Modification Of Human Serum Albumin Research Articles

Quantitative Assessment of the Post-translational Modifications of Human Serum Albumin by Dimethyl Trisulfide.

Some bacteria, such as Fusobacterium nucleatum, act as dimethyl trisulfide (DMTS) producers in the host in vivo. DMTS acts as a sulfane sulfur donor and chemically modifies the sulfhydryl groups. This study explored the post-translational modifications of human serum albumin using DMTS. Quantitative assessments were conducted on mixed disulfides of mercaptoalbumin with mercaptomethane (Alb-SS-CH3) and albumin hydropersulfide (Alb-SSH) as post-translationally modified species. The hydropersulfide group was alkylated with iodoacetamide, resulting in the formation of an albumin-mercaptoacetamide mixed disulfide. The mixed disulfides were subsequently reduced with tris(2-carboxyethyl)phosphine, and the liberated mercaptomethane and mercaptoacetamide were fluorescently labeled with 4-fluoro-7-sulfamoylbenzofurazan (ABD-F). Quantification was performed using HPLC with fluorescence detection. Using this methodology, we examined the formation of Alb-SS-CH3 and Alb-SSH via the reaction between 4% human serum albumin and DMTS at 10-100 µM concentrations. Approximately two molecules of Alb-SS-CH3 and one molecule of Alb-SSH were generated from one DMTS molecule. Moreover, hydrogen sulfide was identified as an intermediate, suggesting its generation and subsequent reaction with intraprotein disulfide bonds, leading to the production of Alb-SSH. These results suggest the production of DMTS in humans in vivo should be involved in the elevation of Alb-SS-CH3 and Alb-SSH contents in plasma samples.

Comparative Analysis of Post-Translational Modifications of Human Serum Albumin Derived from Human Plasma and Recombinant Sources in China.

The variations in sequence, three-dimensional structure, and post-translational modifications (PTMs) of human serum albumin (HSA) are crucial for its physiological functions. This study aims to analyze and compare the disparities in PTMs between HSA derived from human plasma and genetically recombinant sources for clinical treatments in China. Six distinct PTMs, namely acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation, were identified using pan-specific antibodies via Western blot analysis. The samples, comprising human plasma-derived HSA (pHSA) from six different manufacturers and recombinant HSA (rHSA) expressed in yeast and Oryza sativa, underwent detection for various types of PTMs. Additionally, a 4D label-free quantitative proteomic analysis was performed to identify N-glycosylation and the aforementioned PTMs in both pHSA and rHSA samples. This analysis aimed to discern disparities in modification sites and levels. Through Western blot analysis, all six pHSA and two rHSA samples displayed positive bands for albumin (66.5 kDa) across the six PTMs. Subsequent analysis using 4D label-free quantitative proteomics revealed 25 (29) acetylated, 30 (32) succinylated, 41 (50) malonylated, 15 (23) phosphorylated, 36 (30) beta-hydroxybutyrylated, and 27 (34) lactylated modification sites in pHSA and rHSA samples, with no N-glycosylation modification sites detected. The analysis identified 1 acetylation (ALB_K160), 2 beta-hydroxybutyrylation (ALB_K569, ALB_K426), and 3 crotonylation (ALB_K264, ALB_K581, ALB_K560) specific modification sites in pHSA, as well as 3 crotonylation (ALB_K560, ALB_K562, ALB_K75), 1 succinylation (ALB_K490), and 23 phosphorylation specific modification sites in rHSA. In pHSA (rHSA), 2 (6) acetylation, 10 (12) succinylation, 0 (9) crotonylation, 1 (9) phosphorylation, 6 (0) beta-hydroxybutyrylation, and 0 (7) lactylation specific modification sites were found. Moreover, in the shared modification sites between pHSA and rHSA, pHSA exhibited up-regulation of amberylation (16:1) and beta-hydroxybutyrylation (12:2) in more sites, and up-regulation of acetylation (7:11), crotonylation (2:11), phosphorylation (1:8), and lactylation (1:14) in fewer sites compared to rHSA. In clinical practice, both pHSA and rHSA utilized in China commonly display acetylation, succinylation, crotonylation, phosphorylation, beta-hydroxybutyrylation, and lactylation. Notably, there exist distinctions in the site characteristics and modification levels of these alterations between pHSA and rHSA. Further experimental inquiries are imperative to delve into the implications of these disparities in PTMs on the biological functionality, effectiveness, and safety of pHSA and rHSA.

