data indicate that pictobin has therapeutic potential in the treatment of cardiovascular disorders and metastatic disease.

Human proteinase 3 resistance to inhibition extends to alpha-2 macroglobulin.

Polymorphonuclear neutrophils contain at least four serine endopeptidases, namely neutrophil elastase (NE), proteinase 3 (PR3), cathepsin G (CatG), and NSP4, which contribute to the regulation of infection and of inflammatory processes. In physiological conditions, endogenous inhibitors including α2-macroglobulin (α2-M), serpins [α1-proteinase inhibitor (α1-PI)], monocyte neutrophil elastase inhibitor (MNEI), α1-antichymotrypsin, and locally produced chelonianins (elafin, SLPI) control excessive proteolytic activity of neutrophilic serine proteinases. In contrast to human NE (hNE), hPR3 is weakly inhibited by α1-PI and MNEI but not by SLPI. α2-M is a large spectrum inhibitor that traps a variety of proteinases in response to cleavage(s) in its bait region. We report here that α2-M was more rapidly processed by hNE than hPR3 or hCatG. This was confirmed by the observation that the association between α2-M and hPR3 is governed by a kass in the ≤ 105  m-1 ·s-1 range. Since α2-M-trapped proteinases retain peptidase activity, we first predicted the putative cleavage sites within the α2-M bait region (residues 690-728) using kinetic and molecular modeling approaches. We then identified by mass spectrum analysis the cleavage sites of hPR3 in a synthetic peptide spanning the 39-residue bait region of α2-M (39pep-α2-M). Since the 39pep-α2-M peptide and the corresponding bait area in the whole protein do not contain sequences with a high probability of specific cleavage by hPR3 and were indeed only slowly cleaved by hPR3, it can be concluded that α2-M is a poor inhibitor of hPR3. The resistance of hPR3 to inhibition by endogenous inhibitors explains at least in part its role in tissue injury during chronic inflammatory diseases and its well-recognized function of major target autoantigen in granulomatosis with polyangiitis.

Influence of Ascorbic Acid on the Structure and Function of Alpha-2- macroglobulin: Investigations using Spectroscopic and Thermodynamic Techniques.

BACKGROUND: Ascorbic acid is a classic dietary antioxidant which plays an important role in the body of human beings. It is commonly found in various foods as well as taken as dietary supplement. OBJECTIVE: The plasma ascorbic acid concentration may range from low, as in chronic or acute oxidative stress to high if delivered intravenously during cancer treatment. Sheep alpha-2- macroglobulin (α2M), a human α2M homologue is a large tetrameric glycoprotein of 630 kDa with antiproteinase activity, found in sheep's blood. METHODS: In the present study, the interaction of ascorbic acid with alpha-2-macroglobulin was explored in the presence of visible light by utilizing various spectroscopic techniques and isothermal titration calorimetry (ITC). RESULTS: UV-vis and fluorescence spectroscopy suggests the formation of a complex between ascorbic acid and α2M apparent by increased absorbance and decreased fluorescence. Secondary structural changes in the α2M were investigated by CD and FT-IR spectroscopy. Our findings suggest the induction of subtle conformational changes in α2M induced by ascorbic acid. Thermodynamics signatures of ascorbic acid and α2M interaction indicate that the binding is an enthalpy-driven process. CONCLUSION: It is possible that ascorbic acid binds and compromises antiproteinase activity of α2M by inducing changes in the secondary structure of the protein.

Molecular form and concentration of serum α2-macroglobulin in diabetes.

