Clay-Polymer Nanocomposites Mediated Inhibition of Protein Aggregation: Possible Role in the Prevention of Proteinopathies.

BACKGROUND: The transformation of proteins from their native conformation into highly ordered fibrillar structures due to their misfolding and aggregation under particular conditions are described as beta-sheet enriched amyloid fibrils. The accumulation of these fibrils in different body parts is the major cause of several neurological and non-neurological conditions (proteinopathies). OBJECTIVES: To prevent these proteinopathies, inhibition of protein aggregation is considered a promising strategy. Therefore, in this study, we synthesized montmorillonite (MMT) based poly- orthophenylenediamine (PoPD) nanocomposites (NCs) and characterized their size and morphology due to their remarkable biological properties. Further, the effect of these nanocomposites on inhibition of fibril formation was assessed. METHODS: These nanocomposites were evaluated for their anti-amyloidogenic potential on two model proteins of amyloidopathies, i.e., human lysozyme and human serum albumin (HL & HSA), by using several biophysical methods, such as Thioflavin T (ThT) and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence, congo red dye binding assay (CR). Secondary structural content was evaluated by Circular dichroism (CD) spectroscopy. RESULTS: Results demonstrated that synthesized nanocomposites significantly inhibited fibril formation in dose-dependent manner that corresponds to their ability to arrest fibrillation. It is suggested that they may adsorb proteins to protect them against aggregation when they are subjected to aggregating conditions. CONCLUSION: This study offers an opportunity to understand the mechanism of inhibition of fibril formation by nanocomposites, showing that they inhibit amyloid formation and amyloid diseases. Thus, the study concludes that these nanocomposites are promising candidates as therapeutic molecules for proteinopathies and are envisaged to enrich the area of personalized medicine, augmenting the human healthcare system.

Nanoclay based study on protein stability and aggregation and its implication in human health.

Protein aggregation is the major cause of several acute amyloid diseases such as Parkinson's, Huntington's, Alzheimer's, Lysozyme Systemic amyloidosis, Diabetes-II etc. While these diseases have attracted much attention but the cure is still unavailable. In the present study, Human Serum Albumin (HSA) and Human Lysozyme (HL) were used as the model proteins to investigate their aggregations. Nanoclays are hydrous silicates found in clay fraction of soil and known as natural nanomaterials. They have long been used in several applications in health-related products. In the present paper, the different types of nanoclays (MMT K-10, MMT K-30, Halloysite, Bentonite) were used to inhibit the process of HSA and HL aggregation. Aggregation experiments were evaluated using several biophysical tools such as Turbidity measurements, Intrinsic fluorescence, 1-anilino-8-naphthalene sulfonate (ANS), Thioflavin T (Th T), congo red (CR) binding assays and Circular dichroism. Results demonstrated that all the nanoclays inhibit the DTT-induced aggregation. However, bentonite and MMT K-10 were progressively intense and potent as they slowed down nucleation stage which can be perceived using several biophysical techniques. Hence, nanoclays can be used as an artificial chaperone and might provide effective treatment against several protein aggregation related disorders.

Phytochemical analysis and In-vitro Biochemical Characterization of aqueous and methanolic extract of Triphala, a conventional herbal remedy.

Triphala, an Indian ayurvedic triherbal formulation, is an equiproportional mixture of fruits of three herbs, amalaki (Emblica officinalis), haritaki (Terminalia chebula) and bibhitaki (Terminalia bellerica). The present study focused on phytocompounds detection and comparative analysis of various biochemical activities in the aqueous and methanolic extracts of triphala and its constituting herbs. Antioxidant activity was determined by 1, 1-diphenyl-2-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP), super oxide dismutase (SOD), catalase assay. Antibacterial potential was determined by broth dilution and agar well diffusion assays. Results revealed the presence of valuable bioactive compounds such as flavonoids, alkaloids, phenols, etc which might be responsible for biochemical activities. Extracts exhibited satisfactory radical-scavenging activity comparable with ascorbic acid. Methanolic extracts demonstrated higher antioxidant activity compared to aqueous extract. Extracts showed promising antibacterial potential against tested strain comparable to ampicillin. Hence, it can be concluded that triphala may be a promising candidate in pharmaceuticals and future medicine.

Trypsin Inhibitors from Cajanus cajan and Phaseolus limensis Possess Antioxidant, Anti-Inflammatory, and Antibacterial Activity.

Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostic and pharmacological studies. This study aimed to investigate antioxidant, anti-inflammatory, and antimicrobial activities of trypsin inhibitors (TIs) from two plant sources (Cajanus cajan and Phaseolus limensis). TI was purified from C. cajan (PUSA-992) by ammonium sulfate precipitation followed by ion exchange chromatography. TI from Phaseolus limensis (lima bean trypsin inhibitor; LBTI) was procured from Sigma-Aldrich, St. Louis, Missouri, United States. The antioxidant activity was analyzed by ferric ion reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The anti-inflammatory property of TIs was determined by inhibition of albumin denaturation assay. Ascorbic acid and aspirin were used as standards for antioxidant and anti-inflammatory assays, respectively. These TIs were tested against various bacterial and fungal strains. The TIs showed DPPH radical-scavenging activity in a concentration-dependent manner with IC50 values comparable to ascorbic acid. The FRAP values were also observed comparable to ascorbic acid and followed the trend of dose-dependent manner. The half maximal inhibitory concentration (IC50) values of CCTI and LBTI in anti-inflammatory test showed that LBTI is more potent than CCTI. The TIs showed potent antibacterial activity, but apparently no action against fungi. This study has reported the biological properties of CCTI and LBTI for the first time. The results show that TIs possess the ability to inhibit diseases caused by oxidative stress, inflammation, and bacterial infestation.

Panchakola Reduces Oxidative Stress in MCF-7 Breast Cancer and HEK293 Cells.

A large number of studies have proven the efficacy of ayurveda in the field of health and wellness. Panchakola, an ayurvedic formulation, is a general health tonic primarily used to cure fever, inflammation, pain, indigestion, and so on. We investigated effects of panchakola on oxidative stress in MCF-7 breast cancer and human embryonic kidney 293 (HEK293) cells. This work was performed to assess the antineoplastic and free radical-scavenging potential of aqueous extract of panchakola, a polyherbal formulation, in normal and breast cancer cell lines (i.e., HEK and MCF-7, respectively) using MTT assay. Activities of antioxidant enzyme, nitric oxide scavenger, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase were assessed in cell lines incubated with and without panchakola. The outcome was analyzed by spectrophotometer. The results demonstrated increased cytotoxicity in MCF-7 (IC50 16.446 µg/ml) comparable to the results obtained with standard anticancer control (curcumin) with IC50 10.265 µg/ml in MCF-7 cell line. Further, the results obtained from antioxidant assays suggested increased antioxidant activity in MCF-7 cells as compared to normal HEK cells. The results derived from this study suggested panchakola is a strong contender in the field of phytomedicines to fight cancer and free radical-related diseases.

Structural and Biophysical Characterization of Cajanus cajan Protease Inhibitor.

CONTEXT: A large number of studies have proven that Protease inhibitors (PIs), specifically serine protease inhibitors, show immense divergence in regulation of proteolysis by targeting their specific proteases and hence, they play a key role in healthcare. OBJECTIVE: We aimed to access in-vitro anticancer potential of PI from Cajanus cajan (CCPI). Also, crystallization of CCPI was targetted alongwith structure determination and its structure-function relationship. MATERIALS AND METHODS: CCPI was purified from Cajanus cajan seeds by chromatographic techniques. The purity and molecular mass was determined by SDS-PAGE. Anticancer potential of CCPI was determined by MTT assay in normal HEK and cancerous A549 cells. The crystallization screening of CCPI was performed by commercially available screens. CCPI sequence was subject to BLASTp with homologous PIs. Progressive multiple alignment was performed using clustalw2 and was modelled using ab initio protocol of I-TASSER. RESULTS: The results showed ~14kDa CCPI was purified in homogeneity. Also, CCPI showed low cytotoxic effects of in HEK i.e., 27% as compared with 51% cytotoxicity in A549 cells. CCPI crystallized at 16°C using 15% PEG 6000 in 0.1M potassium phosphate buffer (pH 6.0) in 2-3weeks as rod or needles visualized as clusters under the microscope. The molecular modelling revealed that it contains 3 beta sheets, 3 beta hairpins, 2 ß-bulges, 6 strands, 3 helices, 1helix-helix interaction, 41 ß-turns and 27 γ-turns. DISCUSSION AND CONCLUSION: The results indicate that CCPI may help to treat cancer in vivo aswell. Also, this is the first report on preliminary crystallization and structural studies of CCPI.