Albumin of People with Diabetes Mellitus Is More Reduced at Low HbA1c.

Oxidative stress is involved in the development, progression, and complications of diabetes mellitus (DM). Oxidative modification of human serum albumin's cysteine-34 is a marker for oxidative stress-related pathological conditions. We aimed to evaluate the redox state of albumin in patients with DM to investigate possible correlations with age, diabetes duration, and disease control status. Plasma aliquots were collected from 52 participants (26 type 1 and 26 type 2 DM). Patients were divided into two groups according to their glycated hemoglobin levels less than or equal to and greater than 58 mmol/L. Albumin redox state was assessed with high-performance liquid chromatography by fractionating it into human mercaptalbumin (HMA) and human nonmercaptalbumin 1 and 2 (HNA1 and HNA2). Albumin redox fractions were differently related to the age of study participants. In age-matched T1DM and T2DM groups, the albumin redox state was essentially the same. Irreversibly oxidized HNA2 was positively correlated with diabetes duration, especially in the T1DM group. HNA was increased in people with an increased HbA1c (>58 mmol/mol). Our results support the hypothesis that oxidative stress plays a crucial role in DM pathogenesis and emphasize the importance of diabetes control on systemic oxidative burden.

EGFR-targeted humanized single chain antibody fragment functionalized silica nanoparticles for precision therapy of cancer

The combination of highly specific targeting ability and potent killing effect has made antibody-drug conjugates (ADCs) a popular area of focus in the development of anti-cancer drugs. However, the large molecular weight of IgG antibodies (∼ 150 kDa) often faces challenges in penetrating capillaries and stroma in tumor tissue. Moreover, when the drug-antibody ratio (DAR) is too low (DAR < 2) or too high (DAR > 6) it decreases the effectiveness of the ADC and further increases the potential for aggregation, overall clearance of the early system payload, and release rate. In this study, an EGFR-based single-chain antibody fragment (husA)-human serum albumin (HSA)-coupled FITC-labeled mesoporous silica nanoparticle (FMSN-DOX-H-husA) was developed. Chinese hamster ovarian cells express the husA, which is a single chain antibody fragment of the EGFR that has been humanized. The small molecular weight of the single chain antibody allows for shorter penetration into solid tumors and the absence of adverse effects of the Fc fragment. The modification of HSA improves the safety of the antibody nanoparticle couples by both improving the biocompatibility of the nanoparticles, prolonging the circulation time of the nanoparticles, and avoiding early release of the payload. Also, the humanization substantially reduces the immunogenicity. More importantly, the ratio of drug antibodies on nanoparticles was experimentally and computationally derived to be 11.8, providing a more accurate guide for clinical trials. The results of both in vivo and in vitro experiments indicated promising antitumor activity and safety of FMSN-DOX-H-husA. Thus, this antibody-drug conjugate provided a hopeful option for cancer treatment.

Tetrahydrobiopterin as a Trigger for Vitiligo: Phototransformation during UV Irradiation.

