Dual Antifungal and Antiproliferative Activities of a Novel Protein Fraction from a Medicinally Important Herb Trillium govanianum Wall. ex. D. Don.

Antimicrobial resistance of microorganisms and the unwanted side effects of chemoradiation therapy in cancer are major issues in healthcare. In recent times, protein-based drugs have emerged as promising candidates due to their high specificity, less side effects, etc. In this context, the rhizome of Trillium govanianum was first explored for biologically active proteins/peptides. For this, three protein fractions namely Aqueous protein fraction (APF), Hexane-Methanol-treated aqueous protein fraction (HMAPF), and Methanol-treated aqueous protein fraction (MAPF) were prepared and evaluated for antimicrobial and antiproliferative activities. In antifungal activity, HMAPF showed the lowest MIC90 values of 1.56 µg/ml against Candida parapsilosis and Candida glabrata and 3.12 µg/ml against Candida albicans and Candida auris. The antifungal activity was further confirmed by a chitinase assay, a growth kinetics and a proteinase inhibitory assay. Surprisingly, none of the three protein fractions exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus. Moreover, APF exhibited potent antiproliferative and antioxidant activities with IC50 values of 18 µg/ml and 227 µg /ml, respectively. For HMAPF, an IC50 value of 70 µg/ml against the MDA-MB-231 cell line was observed. The present results demonstrate that the protein fractions, particularly HMAPF and APF, might serve as potential sources of a dual antifungal and antiproliferative protein-based drug.

Protecting thermodynamic stability of protein: The basic paradigm against stress and unfolded protein response by osmolytes.

Organic osmolytes are known to play important role in stress protection by stabilizing macromolecules and suppressing harmful effects on functional activity. There is existence of several reports in the literature regarding their effects on structural, functional and thermodynamic aspects of many enzymes and the interaction parameters with proteins have been explored. Osmolytes are compatible with enzyme function and therefore, can be accumulated up to several millimolar concentrations. From the thermodynamic point of view, osmolyte raises mid-point of thermal denaturation (Tm) of proteins while having no significant effect on ΔGD° (free energy change at physiological condition). Unfavorable interaction with the peptide backbone due to preferential hydration is the major driving force for folding of unfolded polypeptide in presence of osmolyte. However, the thermodynamic basis of stress protection and origin of compatibility paradigm has been a debatable issue. In the present manuscript, we attempt to elaborate the origin of stress protection and compatibility paradigm of osmolytes based on the effect on thermodynamic stability of proteins. We also infer that protective effects of osmolytes on ΔGD° (of proteins) could also indicate its potential involvement in unfolded protein response and overall stress biology on macromolecular level.

Comparative milk proteome analysis of Kashmiri and Jersey cattle identifies differential expression of key proteins involved in immune system regulation and milk quality.

BACKGROUND: Exploration of the bioactive components of bovine milk has gained global interest due to their potential applications in human nutrition and health promotion. Despite advances in proteomics profiling, limited studies have been carried out to fully characterize the bovine milk proteome. This study explored the milk proteome of Jersey and Kashmiri cattle at day 90 of lactation using high-resolution mass spectrometry based quantitative proteomics nano-scale LC-MS/Q-TOF technique. Data are available via ProteomeXchange with identifier PXD017412. RESULTS: Proteins from whey were fractionated by precipitation into high and low abundant proteins. A total of 81 high-abundant and 99 low-abundant proteins were significantly differentially expressed between Kashmiri and Jersey cattle, clearly differentiating the two breeds at the proteome level. Among the top differentiating proteins, the Kashmiri cattle milk proteome was characterised by increased concentrations of immune-related proteins (apelin, acid glycoprotein, CD14 antigen), neonatal developmental protein (probetacellulin), xenobiotic metabolising enzyme (flavin monooxygenase 3 (FMO3), GLYCAM1 and HSP90AA1 (chaperone) while the Jersey milk proteome presented higher concentrations of enzyme modulators (SERPINA1, RAC1, serine peptidase inhibitor) and hydrolases (LTF, LPL, CYM, PNLIPRP2). Pathway analysis in Kashmiri cattle revealed enrichment of key pathways involved in the regulation of mammary gland development like Wnt signalling pathway, EGF receptor signalling pathway and FGF signalling pathway while a pathway (T-cell activation pathway) associated with immune system regulation was significantly enriched in Jersey cattle. Most importantly, the high-abundant FMO3 enzyme with an observed 17-fold higher expression in Kashmiri cattle milk seems to be a characteristic feature of the breed. The presence of this (FMO3) bioactive peptide/enzyme in Kashmiri cattle could be economically advantageous for milk products from Kashmiri cattle. CONCLUSION: In conclusion, this is the first study to provide insights not only into the milk proteome differences between Kashmiri and Jersey cattle but also provides potential directions for application of specific milk proteins from Kashmiri cattle in special milk preparations like infant formula.