A review on protein misfolding, aggregation and strategies to prevent related ailments.

This review aims to highlight the fundamental mechanism of protein misfolding leading to protein aggregation and associated diseases. It also aims to anticipate novel therapeutic strategies with which to prevent or treat these highly debilitating conditions linked to these pathologies. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. The core cause of over 20 different human diseases which have now been designated as 'conformational diseases' including neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Parkinson's disease (PD) etc. A comprehensive study on protein misfolding, aggregation, and the outcomes of the effects of cytotoxic aggregates will lead to understand the aggregation-mediated cell toxicity and serves as a foundation for future research in development of promising therapies and drugs. This review has also shed light on the mechanism of protein misfolding which leads to its aggregation and hence the neurodegeneration. From these considerations, one could also envisage the possibility that protein aggregation may be exploited by nature to perform specific physiological functions in differing biological contexts.

Trypsin inhibitors demonstrate antioxidant activities, inhibit A549 cell proliferation, and increase activities of reactive oxygen species scavenging enzymes.

OBJECTIVES: Protease inhibitors are one of the most promising and investigated subjects for their role in pharmacognostical and pharmacological studies. This study aimed to investigate antineoplastic and antioxidant activity of trypsin inhibitors (TIs) isolated from three plant sources and their inhibitory role in the cell line. MATERIALS AND METHODOLOGY: TIs were obtained from different plant sources. Antineoplastic potential on adenocarcinoma human alveolar basal epithelial cell line (A549) and normal Human Embryonic Kidney (HEK) was determined using MTT assay. Activities of antioxidant enzyme, nitric oxide scavenger, superoxide dismutase, glutathione S-transferase, and glutathione peroxidase were assessed in cell lines incubated with and without TIs. The outcome was analyzed by spectrophotometer. RESULTS: TIs showed the higher cytotoxicity on A549 cells as compared to normal HEK cell line. TIs exhibited fair increase in antioxidant enzyme activity in A549 cells as compared to control. This might be one of the strategies of antineoplastic effect in cancer cells. CONCLUSIONS: This study has reported the antioxidant and antineoplastic properties of these TIs for the first time in A549 cells (to the best of our knowledge). The results show that TIs possess ability to prevent cancer and diseases caused due to oxidative stress. Therefore, we conclude that TIs can be used as supplements along with the conventional drugs for increased efficacy in the treatment of diseases such as cardiovascular disease, atherosclerosis, and cancer.

Biophysical insight into structure-function relation of Allium sativum Protease Inhibitor by thermal, chemical and pH-induced modulation using comprehensive spectroscopic analysis.

In this study, we have analyzed the structural and functional changes in the nature of Allium sativum Protease Inhibitor (ASPI) on undergoing various denaturation with variable range of pH, temperature and urea (at pH 8.2). ASPI being anti-tryptic in nature has native molecular mass of ∼15kDa. The conformational stability, functional parameters and their correlation were estimated under different conditions using circular dichroism, fluorescence and activity measurements. ASPI was found to fall in belongs to α+ß protein. It demonstrated structural and functional stability in the pH range 5.0-12.0 and up to70°C temperature. Further decrease in pH and increase in temperature induces unfolding followed by aggregation. Chemical induced denaturation was found to be cooperative and transitions were reversible and sigmoid. Tm (midpoint of denaturation), ΔCp (constant pressure heat capacity change) and ΔHm (van't Hoff enthalpy change at Tm were calculated to be 41.25±0.2°C, 1.3±0.07kcalmol-1K-1 and 61±2kcalmol-1 respectively for thermally denatured ASPI earlier. The reversibility of the protein was confirmed for both thermally and chemically denatured ASPI. The results obtained from trypsin inhibitory activity assay and structural studies are found to be in a significant correlation and hence established structure-function relationship of ASPI.

Purification and characterization of a novel trypsin-like protease from green-seeded chickpea (Cicer arientum).