Vitiligo is a type of hypomelanosis. Tetrahydrobiopterin (H4Bip), the coenzyme of the initial stage of melanogenesis, appears to be a trigger for vitiligo. H4Bip is present in vitiligo in 3-5-fold excess and causes oxidative stress by triggering an autocatalytic cycle of excess hydrogen peroxide synthesis. Using quantum-chemical calculations, we have evaluated the possibility of H4Bip reactions occurring in the dark and under ultraviolet (UV) irradiation, including the formation of dihydropterin dimers. In order to simulate the oxidative stress, oxidative modification of human serum albumin (HSA) has been carried out in the presence of excessive H4Bip using the fluorescence method. The fraction of oxidized protein (FOP) has been calculated. It has been established that there is a strong oxidative modification of amino acids chromophores (tryptophan and tyrosine) in the protein (FOP 0.64). Under UV irradiation of the system (HSA + H4Bip), FOP is reduced to 0.39. Apparently, a part of H4Bip transforms into dihydropterin dimers and does not participate in the oxidative modification of the protein. The data on oxidative modification of HSA are consistent with dynamic light scattering: H4Bip promotes HSA aggregation with the formation of particles with a hydrodynamic radius Rh ≥ 2000 nm, which can become immunogenic.

Controlled Temporal Release of Serum Albumin Immobilized on Gold Nanoparticles.

Proteins adsorbed to gold nanoparticles (AuNPs) form bioconjugates and are critical to many emerging technologies for drug delivery, diagnostics, therapies, and other biomedical applications. A thorough understanding of the interaction between the immobilized protein and AuNP is essential for the bioconjugate to perform as designed. Here, we explore a correlation between the number of solvent-accessible thiol groups on a protein and the protein desorption rate from the AuNP surface in the presence of a competing protein. The chemical modification of human serum albumin (HSA) was carried out to install additional free thiols using Traut's reagent and create a library of HSA analogues by tailoring the molar excess of the Traut's reagent. We pre-adsorbed HSA variants onto the AuNP surface, and the resulting bioconjugates were then exposed to IgG antibody, and protein exchange was monitored as a function of time. We found that the rate of HSA displacement from the AuNP correlated with the experimentally measured number of accessible free thiol groups. Additionally, bioconjugates were synthesized using thiolated analogues of bovine serum albumin (BSA) and suspended in serum as a model for a complex sample matrix. Similarly, desorption rates with serum proteins were modulated with solvent-accessible thiols on the immobilized protein. These results further highlight the key role of Au-S bonds in the formation of protein-AuNP conjugates and provide a pathway to systematically control the number of free thiols on a protein, enabling the controlled release of protein from the surface of AuNP.

Analysis of the binding of warfarin to glyoxal- and methylglyoxal-modified human serum albumin by ultrafast affinity extraction

Ultrafast affinity extraction (UAE) and affinity microcolumns containing immobilized human serum albumin (HSA) were employed to evaluate the effect of advanced stage glycation on HSA and its binding to warfarin, a common site-specific probe for Sudlow site I of this protein. The modification of HSA by glyoxal (GO) and methylglyoxal (MGO) was considered, where GO and MGO are known to be important in the formation of many types of advanced glycation end products. Free drug fractions were measured by UAE for warfarin in solutions containing normal HSA or HSA that had been modified by GO or MGO at levels seen in serum during diabetes. The free fractions measured with the GO-modified HSA gave association equilibrium constants that ranged from 2.42–2.63 × 105 M−1 at pH 7.4 and 37 °C. These values were not significantly different from a value of 2.33 (±0.15) × 105 M−1 that was determined by the same method for warfarin with normal HSA. Similar studies using MGO-modified HSA gave association equilibrium constants for warfarin in the range of 3.07–3.31 × 105 M−1, which were 1.32- to 1.42-fold higher than the value seen for normal HSA (differences that were significant at the 95% confidence level). These results will be valuable in future binding studies based on affinity chromatography or other methods that employ warfarin as a probe to examine drug interactions at Sudlow site I of HSA and modified forms of this protein. This work also illustrates how UAE can be used, with analysis times of only minutes, to detect and measure small changes in the binding by drugs with unmodified or modified forms of a soluble binding agent or protein.

Drug-Induced Self-Assembly Cascade Nanoreactor for Synergistic Tumor Therapy.

