Reza Yousefi | Shiraz University (original) (raw)

Papers by Reza Yousefi

Research paper thumbnail of Efficient expression in the prokaryotic host system, purification and structural analyses of the recombinant human ACE2 catalytic subunit as a hybrid protein with the B subunit of cholera toxin (CTB-ACE2)

Research Square (Research Square), Jul 3, 2023

Angiotensin-converting enzyme 2 (ACE2) has a speci c interaction with the coronavirus spike prote... more Angiotensin-converting enzyme 2 (ACE2) has a speci c interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identi ed with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ∼100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 hrs. Finally, the protein structural features were assessed using several biophysical methods. The uorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The β-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the e cient expression and successful puri cation of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

Research paper thumbnail of Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin

International Journal of Biological Macromolecules

Research paper thumbnail of A novel strategy for production of liraglutide precursor peptide and development of a new long-acting incretin mimic

PLOS ONE

Nowadays, a small number of incretin mimics are used to treat type 2 diabetes mellitus (T2DM) due... more Nowadays, a small number of incretin mimics are used to treat type 2 diabetes mellitus (T2DM) due to their longer half-life. The present study aimed to introduce a novel method for producing the liraglutide precursor peptide (LPP) and developing a potentially new incretin mimic. Here, human αB-crystallin (αB-Cry) was ligated to the LPP at the gene level, and the gene construct was expressed in Escherichia coli with a relatively good efficiency. The hybrid protein (αB-lir) was then purified by a precipitation method followed by anion exchange chromatography. After that, the peptide was released from the carrier protein by a chemical cleavage method yielding about 70%. The LPP was then purified by gel filtration chromatography, and HPLC estimated its purity to be about 98%. Also, the molecular mass of the purified peptide was finally confirmed by mass spectroscopy analysis. Assessment of the secondary structures suggested a dominant α-helical structure for the LPP and a β-sheet rich s...

Research paper thumbnail of Increased chaperone activity of human α‌B-crystallin with incomplete oxidation as a new defense mechanism against oxidative stress

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Research paper thumbnail of Developing a novel exenatide-based incretin mimic (αB-Ex): Expression, purification and structural-functional characterization

Biochimica et Biophysica Acta (BBA) - General Subjects

Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and e... more Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and exenatide have been well received in treating type 2 diabetes mellitus (T2DM) and obesity. In this study, an exenatide analogue, in which methionine at position 14 substituted with leucine, was ligated to human α-crystallin (α-Cry) and then expressed in the bacterial host cells. In the next step, the exenatide analogue was effectively released from the hybrid protein (αB-Ex) and subsequently purified using gel filtration chromatography. The HPLC and electrospray ionization mass spectrometry (ESI-MS) analyses respectively suggested a high purity (more than 97%) and an accurate molecular mass for the exenatide analogue (4168.22 Da and 835.01, z = 5). Also, the molecular mass of the αB-Ex hybrid protein based on the MALDI-TOF analysis was 24,702.162 Da. The secondary structure assessment by the three spectroscopic methods revealed that exenatide analogue and αB-Ex hybrid protein have an α-helix and a β-sheet rich structure, respectively. Also, according to the results of the DLS analysis, the αB-Ex hybrid protein indicated a high tendency to form large oligomeric structures. The NMR assessment suggested that the hybrid protein exists in its folding state. Both exenatide analogue and the αB-Ex hybrid protein revealed a crucial ability to reduce the blood sugar levels in healthy and diabetic mice. They were also capable of inducing insulin secretion to the bloodstream. Overall, our study introduces the αB-Ex hybrid protein as a novel incretin mimic, exerting its biological activity for a longer period of time. It might also be considered a potential drug candidate in the treatment of T2DM.

Research paper thumbnail of A novel method for the chaperone aided and efficient production of human proinsulin in the prokaryotic system

Journal of Biotechnology, 2022

With the rapid spread of diabetes in human society, the demand for insulin and its precursor (pro... more With the rapid spread of diabetes in human society, the demand for insulin and its precursor (proinsulin) continues to rise. Therefore, the introduction of new methods for their production is essential. In the present study, human proinsulin, while ligated to αB-crystallin chaperone, was effectively expressed in the prokaryotic host system and then purified by the ion-exchange chromatography at high purity (>97%). In the next step, human proinsulin with relatively high efficiency was released chemically from the hybrid protein (αB-pIns) and then purified using an appropriate gel filtration column. The SDS-PAGE and HPLC analyses confirmed the high purity, while mass spectroscopy assessment verified the exact molecular mass of the human proinsulin. Using a well-established protocol, the protein was folded in a one-step folding process with a yield of about 70%. The assessment of the secondary structures of the human proinsulin by Raman and FTIR spectroscopy suggested that this protein is rich in α-helix. Also, the conformation of disulfide bonds in the folded proinsulin was confirmed by Raman spectroscopy. The recombinant human proinsulin also demonstrated hypoglycemic activity and mitogenic action (induction of cell proliferation). The method proposed in this work for the production of human proinsulin is easy to run and does not depend on expensive and complex equipment. Thus, it can be used in the industrial production of human proinsulin.

Research paper thumbnail of Analyzing Structure, Chaperone Activity and Aggregation of Human αB-Crystallin in the Presence of Copper Ions and Glutathione

Research paper thumbnail of Insights into Platinum Complex-HSA Bindings: Spectral and Molecular Dynamics Simulation Studies

Physical Chemistry Research, 2021

Mutual interactions of human serum albumin (HSA) with the two binuclear platinum complexes, conta... more Mutual interactions of human serum albumin (HSA) with the two binuclear platinum complexes, containing [(bhq)Pt(dppm)2(Cl)Pt(bhq)(Cl)] (1) and [(bhq)Pt(dppm)2(PhMe)Pt(bhq)(CO2CF3)] (2), in which bhq=benzo[h]quinoline, and dppm=bis(diphenylphosphino) methane, were thoroughly investigated using spectroscopic and molecular modeling techniques. In this respect, fluorescence, Ultraviolet-Visible (UV-Vis) and circular dichroism (CD) spectroscopies, along with the docking and molecular dynamics simulations (MD) were utilized. Analysis of spectroscopic and MD simulation results revealed the structural alterations of HSA, upon binding to the binuclear platinum complexes, while the hydrogen bonding and van der Waals forces were found to mainly contribute to the protein-ligand intermolecular interactions. Results of far-UV CD measurements showed the strong ability of platinum complexes, in reducing the α-helical content of HSA, while other secondary structural features were increased. Due to t...

Research paper thumbnail of The binding assessment with human serum albumin of novel six-coordinate Pt(IV) complexes, containing bidentate nitrogen donor/methyl ligands

Molecular Biology Research Communications, 2015

The interactions between platinum complexes and human serum albumin (HSA) play crucial roles in t... more The interactions between platinum complexes and human serum albumin (HSA) play crucial roles in the distribution, metabolism, and activity of platinum-based anticancer drugs. Octahedral platinum (IV) complexes represent a significant class of anticancer agents that display molecular pharmacological properties different from cisplatin. In this study, the interaction between two Pt(IV) complexes with the general formula [Pt(X)2Me2 (tbu2bpy)], where tbu2bpy = 4,4′-ditert-butyl-2,2′-bipyridine, with two leaving groups of X = Cl (Com1) or Br (Com2), and HSA were investigated, using Ultraviolet-Visible (UV-Vis) spectroscopy, fluorescence spectroscopy, circular dichroism (CD) and molecular docking simulation. The spectroscopic and thermodynamic data revealed that the HSA/Pt(IV) complexes interactions were spontaneous process and Com2 demonstrated stronger interaction and binding constant in comparison with Com1. Also, the results suggest approximately similar structural alteration of HSA i...

Research paper thumbnail of A Comparative study on the chaperone-like activity of camel and bovine β-caseins

Journal of paramedical sciences, 2011

Molecular chaperones are characterized by a general behavior, arresting the exposed hydrophobic s... more Molecular chaperones are characterized by a general behavior, arresting the exposed hydrophobic surfaces of denaturing substrate proteins. In the present study, the capacity of β-caseins (β-CN) from camel and bovine milk in suppression of thermal aggregation process of apo-yeast alcohol dehydrogenase (YADH) was assessed. Apo-I enzyme was prepared by removal of the structural zinc; while apo-II-protein was obtained by depleting conformational and catalytic zinc atoms. Fluorescence spectroscopy using ANS probe revealed greater hydrophobic surface in apo-II ADH. Considerable decrease in aggregation of the heat treated protein molecules was observed upon exposing to β-CNs (camel, bovine). Bovine β-CN afforded more adverse effects on thermal aggregation. A direct correlation between casein's chaperone activity and structural stability of the substrate proteins was displayed. Moreover, an association between casein source and chaperone- like activity is suggested.

Research paper thumbnail of Polyethylene glycol-bonded triethylammonium l-prolinate: a new biodegradable amino-acid-based ionic liquid for the one-pot synthesis of bis(pyrazolyl)methanes as DNA binding agents

New Journal of Chemistry, 2020

[PEG-TEA]LP, as a new biodegradable ionic liquid catalyst, was successfully synthesized, characte... more [PEG-TEA]LP, as a new biodegradable ionic liquid catalyst, was successfully synthesized, characterized, and applied to the one-pot pseudo-five-component synthesis of bis(pyrazolyl)methanes.