α2-Macroglobulin is a highly abundant serum protein involved in the development of atherosclerosis and cardiac hypertrophy. However, its circulating molecular form and exact concentrations in human health/diseases are not known. Blue native-polyacrylamide gel electrophoresis of human serum was used to confirm the native conformation of α2-macroglobulin. We created an enzyme-linked immunosorbent assay suitable for quantifying its circulating molecular form and undertook a cross-sectional study to measure its serum levels in 248 patients with diabetes mellitus and 59 healthy volunteers. The predominant circulating molecular form of α2-macroglobulin was the tetramer, whereas its dimer was detectable in patients with high serum levels of α2-macroglobulin. The serum α2-macroglobulin concentration was not associated with glycated hemoglobin or any other glycemic variable as evaluated from 48-h continuous glucose monitoring, but showed close correlation with left ventricular posterior wall thickness, carotid artery intima-media thickness, urinary albumin:creatinine ratio (ACR) and brachial-ankle pulse wave velocity (baPWV). Multivariate analysis revealed only the ACR and baPWV to be independent variables influencing serum levels of α2-macroglobulin. Thus, an increased ACR and baPWV are associated with higher serum concentrations of α2-macroglobulin, and the latter may contribute to the mechanism by which albuminuria increases the risk of developing cardiovascular diseases.

Alpha-2-Macroglobulin, a Hypochlorite-Regulated Chaperone and Immune System Modulator.

Alpha-macroglobulins are ancient proteins that include monomeric, dimeric, and tetrameric family members. In humans, and many other mammals, the predominant alpha-macroglobulin is alpha-2-macroglobulin (α 2M), a tetrameric protein that is constitutively abundant in biological fluids (e.g., blood plasma, cerebral spinal fluid, synovial fluid, ocular fluid, and interstitial fluid). α 2M is best known for its remarkable ability to inhibit a broad spectrum of proteases, but the full gamut of its activities affects diverse biological processes. For example, α 2M can stabilise and facilitate the clearance of the Alzheimer's disease-associated amyloid beta (Aß) peptide. Additionally, α 2M can influence the signalling of cytokines and growth factors including neurotrophins. The results of several studies support the idea that the functions of α 2M are uniquely regulated by hypochlorite, an oxidant that is generated during inflammation, which induces the native α 2M tetramer to dissociate into dimers. This review will discuss the evidence for hypochlorite-induced regulation of α 2M and the possible implications of this in neuroinflammation and neurodegeneration.

Deciphering the binding of dutasteride with human alpha-2-macroglobulin: Molecular docking and calorimetric approach.

Dutasteride is a pharmacologically important drug employed to treat prostate cancer. Alpha-2-macroglobulin (α2M) is the primary proteinase inhibitor and is abundant in vertebrate plasma. Previous studies have shown that α2M levels were down regulated in prostate cancer. Our results of functional assay shows 50% decrease in the antiproteolytic potential ofα2Mupon its interaction with dutasteride. Fluorescence quenching revealed that dutasteride binds with α2M via static mechanism, resulting in the formation of dutasteride-α2M complex. Synchronous fluorescence studies suggest alteration in the microenvironment around tryptophan residues. Changes in the UV-visible spectra hints at formation of complex between the drug and protein. Secondary structural perturbations in α2M are confirmed by circular dichroism studies. Molecular docking discloses the involvement of hydrogen bonding during the interaction process and suggests the site of interaction of dutasteride on α2M monomer as Asn173, Lys171, Asp1178, Lys1236, His1182, Lys1177, Ser1180 and Lys1240.Isothermal titration calorimetry affirms the binding process to be spontaneous and exothermic. The results of this study may potentially be important should it be shown that dutasteride interacts with α2M under physiological conditions.

Two alpha-2 macroglobulin from Portunus trituberculatus involved in the prophenoloxidase system, phagocytosis and regulation of antimicrobial peptides.