Therapeutic Potential of Medicinal Plant Proteins: Present Status and Future Perspectives.

Biologically active molecules obtained from plant sources, mostly including secondary metabolites, have been considered to be of immense value with respect to the treatment of various human diseases. However, some inevitable limitations associated with these secondary metabolites like high cytotoxicity, low bioavailability, poor absorption, low abundance, improper metabolism, etc., have forced the scientific community to explore medicinal plants for alternate biologically active molecules. In this context, therapeutically active proteins/peptides from medicinal plants have been promoted as a promising therapeutic intervention for various human diseases. A large number of proteins isolated from the medicinal plants have been shown to exhibit anti-microbial, anti-oxidant, anti-HIV, anticancerous, ribosome-inactivating and neuro-modulatory activities. Moreover, with advanced technological developments in the medicinal plant research, medicinal plant proteins such as Bowman-Birk protease inhibitor and Mistletoe Lectin-I are presently under clinical trials against prostate cancer, oral carcinomas and malignant melanoma. Despite these developments and proteins being potential drug candidates, to date, not a single systematic review article has documented the therapeutical potential of the available biologically active medicinal plant proteome. The present article was therefore designed to describe the current status of the therapeutically active medicinal plant proteins/peptides vis-à-vis their potential as future protein-based drugs for various human diseases. Future insights in this direction have also been highlighted.

An anti-cancerous protein fraction from Withania somnifera induces ROS-dependent mitochondria-mediated apoptosis in human MDA-MB-231 breast cancer cells.

Withania somnifera exhibits different pharmacological activities which mainly stem from its broad range of bioactive molecules. Majority of these bioactive molecules, fall into the groupings of alkaloids, steroidal lactones, phenolic compounds and glycoproteins. In this study, we evaluated a novel protein fraction, named here as WSPF, isolated from Withania somnifera roots for its cytotoxic properties against various human cancer cell lines. WSPF exhibited apoptotic activity for each cancer cell line tested, demonstrating significant activity against MDA-MB-231 human breast cancer cells with an IC50 value of 92 µg/mL. WSPF induced mitochondrial-mediated apoptosis of MDA-MB-231 cells via extensive reactive oxygen species generation, dysregulation of Bax/Bcl-2, loss of mitochondrial membrane potential and caspase-3 activation. Additionally, we observed G2/M-phase cell cycle arrest, cleavage of nuclear lamin A/C proteins, and nuclear morphological changes. The present results highlight the anti-cancer properties of WSPF, indicating that the proteins in this fraction can be potential therapeutic agents for triple negative breast cancer treatment.

Effect of an Imposed Contact on Secondary Structure in the Denatured State of Yeast Iso-1-cytochrome c.

There is considerable evidence that long-range interactions stabilize residual protein structure under denaturing conditions. However, evaluation of the effect of a specific contact on structure in the denatured state has been difficult. Iso-1-cytochrome c variants with a Lys54 → His mutation form a particularly stable His-heme loop in the denatured state, suggestive of loop-induced residual structure. We have used multidimensional nuclear magnetic resonance methods to assign 1H and 15N backbone amide and 13C backbone and side chain chemical shifts in the denatured state of iso-1-cytochrome c carrying the Lys54 → His mutation in 3 and 6 M guanidine hydrochloride and at both pH 6.4, where the His54-heme loop is formed, and pH 3.6, where the His54-heme loop is broken. Using the secondary structure propensity score, with the 6 M guanidine hydrochloride chemical shift data as a random coil reference state for data collected in 3 M guanidine hydrochloride, we found residual helical structure in the denatured state for the 60s helix and the C-terminal helix, but not in the N-terminal helix in the presence or absence of the His54-heme loop. Non-native helical structure is observed in two regions that form Ω-loops in the native state. There is more residual helical structure in the C-terminal helix at pH 6.4 when the loop is formed. Loop formation also appears to stabilize helical structure near His54, consistent with induction of helical structure observed when His-heme bonds form in heme-peptide model systems. The results are discussed in the context of the folding mechanism of cytochrome c.