Research paper thumbnail of Synthesis of new curcumin derivatives as influential antidiabetic α-glucosidase and α-amylase inhibitors with anti-oxidant activity

Carbohydrate Research, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions

Data in Brief, 2020

The interaction of αAand αB-crystallins with Cu 2 + ion modulates their structure and chaperone-l... more The interaction of αAand αB-crystallins with Cu 2 + ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αAand αBcrystallins and αAand αB-crystallins modified by peroxynitrite on concentration of Cu 2 + ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu 2 +-binding sites. The microscopic dissociation constant for Cu 2 +-αA-crystallin complex (K diss) was found to be equal to 9.7 μM. For peroxynitrite modified αAcrystallin the K diss value is equal to 17 μM. One subunit of native αB-crystallin contains two non-equivalent Cu 2 +-binding sites. The corresponding microscopic dissociation constants for Cu 2 +-αB-crystallin complexes (K 1 and K 2) were found to be equal to 0.94 and 36 μM. For peroxynitrite modified αBcrystallin the K 1 and K 2 values are equal to 4.3 and 70 μM, respectively.

Research paper thumbnail of The impact of different mutations at Arg54 on structure, chaperone-like activity and oligomerization state of human αA-crystallin: The pathomechanism underlying congenital cataract-causing mutations R54L, R54P and R54C

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2017

A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. ... more A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. While Arg54 is highly conserved within different species, the cataractogenic mutations R54L, R54P and R54C have been recently identified in CRYAA gene, encoding human αA-Cry. The detailed structural and functional aspects, stability andamyloidogenicproperties of αA-Cry were determined upon the above-mentioned missense mutations,using various spectroscopic techniques, gel electrophoresis, electron microscopy, size exclusion HPLC analyses, and chaperone-like activity assay. The different mutations at Arg54 result in diverse structural alterations among mutant proteins. In addition, the mutant proteins displayed reduced thermal stability, increased amyloidogenic properties and attenuated chaperone-like activity against aggregation of γ-Cry, catalase and lysozyme. The mutant proteins were also capable to form larger oligomeric complexes with γ-Cry which is the natural partner of α-Cry in the eye lenses. The most significant structural and functional damages were observed upon R54L mutation which was also accompanied with increased oligomeric size distribution of the mutant protein. The cataractogenic nature of R54P mutation can be explained with its detrimental effect on chaperone-like activity, conformational stability and proteolytic digestibility of the mutant protein. Also, R54C αA-Cry displayed an important intrinsic propensity for disulfide protein cross-linking with significantly reduced chaperone-like activity against all client proteins.These mutations revealed a range of detrimental effects on the structure, stability and functional properties of αA-Cry whichall together canexplain the pathomechanisms underlying development of the associated congenital cataract disorders.

Research paper thumbnail of Protective Effects of Acetylation on the Pathological Reactions of the Lens Crystallins with Homocysteine Thiolactone

PLOS ONE, 2016

Various post-translational lens crystallins modifications result in structural and functional ins... more Various post-translational lens crystallins modifications result in structural and functional insults, contributing to the development of lens opacity and cataract disorders. Lens crystallins are potential targets of homocysteinylation, particularly under hyperhomocysteinemia which has been indicated in various eye diseases. Since both homocysteinylation and acetylation primarily occur on protein free amino groups, we applied different spectroscopic methods and gel mobility shift analysis to examine the possible preventive role of acetylation against homocysteinylation. Lens crystallins were extensively acetylated in the presence of acetic anhydride and then subjected to homocysteinylation in the presence of homocysteine thiolactone (HCTL). Extensive acetylation of the lens crystallins results in partial structural alteration and enhancement of their stability, as well as improvement of αcrystallin chaperone-like activity. In addition, acetylation partially prevents HCTL-induced structural alteration and aggregation of lens crystallins. Also, acetylation protects against HCTL-induced loss of α-crystallin chaperone activity. Additionally, subsequent acetylation and homocysteinylation cause significant proteolytic degradation of crystallins. Therefore, further experimentation is required in order to judge effectively the preventative role of acetylation on the structural and functional insults induced by homocysteinylation of lens crystallins.