The construction of completely biocompatible and biodegradable tumor suppressors by a simple and reliable method is essential for the clinical application of cancer-targeted drugs. Herein, by inserting glucose oxidase (GOx), catalase (CAT), and chlorin e6 (Ce6) into human serum albumin (HSA) assembly molecules, we constructed a cancer-targeted cascade bioreactor for synergistic starvation and photodynamic therapy (PDT). The modification of HSA could block the GOx activity and reduce the cytotoxicity of normal cells and organs. Through active targeting and passive enhanced permeability and retention effect, the loading of AS1411 could promote the cascade bioreactors to effectively target nucleolin-overexpressed tumors. Once internalized by cancer cells, as a result of catalyzing hydrogen peroxide (H2O2) to produce oxygen (O2), the protein nano-cascade reactor promoted microenvironmental oxygenation, which would subsequently lead to an increase in cytotoxic singlet oxygen (1O2) production under light irradiation as well as the decomposition of intracellular glucose. In vitro and in vivo studies showed that the cascaded nanoreactors could significantly enhance therapeutic efficacy through synergistic starvation therapy and enhanced PDT as well as chemotherapy. This cascade strategy will be demonstrated in clinical applications with huge potential.

Glycation end-products specific auto-antibodies in Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which is highly inflammatory. Compared to a healthy control group, SLE patients exhibit a higher concentration of advanced glycation end products (AGEs) and a lower concentration of receptors for AGEs (RAGE) in serum, however, the exact aetiology is still unclear. In the present study, non-enzymatic glycation induced modification of human serum albumin (HSA) has been studied by biophysical techniques. Glycated HSA (G-HSA) was used as an antigen, and serum autoantibody levels were estimated in SLE and normal humans (NH) against it, using direct binding ELISA and competitive inhibition ELISA. Compared to N-HSA, remarkable structural modifications were observed in G-HSA. Modified HSA also showed increased pentosidine fluorescence (213.7 ± 13.4 AU). Glycation of HSA induced a conversion of α-helix and random coil to β-sheet and β-turns. Serum immuno assays results exhibited significantly (p < 0.001) higher binding of G-HSA with serum autoantibodies from SLE patients when compared with native HSA (N-HSA). Furthermore, competitive ELISA results showed significantly (p < 0.001) high percent inhibition of serum IgG from SLE patients with modified antigen. Chronic inflammation with excessive oxidative stress in SLE patients could be a possible reason for structural alterations in blood proteins, generating highly immunogenic unique new-epitopes. These in turn induce the generation of specific autoantibodies against G-HSA that may serve as a potential biomarker for SLE pathogenesis.

Identification of organophosphate modifications by high‐resolution mass spectrometry

AbstractOrganophosphate (OP) compounds exhibit neurotoxicity by binding to serine residues of acetylcholinesterase (AChE) in the cholinergic nervous system and subsequently lead to the accumulation of acetylcholine in neuromuscular junctions of synapses. AChE capable of hydrolyzing choline esters is known to be inhibited in patients with nerve agents poisoning. Since OP is known to be transported by covalent bonding to human serum albumin (HSA), OP‐HSA adducts are considered potential diagnostic markers for OP exposures. In this study, HSA modification sites by OP or OP‐like compounds such as V‐type (VX) and Novichok‐type (A234), insecticide (DFP), and serine protease inhibitor (PMSF) were studied using liquid chromatography‐high‐resolution tandem mass spectrometry. As a result, we have discovered a novel OP‐HSA modification site, Y341.

Lysine modification of human serum albumin and its effect on protein conformation and nalidixic acid binding