The present study describes the purification and physicochemical and biochemical characterization of trypsin-like protease from green-seeded chickpea (Cicer arientum). The crude extract of chickpea trypsin (CpT) was obtained by homogenization followed by differential ammonium sulfate precipitation. The CpT was purified by ion-exchange chromatography on diethylaminoethyl (DEAE) column, pre-equilibrated with 20 mM tris-CaCl2 buffer (pH 8.2) with a flow rate of 0.5 mL min-1. The molecular weight and purity of ∼23 kDa of CpT were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Activity of protease was determined using Nα-benzoyl-DL-arginine-p-nitroanilide as chromogenic substrate and CpT purified showed a specific inhibitor activity of 26978.7697 U mg-1, fold purity of 9.8, and the yield of 70.2%. The characterization was performed for thermal stability, pH profile, and effect of various inhibitors on enzymatic activity. The protein isolated showed stability in the neutral to mild alkaline pH range and thermostability up to 50°C. CpT confirmed its serine nature as it was appreciably inhibited by serine protease inhibitors (maximum 6%), whereas metalloprotease inhibitors barely affected the activity of the enzyme (85%). To the best of our knowledge, it is first reported on purification of protease with trypsin-like properties, from this source.

Nanoparticles-protein interaction: Role in protein aggregation and clinical implications.

This review helps to understand protein misfolding events, which results in protein aggregation, and hence to related neurodegenerative diseases. Many chaperones and folding factors are found inside the cell system for the proper folding of protein. If protein gets misfolded, it may accumulate in cells and can lead to several fatal diseases. In some cases, misfolded proteins aggregated in form of loop-sheet polymer and amyloid fibril when they escape the degradation process and leads to neurodegenerative disorders. Nanoparticles (NPs) are nano-sized materials, can be formulated by using organic molecules such as gelatin, chitosan, inorganic molecules, metals such as iron, gold, silver, etc. NPs unite with proteins and form a dynamic nanoparticle-protein (NP-P) corona. Conformational changes may be induced in adsorbed protein by this NP-P corona which might change overall bio-reactivity of NP. They can influence correct folding of unfolded or misfolded protein and prevent their aggregation which may be helpful in the cure of neurodegenerative disorders. Due to high area:size ratio, NPs have higher advantages over bulk materials. Hence, the effect of NPs on the proper protein folding opens new gateways to produce a biologically active three dimensional biomolecule.

Allium sativum Protease Inhibitor: A Novel Kunitz Trypsin Inhibitor from Garlic Is a New Comrade of the Serpin Family.

PURPOSE: This study was aimed to purify and characterize the Protease inhibitor (PI) from a plant Allium sativum (garlic) with strong medicinal properties and to explore its phytodrug potentials. METHODS: Allium sativum Protease Inhibitor (ASPI) was purified using ammonium sulphate fractionation and Fast Protein Liquid Chromatography on anion exchanger Hi-Trap DEAE column. The purified protein was analyzed for its purity and molecular weight by SDS PAGE. The confirmation of presence of trypsin inhibiting PI was performed by MALDI TOF-TOF and analyzed by MASCOT database. The ASPI was further investigated for its kinetic properties and stability under extreme conditions of pH, temperature and chemical denaturants. Secondary structure was determined by Circular Dichorism (CD) spectroscopy. RESULTS: ASPI of ~15 kDa inhibited trypsin and matched "truncated kunitz Trypsin Inhibitor (Glycine max)" in MASCOT database. The purified ASPI showed 30376.1371 U/mg specific activity with a fold purity of 159.92 and yield ~93%. ASPI was quite stable in the range of pH 2-12 showing a decline in the activity around pH 4-5 suggesting that the pI value of the protein as ASPI aggregates in this range. ASPI showed stability to a broad range of temperature (10-80°C) but declined beyond 80°C. Further, detergents, oxidizing agents and reducing agents demonstrated change in ASPI activity under varying concentrations. The kinetic analysis revealed sigmoidal relationship of velocity with substrate concentration with Vmax 240.8 (µM/min) and Km value of 0.12 µM. ASPI showed uncompetitive inhibition with a Ki of 0.08±0.01 nM). The Far UV CD depicted 2.0% α -helices and 51% ß -sheets at native pH. CONCLUSIONS: To conclude, purified ~15 kDa ASPI exhibited fair stability in wide range of pH and temperature Overall, there was an increase in purification fold with remarkable yield. Chemical modification studies suggested the presence of lysine and tryptophan residues as lead amino acids present in the reactive sites. Therefore, ASPI with trypsin inhibitory property has the potential to be used as a non-cytotoxic clinical agents.

Characterization, biomedical and agricultural applications of protease inhibitors: A review.

This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid ß-peptide (Aß) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles.