Important Carrier Protein Research Articles

Comparative study on interactions of phillyrin and phillygenol with lysozyme: Spectroscopy, differential scanning calorimetry and molecular modeling approaches

Phillyrin (PR) and its derivative phillygenol (PG) are major active extracts of the Chinese medicine Forsythiae Fructus. Lysozyme, an important carrier protein, has a variety of physiological along with pharmaceutical functions. Herein, the interactions of PR and PG with hen egg white lysozyme (HEWL) are investigated in details by spectroscopic analyses, differential scanning calorimetry (DSC) tests, and molecular modeling approaches. Fluorescence measurement and UV–visible absorption difference spectroscopy reveal that an “untypical static quenching” takes place pointing towards the formation of ground state complex. The binding constants of HEWL-PG are greater than that of HEWL-PR. Moreover, the major driving force for the binding process is elucidated as hydrophobic interactions for both cases. Circular dichroism spectroscopy and three-dimensional fluorescence indicate that PR and PG decrease the helical structure of the HEWL and hydrophobicity surrounding Trp and Tyr residues. The thermal stability of HEWL decreases in the presence of PR and PG by DSC. Docking study infers that both PR and PG could bind to the hydrophobic cavity of HEWL and supports the higher binding of PG than PR. Molecular dynamics study illustrates that PR has higher relative position change, which may attribute to its lower binding ability. In terms of the radius of gyration (Rg), PG causes the protein structure to swell even more. The average hydrogen bonds number of HEWL-PG is less than that of HEWL-PR and more changes of the residual fluctuation are observed in HEWL-PG system. Furthermore, MM-PBSA analysis exhibits that HEWL-PG has lower binding energy. Collectively, these results support that both PR and PG could bind to HEWL, but PG possesses higher affinity which leads to stronger secondary structural changes of HEWL than PR. These findings are beneficial to comprehensively understand the binding characteristic of PR and PG with HEWL, providing experimental basis for pharmacokenitics of PR and PG.

Elucidation of the interaction mechanism of olmutinib with human α-1 acid glycoprotein: insights from spectroscopic and molecular modeling studies

Olmutinib, the third-generation tyrosine kinase inhibitor, is applied in treating non-small cell lung cancer (NSCLC). The aim of this study is to elucidate the interaction mechanism of olmutinib with human α-1 acid glycoprotein (HAG), an important carrier protein, by mean of multi-spectroscopic and molecular simulation techniques. Fluorescence spectral results confirmed that the fluorescence of this carrier protein can be quenched by olmutinib in the static quenching mode, and this anticancer drug possesses a moderate binding affinity on HAG. The evidence from thermodynamic analysis, replacement interaction with ANS and sucrose, and computational simulation results showed that hydrogen bonding, hydrophobic interactions, and van der Waals forces involved the olmutinib-HAG complexation process. The results from UV-vis, 3D fluorescence and synchronous fluorescence spectroscopy proved that binding anticancer drug olmutinib caused the alteration in the microenvironment around Trp residues. And, circular dichroism spectral results provided the support for the conformational alterations in the carrier protein. The data also proved that olmutinib preferably bound to the hydrophobic cavity of HAG and the binding distance between the two was 2.21 nm. In addition, it can be found that the presence of some metal ions such as Zn2+, Ca2+, Ni2+ and Cu2+ would exert a certain extent effect on the olmutinib-HAG complexation process. Communicated by Ramaswamy H. Sarma

Albumin is the major carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in human plasma

Perfluoroalkyl (PFAS) substances are widespread in the environment and in organisms. The fact that exposure to PFAS is associated with elevated cholesterol levels is a major concern for human health. Previous investigations, in which bovine serum albumin was frequently studied, indicate that PFOS, PFOA and PFNA bind to serum albumin. However, it is critical to know whether these and other PFAS have a preference for the protein or the lipid fraction in native human blood fractions. For this reason, blood samples from four young healthy volunteers (two women, two men, 23–31 years old) were used for protein size separation and fractionation by the Cohn method in combination with serial ultracentrifugation. The plasma fractions were analyzed for 11 PFAS using high-performance tandem mass spectrometry (HPLC-MS/MS). Although the data are based on a small sample, they clearly show that albumin is the most important carrier protein for PFOS, PFOA, PFHxS, PFNA and PFDA in native human plasma. These five compounds have very little or no affinity for lipoproteins. The confirmation of their transport through albumin is important for the epidemiology of PFAS. The present results must be verified by the examination of a larger number of persons.

In vitro interaction of organophosphate metabolites with bovine serum albumin: A comparative 1H NMR, fluorescence and molecular docking analysis

Since the exposure of organophosphate pesticides are known to cause severe health consequences, it is important to understand the molecular interaction of these pesticides metabolites with vital biomolecules, especially with the proteins. Here, considering bovine serum albumin (BSA) as a model protein, we have examined its interaction with two selected organophosphate metabolites, 3,5,6-trichloro-2-pyridinol (TCPy) and paraoxon methyl (PM). TCPy and PM are resultant metabolites of two most widely used organophosphate pesticides chlorpyrifos and parathion respectively. 1H NMR line broadening, selective spin-lattice relaxation rate measurements, saturation transfer difference (STD) NMR of both TCPy and PM were carried out in the presence and absence of BSA. The obtained values of the affinity index (A), binding constants (Ka) and thermodynamic parameters indicated strong organophosphates-BSA interaction. Further, fluorescence quenching data on TCPy-BSA and PM-BSA interactions strongly supported the NMR results, besides providing the stoichiometry of these complexes. Molecular docking analysis unraveled viable, strong hydrogen bonds and electrostatic interactions in TCPy-BSA and PM-BSA complexes. This study also revealed substantial time-dependent changes in the 1H NMR intensity of PM in the presence of BSA, which suggests faster degradation of PM with increasing protein concentration during protein-metabolite interactions. The hydrolysis is attributed to the esterase-like action of BSA. The result provides key insights into the direct interaction of the organophosphate metabolites with a biologically important carrier protein, serum albumin.

Probing the interaction of anticancer drug temsirolimus with human serum albumin: molecular docking and spectroscopic insight

The binding interaction between temsirolimus, an important antirenal cancer drug, and HSA, an important carrier protein was scrutinized making use of UV and fluorescence spectroscopy. Hyper chromaticity observed in UV spectroscopy in the presence of temsirolimus as compared to free HSA suggests the formation of complex between HSA and temsirolimus. Fluorescence quenching experiments clearly showed quenching in the fluorescence of HSA in the presence of temsirolimus confirming the complex formation and also confirmed that static mode of interaction is operative for this binding process. Binding constant values obtained through UV and fluorescence spectroscopy reveal strong interaction; temsirolimus binds to HSA at 298 K with a binding constant of 2.9 × 104 M−1implying the strength of interaction. The negative Gibbs free energy obtained through Isothermal titration calorimetry as well as quenching experiments suggests that binding process is spontaneous. Molecular docking further provides an insight of various residues that are involved in this binding process; showing the binding energy to be -12.9 kcal/mol. CD spectroscopy was retorted to analyze changes in secondary structure of HSA; increased intensity in presence of temsirolimus showing changes in secondary structure of HSA induced by temsirolimus. This study is of importance as it provides an insight into the binding mechanism of an important antirenal cancer drug with an important carrier protein. Once temsirolimus binds to HSA, it changes conformation of HSA which in turn can alter the functionality of this important carrier protein and this altered functionality of HSA can be highlighted in variety of diseases.

An insight to the binding of ellagic acid with human serum albumin using spectroscopic and isothermal calorimetry studies

Ellagic acid (EA), a natural polyphenol evidence several pharmacological benefits. The binding profile of EA with human serum albumin (HSA) has been explored and investigated by Isothermal titration calorimetry (ITC), circular dichroism (CD) spectroscopy, time-correlated single-photon counting (TCSPC), absorbance spectroscopy, steady-state fluorescence spectroscopy, and modelling studies. The ITC data analysis revealed the binding Constant (Ka), ΔH, ΔS and ΔG values to be 15.5×104M−1, −116.2±18.1Kcalmol−1, −366calmol−1K−1 and −7.13Kcal mol−1 respectively with a unique binding site at HSA. EA effectively quenched the intrinsic fluorescence of HSA by static quenching, whereas TCSPC data also revealed association of dynamic quenching also. Thermodynamic analysis confirmed that hydrophobic and mainly hydrogen bonding interaction played important role in stabilizing the HSA-EA complex. It further dictates the binding reaction to be enthalpy driven. The secondary structure of HSA was altered upon binding with EA. CD spectroscopic data indicated the fraction of alpha helicity to be decreased from 52% to 40% upon binding to EA. This study will provide an insight on evaluation of this bioactive interaction during transport and releasing efficiency at the target site in human physiological system since HSA is the most important carrier protein in blood serum.

Germ line knockout of IGFBP-3 reveals influences of the gene on mammary gland neoplasia.

Insulin-like growth factor binding protein-3 (IGFBP-3) is an important carrier protein for insulin-like growth factors (IGFs) in the circulation. IGFBP-3 antagonizes the growth-promoting and anti-apoptotic activities of IGFs in experimental systems, but in certain contexts can increase IGF bioactivity, probably by increasing its half-life. The goal of this study was to investigate the role of IGFBP-3 in breast carcinogenesis and breast cancer metastasis. In the first part of the study, we exposed IGFBP-3 knockout and wild-type female mice to dimethylbenz[a]anthracene (DMBA) and followed them for appearance of primary tumors for up to 13 months. In the second part, mice of each genotype received an IV injection of 4T1 mammary carcinoma cells and then lung nodules were counted. Our results show that IGFBP-3 knockout mice developed breast tumors significantly earlier than the wild-type (13.9 ± 1.1 versus 22.5 ± 3.3 weeks, respectively, P = 0.0144), suggesting tumor suppression activity of IGFBP-3. In tumors of IGFBP-3 knockout mice, levels of phospho-AKT(Ser473) were increased compared to wild-type mice. The lung metastasis assay showed significantly more and larger lung nodules in IGFBP-3 knockout mice than in wild-type mice. While we observed increased levels of IGFBP-5 protein in the IGFBP-3 knockout mice, our findings suggest that this was not sufficient to completely compensate for the absence of IGFBP-3. Even though knockout of IGFBP-3 is associated with only a subtle phenotype under control conditions, our results reveal that loss of this gene has measurable effects on breast carcinogenesis and breast cancer metastasis.