In order to investigate the involvement of lysine residues of human serum albumin (HSA) in nalidixic acid (NA) binding, various modified preparations of HSA such as 44% carbamylated (C44), 83% carbamylated (C83) and 85% acetylated (A85) were made by treating the HSA solution with a different molar excess of potassium cyanate and acetic anhydride. The extent of modification, charge homogeneity and conformational changes of these derivatives were checked by TNBSA reaction method, polyacrylamide gel electrophoresis (PAGE) and gel filtration using Sephacryl S-200 HR column, respectively. Binding of NA to HSA and its derivatives was examined using fluorescence quenching titration method to determine the binding constant. The emergence of a single band in PAGE and single symmetrical peak in gel filtration results confirmed the charge and size homogeneity of these derivatives. Hydrodynamic properties such as Stokes radius and frictional ratio, as obtained from the analytical gel filtration results suggested molecular expansion in C83 and A85 HSAs while C44 HSA retained the native conformation. Addition of NA to both native and modified HSA derivatives quenched the fluorescence intensity of the protein at 344 ​nm to a different extent. Whereas the values of the Stern-Volmer constant (KSV) and bimolecular quenching rate constant (kq) suggested, NA-HSA complex formation, binding constant (Ka) value suggested an intermediate binding affinity between NA and HSA. Furthermore, the decrease in the Ka value with the extent of modification was indicative of the involvement of lysine residues in NA-HSA interaction.

Characterization of human serum albumin modified by hair dye component, 4-chloro-1,2-phenylenediamine: Role in protein aggregation, redox biology and cytotoxicity

Hair dyes and their components have been described as mutagenic and carcinogenic. They penetrate the skin, undergo auto-oxidation, yield protein reactive derivatives and modify proteins in an irreversible manner. Investigations into the impact of aromatic diamines on protein structures are expected to have a significant clinical value. This study documents the strong potential of 4-chloro-1, 2-phenylenediamine (4-Cl-o-PDA), a hair dye component, in the modification of human serum albumin (HSA). HSA was modified in vitro by 4-Cl-o-PDA and changes in tertiary and secondary structural characteristics were analyzed by UV–visible, fluorescence and far UV circular dichroism, fourier transform infrared spectroscopical techniques and ANS binding assay. Interaction of 4-Cl-o-PDA and HSA was studied by molecular docking and drug displacement assay. Formation of aggregates and inhibitory effect of antioxidant curcumin was analyzed by ThT binding assay, ThT fluorescence and electron microscopy. Changes in oxidation status of HSA were studied by thiol estimation and NBT assay. Effect of 4-Cl-o-PDA on cellular redox status, DNA integrity and cytotoxicity was analyzed in lymphocytes using dichlorofluorescein method, comet assay and MTT assay. We observed gross structural damage in HSA modified by 4-Cl-o-PDA. Modification led to unmasking of hydrophobic clusters, loss of its alpha helix and an increase in beta strand and unordered structure in HSA. 4-Cl-o-PDA strongly binds at sub-domain IB of HSA. HSA modified by 4-Cl-o-PDA formed amyloid fibrillar aggregates, whose formation was inhibited by antioxidant treatment. Modified HSA showed reduction of free sulfydryl content. 4-Cl-o-PDA-HSA led to the generation of reactive oxygen species in human lymphocytes. Modified HSA caused an increase in the tail length of DNA indicating enhanced single strand DNA breaks in cells. Modified protein was found to be genotoxic and cytotoxic. This works offers insights in the role of hair dye components in the biological world.

Elucidation of molecular interaction of bioactive flavonoid luteolin with human serum albumin and its glycated analogue using multi-spectroscopic and computational studies

Human serum albumin (HSA), an abundant protein in human plasma, which is associated with the transportation of numerous drugs, fatty acids to their targets and regulates the blood pH levels, is exposed to non-enzymatic glycation by reducing sugars. The existence of elevated levels of glucose during diabetes mellitus mediates the glycation of HSA which leads to structural and functional modification of the protein. The aim of our present study is to determine the effect of non-enzymatic glycation on the binding of a bioactive flavonoid luteolin to HSA using multi-spectroscopic and computational studies. The intrinsic fluorescence exhibited by the proteins (HSA and gHSA) were quenched by luteolin through static quenching mechanism. The binding constant for the interaction of HSA with luteolin was found higher as compared to that of gHSA-Luteolin at the experimental temperatures (290, 300 and 310 K). The ∆G values for the complexes formed between HSA/gHSA and luteolin were observed to be negative, indicating the spontaneity of the binding processes. Both enthalpy and entropy factors contribute to the binding of luteolin to HSA, whereas, enthalpy factors played a major role in the binding of luteolin to gHSA. A reduction in the α-helical content of HSA-Luteolin complex was observed, but no significant change was found for gHSA-Luteolin complex. Site probe displacement and related binding studies indicated that luteolin binds to both Subdomain IIA and IIIA of HSA and gHSA but with different affinities. Further molecular docking and MD simulation studies confirmed that luteolin preferably binds to the Subdomain IIA of HSA and IIIA of gHSA. These information, in turn, would give a better understanding of the functional changes occurred due to structural modification of HSA induced by glycation and may have some useful impact on the field of pharmaceutical sciences.