Research paper thumbnail of Comparative study on the interaction of two binuclear Pt (II) complexes with human serum albumin: Spectroscopic and docking simulation assessments

Journal of Photochemistry and Photobiology B: Biology, 2016

Human serum albumin (HSA) principally tasks as a transport carrier for a vast variety of natural ... more Human serum albumin (HSA) principally tasks as a transport carrier for a vast variety of natural compounds and pharmaceutical drugs. In the present study, two structurally related binuclear Pt (II) complexes containing cis, cis-[Me2Pt (μ-NN) (μ-dppm) PtMe2] (1), and cis, cis-[Me2Pt(μ-NN)(μ dppm) Pt((CH2)4)] (2) in which NN=phthalazine and dppm=bis (diphenylphosphino) methane were used to investigate their interaction with HSA, using UV-Vis absorption spectroscopy, fluorescence, circular dichroism and molecular dynamic analyses. The spectroscopic results suggest that upon binding to HSA, the binuclear Pt (II) complexes could effectively induce structural alteration of this protein. These complexes can bind to HSA with the binding affinities of the following order: complex 2>complex 1. Moreover, the thermodynamic parameters of binding between these complexes and HSA suggested the existence of entropy-driven spontaneous interaction, which mostly dominated with the hydrophobic forces. The ANS fluorescence results also indicated that two binuclear Pt (II) complexes were competing for the binding to the hydrophobic regions on HSA. In addition, competitive displacement assay and docking simulation study revealed that complexes 1 and 2 bind to the drug binding sites II and I on HSA, respectively. Furthermore, complex 2, with the higher binding affinity for HSA, shows more denaturing effect on this protein. Considering the protein structural damages in the pathway of harmful side effects of platinum drugs, complex 1 with the moderate binding affinity and low denaturing effect might be of high significance.

Research paper thumbnail of Investigation on the Surface Hydrophobicity and Aggregation Kinetics of Human Calprotectin in the Presence of Calcium

Research paper thumbnail of The structural alteration and aggregation propensity of glycated lens crystallins in the presence of calcium: Importance of lens calcium homeostasis in development of diabetic cataracts

Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, Jan 5, 2017

The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an imp... more The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an important risk factor for development of cataract diseases. In the current study, the impact of elevated levels of calcium ions were investigated on structure and aggregation propensity of glycated lens crystallins using gel electrophoresis and spectroscopic assessments. The glycated proteins indicated significant resistance against calcium-induced structural insults and aggregation. While, glycated crystallins revealed an increased conformational stability; a slight instability was observed for these proteins upon interaction with calcium ions. Also, in the presence of calcium, the proteolytic pattern of native crystallins was altered and that of glycated protein counterparts remained almost unchanged. According to results of this study it is suggested that the structural alteration of lens crystallins upon glycation may significantly reduce their calcium buffering capacity in eye lenses. ...

Research paper thumbnail of Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins

International journal of biological macromolecules, Jan 14, 2016

The main components of sunlight reaching the eye lens are UVA and visible light exerting their ph... more The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300-400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned st...

Research paper thumbnail of Anticancer activity assessment of two novel binuclear platinum (II) complexes

Journal of Photochemistry and Photobiology B: Biology, 2016

In the current study, two binuclear Pt (II) complexes, containing cis, cis-[Me 2 Pt (µ-NN) (µ-dpp... more In the current study, two binuclear Pt (II) complexes, containing cis, cis-[Me 2 Pt (µ-NN) (µ-dppm) PtMe 2 ] (1), and cis,cis-[Me 2 Pt(µ-NN)(µ dppm) Pt((CH 2) 4)] (2) in which NN = phthalazine and dppm = bis (diphenylphosphino) methane were evaluated for their anticancer activities and DNA/purine nucleotide binding properties. These Pt (II) complexes, with the non-classical structures, demonstrated a significant anticancer activity against Jurkat and MCF-7 cancer cell lines. The results of ethidium bromide/acridine orange staining and Caspase-III activity suggest that these complexes were capable to stimulate an apoptotic mechanism of cell death in the cancer cells. Using different biophysical techniques and docking simulation analysis, we indicated that these complexes were also capable to interact efficiently with DNA via a non-intercalative mechanism. According to our results, substitution of cyclopentane (in complex 2) with two methyl groups (in complex 1) results in significant improvement of the complex ability to interact with DNA and subsequently to induce the anticancer activity. Overall, these binuclear Pt (II) complexes are promising group of the non-classical potential anticancer agents which can be considered as molecular templates in designing of highly efficient platinum anticancer drugs.

Research paper thumbnail of Efficient expression in the prokaryotic host system, purification and structural analyses of the recombinant human ACE2 catalytic subunit as a hybrid protein with the B subunit of cholera toxin (CTB-ACE2)

Research Square (Research Square), Jul 3, 2023

Angiotensin-converting enzyme 2 (ACE2) has a speci c interaction with the coronavirus spike prote... more Angiotensin-converting enzyme 2 (ACE2) has a speci c interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identi ed with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ∼100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 hrs. Finally, the protein structural features were assessed using several biophysical methods. The uorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The β-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the e cient expression and successful puri cation of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

Research paper thumbnail of Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin

International Journal of Biological Macromolecules

Research paper thumbnail of A novel strategy for production of liraglutide precursor peptide and development of a new long-acting incretin mimic

PLOS ONE

Nowadays, a small number of incretin mimics are used to treat type 2 diabetes mellitus (T2DM) due... more Nowadays, a small number of incretin mimics are used to treat type 2 diabetes mellitus (T2DM) due to their longer half-life. The present study aimed to introduce a novel method for producing the liraglutide precursor peptide (LPP) and developing a potentially new incretin mimic. Here, human αB-crystallin (αB-Cry) was ligated to the LPP at the gene level, and the gene construct was expressed in Escherichia coli with a relatively good efficiency. The hybrid protein (αB-lir) was then purified by a precipitation method followed by anion exchange chromatography. After that, the peptide was released from the carrier protein by a chemical cleavage method yielding about 70%. The LPP was then purified by gel filtration chromatography, and HPLC estimated its purity to be about 98%. Also, the molecular mass of the purified peptide was finally confirmed by mass spectroscopy analysis. Assessment of the secondary structures suggested a dominant α-helical structure for the LPP and a β-sheet rich s...

Research paper thumbnail of Increased chaperone activity of human α‌B-crystallin with incomplete oxidation as a new defense mechanism against oxidative stress

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

Research paper thumbnail of Developing a novel exenatide-based incretin mimic (αB-Ex): Expression, purification and structural-functional characterization

Biochimica et Biophysica Acta (BBA) - General Subjects

Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and e... more Among the various treatments, GLP-1 receptor agonists (incretin mimics) such as liraglutide and exenatide have been well received in treating type 2 diabetes mellitus (T2DM) and obesity. In this study, an exenatide analogue, in which methionine at position 14 substituted with leucine, was ligated to human α-crystallin (α-Cry) and then expressed in the bacterial host cells. In the next step, the exenatide analogue was effectively released from the hybrid protein (αB-Ex) and subsequently purified using gel filtration chromatography. The HPLC and electrospray ionization mass spectrometry (ESI-MS) analyses respectively suggested a high purity (more than 97%) and an accurate molecular mass for the exenatide analogue (4168.22 Da and 835.01, z = 5). Also, the molecular mass of the αB-Ex hybrid protein based on the MALDI-TOF analysis was 24,702.162 Da. The secondary structure assessment by the three spectroscopic methods revealed that exenatide analogue and αB-Ex hybrid protein have an α-helix and a β-sheet rich structure, respectively. Also, according to the results of the DLS analysis, the αB-Ex hybrid protein indicated a high tendency to form large oligomeric structures. The NMR assessment suggested that the hybrid protein exists in its folding state. Both exenatide analogue and the αB-Ex hybrid protein revealed a crucial ability to reduce the blood sugar levels in healthy and diabetic mice. They were also capable of inducing insulin secretion to the bloodstream. Overall, our study introduces the αB-Ex hybrid protein as a novel incretin mimic, exerting its biological activity for a longer period of time. It might also be considered a potential drug candidate in the treatment of T2DM.

Research paper thumbnail of A novel method for the chaperone aided and efficient production of human proinsulin in the prokaryotic system

Journal of Biotechnology, 2022

With the rapid spread of diabetes in human society, the demand for insulin and its precursor (pro... more With the rapid spread of diabetes in human society, the demand for insulin and its precursor (proinsulin) continues to rise. Therefore, the introduction of new methods for their production is essential. In the present study, human proinsulin, while ligated to αB-crystallin chaperone, was effectively expressed in the prokaryotic host system and then purified by the ion-exchange chromatography at high purity (>97%). In the next step, human proinsulin with relatively high efficiency was released chemically from the hybrid protein (αB-pIns) and then purified using an appropriate gel filtration column. The SDS-PAGE and HPLC analyses confirmed the high purity, while mass spectroscopy assessment verified the exact molecular mass of the human proinsulin. Using a well-established protocol, the protein was folded in a one-step folding process with a yield of about 70%. The assessment of the secondary structures of the human proinsulin by Raman and FTIR spectroscopy suggested that this protein is rich in α-helix. Also, the conformation of disulfide bonds in the folded proinsulin was confirmed by Raman spectroscopy. The recombinant human proinsulin also demonstrated hypoglycemic activity and mitogenic action (induction of cell proliferation). The method proposed in this work for the production of human proinsulin is easy to run and does not depend on expensive and complex equipment. Thus, it can be used in the industrial production of human proinsulin.