Amelioration of hyperglycaemia and modulation of antioxidant status by Alcea rosea seeds in alloxan-induced diabetic rats.

CONTEXT: Alcea rosea L. (Malvaceae) has various medicinal uses including anticancer, anti-inflammatory and analgesic properties. However, there is no report on its antidiabetic activity. OBJECTIVE: Alcea rosea seed extracts were evaluated for antihyperglycaemic and antioxidative potential in diabetic rats. MATERIALS AND METHODS: Single intra-peritoneal injection of alloxan (130 mg/kg b.w.) was used for induction of diabetes in Albino Wistar rats. Antihyperglycaemic and antioxidant activities of methanol and aqueous extracts of Alcea rosea seed (100 and 300 mg/kg b.w.), administered orally on daily basis for 15 days, were assessed in vivo for fasting blood glucose level and antioxidant status of liver and pancreas. Metformin was used as a positive control. RESULTS: Aqueous and methanol extracts (300 mg/kg b.w.) decreased blood glucose level in diabetic rats by 24% and 46%, respectively. Administration of aqueous and methanol extracts at 300 mg/kg b.w. significantly (p < 0.01) modulated the antioxidant status of liver in diabetic rats by increasing levels of GR (22.5 ± 1.0, 24.4 ± 1.02 µg GSSG utilized/min/mg of protein), GPx (20.7 ± 1.2, 23.6 ± 2.04 µg GSH utilized/min/mg of protein), SOD (36.1 ± 1.7, 39.05 ± 1.5 units/mg of protein) and CAT (1744.5 ± 132.5, 1956.6 ± 125.2 nmol H2O2 decomposed/min/mg of protein), respectively. Similar results were observed for pancreas. DISCUSSION AND CONCLUSIONS: Antihyperglycaemic and antioxidative potentials of Alcea rosea seeds suggest its usefulness in management of diabetes and its complications. This is the first report on antidiabetic activity of this plant.

Isolation, molecular characterization and prevalence of Clostridium perfringens in sheep and goats of Kashmir Himalayas, India.

AIM: The study was conducted to report the occurrence of the Clostridium perfringens in sheep and goats of the Kashmir valley for the 1st time and to characterize them molecularly with respect to toxin genes to determine the prevalence of the various toxinotypes. MATERIALS AND METHODS: A total of 177 samples (152 from sheep and 25 from goats) collected from healthy, diarrheic animals, and morbid material of animals suspected to have died of enterotoxaemia were screened for C. perfringens toxinotypes. The presumptive positive isolates were confirmed using 16S rRNA gene-based polymerase chain reaction (PCR). All the confirmed isolates were screened for six toxin genes, namely; cpa, cpb, etx, cpi, cpb2, and cpe using a multiplex PCR. RESULTS: The PCR amplification of 16S rRNA gene revealed that out of 177 samples collected, 125 (70.62%) were found positive for C. perfringens, of which 110 (72.36%) were from sheep and 15 (60%) were from goats. The highest prevalence of C. perfringens toxinotype D was observed in lambs (56.16%) and kids (46.16%) followed by 3.84% in adult sheep while it was absent in samples obtained from adult goats. The multiplex PCR revealed that 67 (60.90%) isolates from sheep and 8 (53.33%) isolates from goats belonged to toxinotype A, while 43 (39.09%) isolates from sheep and 7 (46.66%) isolates from goats were detected as toxinotype D. None of the isolates was found to be toxinotype B, C, or E. All the C. perfringens toxinotype A isolates from sheep were negative for both cpb2 and cpe genes, however, 27.90% toxinotype D isolates from sheep carried cpb2 gene, and 6.97% possessed cpe gene. In contrast, 12.50% C. perfringens toxinotype A isolates from goats harbored cpb2 and cpe genes while 14.28% isolates belonging to toxinotype D carried cpb2 and cpe genes, respectively. CONCLUSION: The high prevalence of C. perfringens was observed, even in day-old lambs. The toxinotypes A and D are prevalent in both sheep and goats. The severity of disease and mortality may be associated with the presence of minor toxins in both the detected toxinotypes.

A Current Perspective on the Inhibition of Cholinesterase by Natural and Synthetic Inhibitors.