In Vitro Investigations of Acetohexamide Binding to Glycated Serum Albumin in the Presence of Fatty Acid.

The interaction of drugs with human serum albumin (HSA) is an important element of therapy. Albumin affects the distribution of the drug substance in the body, as well as its pharmacokinetic and pharmacodynamic properties. On the one hand, inflammation and protein glycation, directly associated with many pathological conditions and old age, can cause structural and functional modification of HSA, causing binding disorders. On the other hand, the widespread availability of various dietary supplements that affect the content of fatty acids in the body means that knowledge of the binding activity of transporting proteins, especially in people with chronic diseases, e.g., diabetes, will achieve satisfactory results of the selected therapy. Therefore, the aim of the present study was to evaluate the effect of a mixture of fatty acids (FA) with different saturated and unsaturated acids on the affinity of acetohexamide (AH), a drug with hypoglycaemic activity for glycated albumin, simulating the state of diabetes in the body. Based on fluorescence studies, we can conclude that the presence of both saturated and unsaturated FA disturbs the binding of AH to glycated albumin. Acetohexamide binds more strongly to defatted albumin than to albumin in the presence of fatty acids. The competitive binding of AH and FA to albumin may influence the concentration of free drug fraction and thus its therapeutic effect.

Streptococcus pneumoniae Surface Adhesin PfbA Exhibits Host Specificity by Binding to Human Serum Albumin but Not Bovine, Rabbit and Porcine Serum Albumins.

PfbA (Plasmin(ogen) and Fibronectin Binding protein A) is an adhesin present on the surface of Streptococcus pneumoniae. Initial studies characterized PfbA as plasmin(ogen) and fibronectin binding protein and later it was found that it binds with many other proteins of the extracellular matrix such as fibrinogen, collagen and laminin. It also binds to blood protein human serum albumin (HSA). Interestingly, PfbA exhibits no binding with serum albumins of bovine (BSA), rabbit (RSA) and porcine (PSA) which are sequentially and structurally homologous to HSA. This suggests that PfbA is likely involved in host specificity. Therefore, to get more insights into this aspect, a detailed analysis, which includes the interaction of rPfbA with HSA/BSA/RSA/PSA at different pHs by bio-layer interferometry, comparison of sequences and surface electrostatic potential of HSA/BSA/RSA/PSA, lysine modification of HSA by succinylation and subsequent interaction analysis of succinylated HSA with rPfbA and the secondary structural content estimation by FT-IR spectroscopy was carried out. Since large protrusions are another important geometric feature of protein surfaces, the property was also analyzed for HSA/BSA/RSA/PSA. The results of the above studies clearly suggest that the rPfbA exhibits host specificity by selectively binding only to HSA and not with its homologous BSA/RSA/PSA. Since the three dimensional structures of these albumins are highly similar, it is likely that rPfbA utilizes the differences in the surface electrostatic charge in combination with surface protrusions of HSA/BSA/RSA/PSA for the selective molecular recognition process and this feature may be important in the pathogenesis of pneumococcal infection.

Human serum albumin presents isoform variants with altered neonatal Fc receptor interactions.