Research paper thumbnail of Analyzing Structure, Chaperone Activity and Aggregation of Human αB-Crystallin in the Presence of Copper Ions and Glutathione

Research paper thumbnail of Insights into Platinum Complex-HSA Bindings: Spectral and Molecular Dynamics Simulation Studies

Physical Chemistry Research, 2021

Mutual interactions of human serum albumin (HSA) with the two binuclear platinum complexes, conta... more Mutual interactions of human serum albumin (HSA) with the two binuclear platinum complexes, containing [(bhq)Pt(dppm)2(Cl)Pt(bhq)(Cl)] (1) and [(bhq)Pt(dppm)2(PhMe)Pt(bhq)(CO2CF3)] (2), in which bhq=benzo[h]quinoline, and dppm=bis(diphenylphosphino) methane, were thoroughly investigated using spectroscopic and molecular modeling techniques. In this respect, fluorescence, Ultraviolet-Visible (UV-Vis) and circular dichroism (CD) spectroscopies, along with the docking and molecular dynamics simulations (MD) were utilized. Analysis of spectroscopic and MD simulation results revealed the structural alterations of HSA, upon binding to the binuclear platinum complexes, while the hydrogen bonding and van der Waals forces were found to mainly contribute to the protein-ligand intermolecular interactions. Results of far-UV CD measurements showed the strong ability of platinum complexes, in reducing the α-helical content of HSA, while other secondary structural features were increased. Due to t...

Research paper thumbnail of The binding assessment with human serum albumin of novel six-coordinate Pt(IV) complexes, containing bidentate nitrogen donor/methyl ligands

Molecular Biology Research Communications, 2015

The interactions between platinum complexes and human serum albumin (HSA) play crucial roles in t... more The interactions between platinum complexes and human serum albumin (HSA) play crucial roles in the distribution, metabolism, and activity of platinum-based anticancer drugs. Octahedral platinum (IV) complexes represent a significant class of anticancer agents that display molecular pharmacological properties different from cisplatin. In this study, the interaction between two Pt(IV) complexes with the general formula [Pt(X)2Me2 (tbu2bpy)], where tbu2bpy = 4,4′-ditert-butyl-2,2′-bipyridine, with two leaving groups of X = Cl (Com1) or Br (Com2), and HSA were investigated, using Ultraviolet-Visible (UV-Vis) spectroscopy, fluorescence spectroscopy, circular dichroism (CD) and molecular docking simulation. The spectroscopic and thermodynamic data revealed that the HSA/Pt(IV) complexes interactions were spontaneous process and Com2 demonstrated stronger interaction and binding constant in comparison with Com1. Also, the results suggest approximately similar structural alteration of HSA i...

Research paper thumbnail of A Comparative study on the chaperone-like activity of camel and bovine β-caseins

Journal of paramedical sciences, 2011

Molecular chaperones are characterized by a general behavior, arresting the exposed hydrophobic s... more Molecular chaperones are characterized by a general behavior, arresting the exposed hydrophobic surfaces of denaturing substrate proteins. In the present study, the capacity of β-caseins (β-CN) from camel and bovine milk in suppression of thermal aggregation process of apo-yeast alcohol dehydrogenase (YADH) was assessed. Apo-I enzyme was prepared by removal of the structural zinc; while apo-II-protein was obtained by depleting conformational and catalytic zinc atoms. Fluorescence spectroscopy using ANS probe revealed greater hydrophobic surface in apo-II ADH. Considerable decrease in aggregation of the heat treated protein molecules was observed upon exposing to β-CNs (camel, bovine). Bovine β-CN afforded more adverse effects on thermal aggregation. A direct correlation between casein's chaperone activity and structural stability of the substrate proteins was displayed. Moreover, an association between casein source and chaperone- like activity is suggested.

Research paper thumbnail of Polyethylene glycol-bonded triethylammonium l-prolinate: a new biodegradable amino-acid-based ionic liquid for the one-pot synthesis of bis(pyrazolyl)methanes as DNA binding agents

New Journal of Chemistry, 2020

[PEG-TEA]LP, as a new biodegradable ionic liquid catalyst, was successfully synthesized, characte... more [PEG-TEA]LP, as a new biodegradable ionic liquid catalyst, was successfully synthesized, characterized, and applied to the one-pot pseudo-five-component synthesis of bis(pyrazolyl)methanes.