Redox Albuminomics: Oxidized Albumin in Human Diseases

Albumin is the major contributor to colloid oncotic pressure and also serves as an important carrier protein of many endogenous and exogenous molecules throughout the body. In blood and extravascular fluids, albumin is susceptible to different oxidative modifications, especially thiol oxidation and carbonylation. Because of its metal-binding properties and the redox properties of its Cys34 thiol, albumin displays an important antioxidant activity. As albumin is the predominant protein in most body fluids, its Cys34 represents the largest fraction of free thiols within body fluids. Evidence that albumin oxidation takes place in vivo has been reported only recently. Different redox proteomic, mass spectrometric, and chromatographic techniques have shown albumin redox modifications in various human pathophysiological conditions. As a whole, most data here presented demonstrate that massive albumin oxidation occurs in vivo in different biological fluids and, to some extent, that this process is correlated to organ dysfunction. Recent reports suggest that the albumin redox state may serve as a global biomarker for the redox state in the body in various human diseases. However, further study is required to elucidate the exact relationship between albumin oxidation and pathology. In addition, it is unknown if some albumin oxidized forms may also have diagnostic uses. Application of specific redox proteomics techniques for the characterization of oxidized albumin forms in screening studies is required. A further challenge will be to analyze how these oxidative albumin modifications are related to real impact to the body.

Apical Sodium Dependent Bile Acid Transporter (ASBT, SLC10A2): A Potential Prodrug Target

A major hurdle impeding the successful clinical development of drug candidates can be poor intestinal permeability. Low intestinal permeability may be enhanced by a prodrug approach targeting membrane transporters in the small intestine. Transporter specificity, affinity, and capacity are three factors in targeted prodrug design. The human apical sodium dependent bile acid transporter (SLC10A2) belongs to the solute carrier family (SLC) of transporters and is an important carrier protein expressed in the small intestine. In spite of its appearing to be an excellent target for prodrug design, few studies have targeted human apical sodium dependent bile acid transporter (hASBT) to improve oral bioavailability. This review discusses bile acids including their chemistry and their absorptive disposition. Additionally, hASBT-mediated prodrug targeting is discussed, including QSAR, in vitro models for hASBT assay, and the current progress in utilizing hASBT as a drug delivery target.

Apolipoprotein-E genotype and the risk of developing cholelithiasis following bariatric surgery: a clue to prevention of routine prophylactic cholecystectomy.

Obesity and especially rapid weight loss following bariatric surgery are known risk factors for cholelithiasis. Since the risk may be high, prophylactic cholecystectomy has been advocated. Apolipoprotein (Apo) E, an important carrier protein in cholesterol metabolism and trafficking, is believed to play a role in gallstone pathogenesis. In particular, the Apo E4 allele has been suggested to be associated with cholesterol cholelithiasis. The aim of this study was to assess the incidence of postoperative cholelithiasis in our patient population and to determine a possible correlation with the Apo-E genotype. 134 morbidly obese patients undergoing gastric restrictive surgery [laparoscopic assisted gastric banding (LAGB) or silastic ring vertical gastroplasty (SRVG)] had abdominal ultrasound before and 6 to 12 months after operation, to determine the presence of gallstones. None of the patients enrolled in the study had gallstones before surgery. They did not have a prophylactic cholecystectomy or receive bile salt treatment. Apo-E genotypes were determined by Polymerase Chain Reaction restriction enzyme analysis. 10 patients (7.5%) developed postoperative cholelithiasis. The incidence of cholelithiasis in each ApoE genotype was: E2/E3--1/20 (5%), E3/E3--3/91 (3%), E3/E4--6/21 (29%), and E4/E4--0/2. ApoE allele frequencies in the study population were identical to those of a healthy control population. The mean BMI dropped from 43.6 to 29.4 kg/m2. The occurrence of postoperative gallstones was low in our population. However, in subjects with the Apo-E3/E4 genotype, the incidence is of practical significance. These data suggest that Apo-E genotyping may be useful in selecting patients for gallstone prevention (surgical or medical) when undergoing bariatric surgery. Further testing in larger patient populations may be able to give more definite guidelines in the future.

Hemopexin as a carrier protein of tumor-localizing Ga-metalloporphyrin-ATN-2

During size exclusion HPLC, ATN-2 binding protein separated from human and mouse sera, SCCVII and colon 26 tumor tissues were found in fraction 13 (A: estimated molecular weight 70 000). Fraction 13(A) of human sera was exclusively reactive to the human hemopexin antibody. During two-dimensional electrophoresis and amino acid sequence analysis, Fraction 13(A) of C3H/He mouse sera was found to have partial homology with the mouse hemopexin precursor. Glycoprotein with the same domain structure of hemopexin has been proposed to be an important carrier protein that forms the tumor-localizing activity of water-soluble porphyrin.