Human serum albumin (HSA) is the most abundant protein in plasma and presents the particularity, with IgG, to have an extraordinary long serum half-life conferred by its interaction with the neonatal Fc receptor (FcRn). If the impact of IgG post-translational modifications (PTMs) on FcRn binding is well documented, it is far less reported for HSA despite numerous PTMs occurring on the protein in plasma. HSA is susceptible to numerous degradation reactions in plasma, because of aging, oxidative stress or liver and pancreas related pathologies. In the present study, we combined FcRn affinity chromatography and mass spectrometry to investigate the impact of HSA PTMs upon FcRn binding. This methodology presents the advantage to distinguish the effect of a single modification from a plasma HSA preparation made of a mixture of different isoforms. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation, which are modifications related to several pathological conditions, were demonstrated to act negatively on HSA-FcRn interaction. The HSA-FcRn binding alteration generated by these modifications is consistent with their vicinity with the interaction interface of the two proteins. Results were discussed regarding altered half-life of HSA observed in several disease states and pave the way toward new understandings of the hypoalbuminemia pathogenesis. SIGNIFICANCE STATEMENT: In this study, we investigated the impact of several post-translational modifications of HSA toward its ability to bind to the neonatal Fc receptor using in vitro affinity chromatography, mass spectrometry, and surface plasmon resonance. Cys34 oxidation, Lys525 glycation, and Leu585 C-terminal truncation were demonstrated to decrease HSA-FcRn binding. These modifications occurring in circulating HSA were discussed in relation to several pathologies as well as for the use of HSA as a therapeutic protein.

Artificial Viral Capsid Dressed Up with Human Serum Albumin.

Capsid of tomato bushy stunt virus consists of an outer coat protein shell decorated on an internal skeleton comprising a β-annulus motif. We mimicked this capsid structure with our artificial viral capsid dressed up with protein. We synthesized the β-annulus peptide bearing a Cys at the C-terminal side and linked it with Cys34 of the human serum albumin (HSA) via a bismaleimide linker. The β-annulus peptide-HSA conjugate self-assembled into spherical structures of a 50-70 nm size range in the Tris-HCl buffer, with the ζ-potential of assemblies of such conjugate revealing that HSA proteins were displayed on the outer surface of the artificial viral capsid. Interestingly, the critical aggregation concentration (CAC) of the conjugate intheTris-HCl buffer at 25 °C was approximately 0.01 μM, or 1/2500 lower than that of the unmodified β-annulus peptides, suggesting that the artificial viral capsids were stabilized via HSA modification. The present strategy of constructing protein nanocapsule by self-assembly of a β-annulus peptide-protein conjugate is simpler than that of previously reported protein nanocapsules.

Human serum albumin-based probes for molecular targeting of macrophage scavenger receptors.

Background: Inflammation and accumulation of macrophages are key features of unstable atherosclerotic plaques. The ability of macrophages to take up molecular probes can be exploited in new clinical imaging methods for the detection of unstable atherosclerotic lesions. We investigated whether modifications of human serum albumin (HSA) could be used to target macrophages efficiently in vitro.Materials and methods: Maleylated and aconitylated HSA were compared with unmodified HSA. Fluorescent or radiolabeled (89Zr) modified HSA was used in in vitro experiments to study cellular uptake by differentiated THP-1 cells and primary human macrophages. The time course of uptake was evaluated by flow cytometry, confocal microscopy, real-time microscopy and radioactivity measurements. The involvement of scavenger receptors (SR-A1, SR-B2, LOX-1) was assessed by knockdown experiments using RNA interference, by blocking experiments and by assays of competition by modified low-density lipoprotein.Results: Modified HSA was readily taken up by different macrophages. Uptake was mediated nonexclusively via the scavenger receptor SR-A1 (encoded by the MSR1 gene). Knockdown of CD36 and ORL1 had no influence on the uptake. Modified HSA was preferentially taken up by human macrophages compared with other vascular cell types such as endothelial cells and smooth muscle cells.Conclusions: Modified 89Zr-labeled HSA probes were recognized by different subsets of polarized macrophages, and maleylated HSA may be a promising radiotracer for radionuclide imaging of macrophage-rich inflammatory vascular diseases.