Research paper thumbnail of Synthesis of new curcumin derivatives as influential antidiabetic α-glucosidase and α-amylase inhibitors with anti-oxidant activity

Carbohydrate Research, 2020

This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Research paper thumbnail of Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions

Data in Brief, 2020

The interaction of αAand αB-crystallins with Cu 2 + ion modulates their structure and chaperone-l... more The interaction of αAand αB-crystallins with Cu 2 + ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αAand αBcrystallins and αAand αB-crystallins modified by peroxynitrite on concentration of Cu 2 + ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu 2 +-binding sites. The microscopic dissociation constant for Cu 2 +-αA-crystallin complex (K diss) was found to be equal to 9.7 μM. For peroxynitrite modified αAcrystallin the K diss value is equal to 17 μM. One subunit of native αB-crystallin contains two non-equivalent Cu 2 +-binding sites. The corresponding microscopic dissociation constants for Cu 2 +-αB-crystallin complexes (K 1 and K 2) were found to be equal to 0.94 and 36 μM. For peroxynitrite modified αBcrystallin the K 1 and K 2 values are equal to 4.3 and 70 μM, respectively.

Research paper thumbnail of The impact of different mutations at Arg54 on structure, chaperone-like activity and oligomerization state of human αA-crystallin: The pathomechanism underlying congenital cataract-causing mutations R54L, R54P and R54C

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2017

A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. ... more A major part of cataractogenic mutations in human αA-Crystallin (αA-Cry) occurs at Arg residues. While Arg54 is highly conserved within different species, the cataractogenic mutations R54L, R54P and R54C have been recently identified in CRYAA gene, encoding human αA-Cry. The detailed structural and functional aspects, stability andamyloidogenicproperties of αA-Cry were determined upon the above-mentioned missense mutations,using various spectroscopic techniques, gel electrophoresis, electron microscopy, size exclusion HPLC analyses, and chaperone-like activity assay. The different mutations at Arg54 result in diverse structural alterations among mutant proteins. In addition, the mutant proteins displayed reduced thermal stability, increased amyloidogenic properties and attenuated chaperone-like activity against aggregation of γ-Cry, catalase and lysozyme. The mutant proteins were also capable to form larger oligomeric complexes with γ-Cry which is the natural partner of α-Cry in the eye lenses. The most significant structural and functional damages were observed upon R54L mutation which was also accompanied with increased oligomeric size distribution of the mutant protein. The cataractogenic nature of R54P mutation can be explained with its detrimental effect on chaperone-like activity, conformational stability and proteolytic digestibility of the mutant protein. Also, R54C αA-Cry displayed an important intrinsic propensity for disulfide protein cross-linking with significantly reduced chaperone-like activity against all client proteins.These mutations revealed a range of detrimental effects on the structure, stability and functional properties of αA-Cry whichall together canexplain the pathomechanisms underlying development of the associated congenital cataract disorders.

Research paper thumbnail of Protective Effects of Acetylation on the Pathological Reactions of the Lens Crystallins with Homocysteine Thiolactone

PLOS ONE, 2016

Various post-translational lens crystallins modifications result in structural and functional ins... more Various post-translational lens crystallins modifications result in structural and functional insults, contributing to the development of lens opacity and cataract disorders. Lens crystallins are potential targets of homocysteinylation, particularly under hyperhomocysteinemia which has been indicated in various eye diseases. Since both homocysteinylation and acetylation primarily occur on protein free amino groups, we applied different spectroscopic methods and gel mobility shift analysis to examine the possible preventive role of acetylation against homocysteinylation. Lens crystallins were extensively acetylated in the presence of acetic anhydride and then subjected to homocysteinylation in the presence of homocysteine thiolactone (HCTL). Extensive acetylation of the lens crystallins results in partial structural alteration and enhancement of their stability, as well as improvement of αcrystallin chaperone-like activity. In addition, acetylation partially prevents HCTL-induced structural alteration and aggregation of lens crystallins. Also, acetylation protects against HCTL-induced loss of α-crystallin chaperone activity. Additionally, subsequent acetylation and homocysteinylation cause significant proteolytic degradation of crystallins. Therefore, further experimentation is required in order to judge effectively the preventative role of acetylation on the structural and functional insults induced by homocysteinylation of lens crystallins.

Research paper thumbnail of Comparative study on the interaction of two binuclear Pt (II) complexes with human serum albumin: Spectroscopic and docking simulation assessments