Alpha-2 macroglobulin (A2M) is a ubiquitous protease inhibitor involved in the innate host defense system. Herein, two distinct A2M genes (designated as PtA2M-1 and PtA2M-2, respectively) were isolated from the swimming crab Portunus trituberculatus. PtA2M-1 and PtA2M-2 encoded proteins with 1541 or 1516 amino acids, respectively, containing the typically functional domains of A2M. Unlike highly expressed in hemocytes of most arthropods, PtA2M-1 and PtA2M-2 were predominantly detected in gill, eyestalk and digestive tracks. During the embryonic stages, PtA2Ms were found to be expressed most highly in fertilized eggs, suggesting their maternal origin. After challenged with Vibrio alginolyticus, the transcripts of PtA2Ms showed similar time-dependent response expression pattern, while PtA2M-1 was more sensitive to Micrococcus luteus and Pichia pastoris infection than PtA2M-2. Knockdown of PtA2M-1 or PtA2M-2 could significantly enhance the expression of prophenoloxidase (proPO) associated genes (PtproPO and PtPPAF) and serine protease related genes (PtcSP1-3 and PtSPH), however, PtLSZ and the phagocytosis-related genes (PtMyosin and PtRab5) were effectively inhibited. These results were further supported by the PO and lysozyme activities in hemolymph of the PtA2M-1- or PtA2M-2-silenced crabs. In addition, PtA2M-1 and PtA2M-2 could regulate the expression of antimicrobial peptide (AMP) genes (PtALF1-3, PtCrustin1 and PtCrustin3) through the Toll and NF-κB pathways. Our findings together suggest that PtA2Ms might function in crab host defense via regulating the proPO system, phagocytosis and the expression of AMP genes.

Biophysical analysis of interaction between curcumin and alpha-2-macroglobulin.

Alpha-2-macroglobulin (α2M) is large glycoprotein present in the body fluids of vertebrates. It is an antiproteinase that inhibits a broad spectrum of proteases without the direct blockage of the protease active site. Curcumin, a yellow spice commonly used in India and several Asian countries, is reported to have anti-tumor and anti-inflammatory effects because of its antioxidant properties. In the present study, we have explored the interaction of curcumin with α2M using various technique such as antiproteinase activity assay, spectroscopy. Changes in the secondary structure of α2M following interaction with curcumin was investigated by CD and FT-IR spectroscopy. Thermodynamics of curcumin-α2M binding were also analyzed by isothermal titration calorimetry to identify the number of binding sites, changes in enthalpy, entropy and Gibbs free energy changes for this interaction. Thermodynamics parameters reveal that the binding is exothermic in nature. Our results suggest that the binding of curcumin with α2M induces a conformational change in the native form of protein that compromises its anti-proteinase activity. This exothermic and spontaneous interaction leads to alteration in the ß-sheet content of the protein leading to subtle changes in conformational status of the protein leading possibly to loss in the antiproteinase potential of the inhibitor.

Serum leucine-rich α2-glycoprotein is elevated in patients with systemic lupus erythematosus and correlates with disease activity.

BACKGROUND: We evaluated whether serum leucine-rich α2-glycoprotein (LRG) is associated with disease activity in patients with systemic lupus erythematosus (SLE). METHODS: We measured serum LRG in 194 SLE patients. SLE disease activity index-2000 (SLEDAI-2 K) was used to assess SLE activity, and patients with SLEDAI-2 K ≥5 were defined as having active SLE. Correlation between serum LRG, SLEDAI-2 K, and laboratory variables was estimated by Pearson's correlation analysis. The optimal serum LRG cut-off value for predicting active SLE was calculated using receiver operator characteristic (ROC) curve, and multivariable logistic regression was used to determine the odds ratio (OR) of laboratory variables. RESULTS: In total, 74 (38.1%) and 120 (61.9%) patients were classified as active and inactive SLE, respectively. Serum LRG was higher in patients with active SLE than in inactive SLE and healthy controls (26.6 vs. 14.4 vs. 1.2 ng/ml, p < .001). Serum LRG significantly correlated with SLEDAI-2 K (r = 0.340, p < .001) and laboratory variables. ROC analysis revealed that optimal serum LRG cut-off value for active SLE was >45.7 ng/ml. In multivariable logistic regression analysis, serum LRG >45.7 ng/ml (OR 4.089, 95% confidence interval 1.351, 12.376, p = .013) was an independent predictor of active SLE. CONCLUSIONS: Serum LRG might be a biomarker for estimating SLE disease activity.