Cysteinylated and glycated human serum albumin levels are increased in human heart failure

Cysteinylated human serum albumin (Cys-HSA) is a post-translational modification of human serum albumin (HSA) where the sulfhydryl group (SH) of cysteine in position 34 (Cys34) becomes oxidized by a cysteine [1] and this oxidation easily occurs when exposed to oxidative stress [2]. The degree of oxidized Cys34 in HSA is correlated with oxidative stress related pathological conditions [3]. Indeed, increased Cys-HSA levels have been detected in patients undergoing end stage renal disease [4], coronary artery disease [2], chronic liver disease or diabetes mellitus [3] as well as in pregnant women with intrauterine growth restriction [5]. Functional loss of HSA due to post-translational modification could influence homeostasis, which may contribute to the progression of chronic diseases. For example, Oettl et al. reported that, in advanced liver disease, oxidative damage impairs the binding properties of HSA resulting in increased tissue distribution of toxic endogenous compounds, and thus enhancing the risk of tissue damage related to complications [6]. Similarly, the loss of significant oxidant buffering capacity of HSA due to Cys-HSA may lead pregnancy complications such as intrauterine growth restriction [5]. In this study we examined for the first time the extent of Cys-HSA in plasma of patients with heart failure [NYHA class III and IV (n class III =10, n class IV = 10)] compared with healthy subjects (n=11) by using a direct infusion ESI-MS. Blood samples were collected in citrate tubes and centrifuged immediately after collection. Plasma fraction was stored at -80 °C. Before the analysis, plasma samples were diluted 1:200 in water containing 50% acetonitrile and 0.1% formic acid and infused into a triple quadrupole mass spectrometer (Xevo TQ-S from Waters). The relative content of the HSA isoforms (Cys-HSA and glycated-HSA) were determined by measuring their relative intensities. Results showed a significant increase of Cys-HSA in heart failure (HF) patients (13.11%±5 and 17.31%±3.6 for NYHA III and IV, respectively) with respect to age-matched healthy subjects (9.87%±3). Furthermore, the levels of glycated-HSA were higher in HF patients (7.72%±1.49 and 8.04%±0.85, for NYHA III and IV, respectively) in comparison with age-matched healthy subjects (6.535%+0.54). Finally, HF patients showed a significant decrease in the total level of HSA as expected [7]. In conclusion, this study revealed an increased level of cysteinylation and glycation of HSA in patients with HF expanding the current knowledge that different residues on HSA can undergo certain post-translational modifications in specific environments in oxidative stress-related diseases. Future studies will establish the utility of monitoring the redox status of Cys34 in HSA as a marker for oxidative stress in HF and its potential role in the onset and progression of the disease. Finally, since HSA has an established role as a blood stream carrier, it will be interesting and exciting to test how these modifications can affect drug delivery of various pharmacological treatments in HF.

Impact of Hydroxyl Radical Modified-Human Serum Albumin Autoantigens in Systemic Lupus Erythematosus.

Free radicals are important mediators for cell toxicity and pathogenesis of diseases. Reactive oxygen species (ROS) have been generated broadly in inflammatory diseases including autoimmune diseases. ROS have been not only associated with the initiation and progression of the autoimmune response but also in amplification and exploring to novel epitopes, through the unveiling of antigenic determinants. This review explores the involvement of ROS in the pathophysiology of non-organ specific autoimmune diseases like systemic lupus erythematosus (SLE). The modification of human serum albumin through hydroxyl radical is thought to be responsible for the induction of autoantibodies against modified human serum albumin. In the light of overwhelming evidence suggesting the association with oxidative damage in autoimmunity, the administration of antioxidants could be a viable alternative for the neutralization of free radicals that are involved in eliciting autoimmune disease. In this review, we have discussed their pro-oxidant as well anti-oxidant properties which are capable of differentially modulating the autoimmune response.