Journal of Photochemistry and Photobiology B: Biology, 2016

Human serum albumin (HSA) principally tasks as a transport carrier for a vast variety of natural ... more Human serum albumin (HSA) principally tasks as a transport carrier for a vast variety of natural compounds and pharmaceutical drugs. In the present study, two structurally related binuclear Pt (II) complexes containing cis, cis-[Me2Pt (μ-NN) (μ-dppm) PtMe2] (1), and cis, cis-[Me2Pt(μ-NN)(μ dppm) Pt((CH2)4)] (2) in which NN=phthalazine and dppm=bis (diphenylphosphino) methane were used to investigate their interaction with HSA, using UV-Vis absorption spectroscopy, fluorescence, circular dichroism and molecular dynamic analyses. The spectroscopic results suggest that upon binding to HSA, the binuclear Pt (II) complexes could effectively induce structural alteration of this protein. These complexes can bind to HSA with the binding affinities of the following order: complex 2>complex 1. Moreover, the thermodynamic parameters of binding between these complexes and HSA suggested the existence of entropy-driven spontaneous interaction, which mostly dominated with the hydrophobic forces. The ANS fluorescence results also indicated that two binuclear Pt (II) complexes were competing for the binding to the hydrophobic regions on HSA. In addition, competitive displacement assay and docking simulation study revealed that complexes 1 and 2 bind to the drug binding sites II and I on HSA, respectively. Furthermore, complex 2, with the higher binding affinity for HSA, shows more denaturing effect on this protein. Considering the protein structural damages in the pathway of harmful side effects of platinum drugs, complex 1 with the moderate binding affinity and low denaturing effect might be of high significance.

Research paper thumbnail of Investigation on the Surface Hydrophobicity and Aggregation Kinetics of Human Calprotectin in the Presence of Calcium

Research paper thumbnail of The structural alteration and aggregation propensity of glycated lens crystallins in the presence of calcium: Importance of lens calcium homeostasis in development of diabetic cataracts

Spectrochimica acta. Part A, Molecular and biomolecular spectroscopy, Jan 5, 2017

The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an imp... more The imbalance of the calcium homeostasis in the lenticular tissues of diabetic patients is an important risk factor for development of cataract diseases. In the current study, the impact of elevated levels of calcium ions were investigated on structure and aggregation propensity of glycated lens crystallins using gel electrophoresis and spectroscopic assessments. The glycated proteins indicated significant resistance against calcium-induced structural insults and aggregation. While, glycated crystallins revealed an increased conformational stability; a slight instability was observed for these proteins upon interaction with calcium ions. Also, in the presence of calcium, the proteolytic pattern of native crystallins was altered and that of glycated protein counterparts remained almost unchanged. According to results of this study it is suggested that the structural alteration of lens crystallins upon glycation may significantly reduce their calcium buffering capacity in eye lenses. ...

Research paper thumbnail of Preventive role of lens antioxidant defense mechanism against riboflavin-mediated sunlight damaging of lens crystallins

International journal of biological macromolecules, Jan 14, 2016

The main components of sunlight reaching the eye lens are UVA and visible light exerting their ph... more The main components of sunlight reaching the eye lens are UVA and visible light exerting their photo-damaging effects indirectly by the aid of endogenous photosensitizer molecules such as riboflavin (RF). In this study, lens proteins solutions were incubated with RF and exposed to the sunlight. Then, gel mobility shift analysis and different spectroscopic assessments were applied to examine the structural damaging effects of solar radiation on these proteins. Exposure of lens proteins to direct sunlight, in the presence of RF, leads to marked structural crosslinking, oligomerization and proteolytic instability. These structural damages were also accompanied with reduction in the emission fluorescence of Trp and Tyr and appearance of a new absorption peak between 300-400nm which can be related to formation of new chromophores. Also, photo-oxidation of lens crystallins increases their oligomeric size distribution as examined by dynamic light scattering analysis. The above mentioned st...

Research paper thumbnail of Anticancer activity assessment of two novel binuclear platinum (II) complexes

Journal of Photochemistry and Photobiology B: Biology, 2016

In the current study, two binuclear Pt (II) complexes, containing cis, cis-[Me 2 Pt (µ-NN) (µ-dpp... more In the current study, two binuclear Pt (II) complexes, containing cis, cis-[Me 2 Pt (µ-NN) (µ-dppm) PtMe 2 ] (1), and cis,cis-[Me 2 Pt(µ-NN)(µ dppm) Pt((CH 2) 4)] (2) in which NN = phthalazine and dppm = bis (diphenylphosphino) methane were evaluated for their anticancer activities and DNA/purine nucleotide binding properties. These Pt (II) complexes, with the non-classical structures, demonstrated a significant anticancer activity against Jurkat and MCF-7 cancer cell lines. The results of ethidium bromide/acridine orange staining and Caspase-III activity suggest that these complexes were capable to stimulate an apoptotic mechanism of cell death in the cancer cells. Using different biophysical techniques and docking simulation analysis, we indicated that these complexes were also capable to interact efficiently with DNA via a non-intercalative mechanism. According to our results, substitution of cyclopentane (in complex 2) with two methyl groups (in complex 1) results in significant improvement of the complex ability to interact with DNA and subsequently to induce the anticancer activity. Overall, these binuclear Pt (II) complexes are promising group of the non-classical potential anticancer agents which can be considered as molecular templates in designing of highly efficient platinum anticancer drugs.