Majority of the observed cognitive and behavioral changes in Alzheimer's disease are postulated to be due to the deficiencies in cholinergic pathways of the brain. Enhancement of cholinergic transmission may thus stimulate the cholinergic receptors or prolong the availability of acetylcholine in synaptic cleft and hence improve the Alzheimer's disease associated symptoms. Of these two, the inhibition of cholinesterases (Acetylcholinesterase and Butyrylcholinesterase) by cholinesterase inhibitors is suggested to be a promising strategy. In this regard, various agents both natural and non-natural have been evaluated for the inhibition of cholinesterases. Phytochemical studies of some of the medicinal plants have shown the presence of many valuable compounds that show a wide range of pharmacological activity against cholinesterase enzymes. Interestingly, a good number of potent synthetic inhibitors of cholinesterase enzymes reported so far are natural-product based. This article aims to provide a comprehensive overview of both natural and synthetic cholinesterase inhibitors reported so far. Presenting a comparative overview of synthetic and natural cholinesterase inhibitors may provide some leads for the synthesis of new cholinesterase inhibitors from medicinal plants. Structural activity relationship of the active cholinesterase inhibitors is also discussed with some insights from simulation studies. Insights for possible future research have also been highlighted.

Human sex hormone-binding globulin as a potential target of alternate plasticizers: an in silico study.

BACKGROUND: Currently, alternate plasticizers are used to replace phthalate plasticizers in children's toys, medical equipments and food packaging, due to the adverse effects of phthalate compounds on human health and laws prohibiting their use. Current information regarding the safety and potential adverse effects of alternate plasticizers is limited and recent studies have found alternate plasticizers to display similar characteristics to those observed in phthalate plasticizers. This study was undertaken to evaluate and predict the potential endocrine disrupting activity of the three most commonly used alternate plasticizers: di(2-ethylhexyl)terephthalate (DEHT), tris(2-ethylhexyl)trimellitate (TOTM), and diisononyl hexahydrophthalate (DINCH) against human sex hormone-binding globulin (SHBG) using in silico approaches. MATERIALS AND METHODS: The crystal structure of human SHBG (Id: 1D2S) was retrieved from Protein Data Bank. PubChem database was searched for the structures of alternate plasticizers, DEHT, TOTM, and DINCH. Docking was performed using Glide (Schrodinger) Induced Fit Docking module. RESULTS: Induced Fit Docking of three alternate plasticizer compounds indicated that each of the three compounds fitted well into the steroid binding pocket of SHBG. Docking displays showed interactions of alternate plasticizers with 25-30 amino-acid residues of SHBG; 18-20 amino residues overlapped between the natural ligand, DHT, and the three compounds (commonality of 82-91 %). The hydrogen-bonding interaction of the amino-acid residue, Asn-82, of SHBG was also present in displays of DHT and all the three alternate phthalates. The binding affinity of all the three alternate phthalates was higher than DHT; maximum in DINCH followed by TOTM and DEHT. CONCLUSION: Our results suggested that the three alternate plasticizers have potential to engage the important interacting residues of SHBG and thus interfere in its steroid homeostatic function.

Alanine Counteracts the Destabilizing Effect that Urea has on RNase-A.

BACKGROUND: It is generally believed that organisms use and accumulate methylamine osmolytes to prevent urea's damaging effect on protein stability and activity. However, urea-rich cells not only accumulate methylamines but also many other methylated and non-methylated compounds as well. But, so far it is not known whether osmolytes that are not accumulated in urea-rich cells could also confer urea-counteracting properties. OBJECTIVE: We investigated the behavior of a non-methylamine osmolyte, alanine for its counteracting effect against urea denaturation of a model protein, ribonuclease A (RNase-A). METHODS: We have measured structure and thermodynamic parameters (Tm, ΔHm, and ΔGD°) of RNase-A in the presence of alanine, urea and their combination. The results were also compared with the ability of glycine (osmolyte lacking one methyl group when compared with alanine) to counter urea's effect on protein stability. RESULTS: We observed that alanine but not glycine counteracts urea's harmful effect on RNase-A stability. DISCUSSION: The results indicated that alanine (in addition to methylamine osmolytes) may serve as an alternate urea-counteractant. Since glycine fails to protect RNase-A from urea's destabilizing effect, it seems that methylation to glycine might have some evolutionary significance to protect proteins against harmful effects of urea.

Unique Medicinal Properties of Withania somnifera: Phytochemical Constituents and Protein Component.

Withania somnifera is an important medicinal herb that has been widely used for the treatment of different clinical conditions. The overall medicinal properties of Withania somnifera make it a viable therapeutic agent for addressing anxiety, cancer, microbial infection, immunomodulation, and neurodegenerative disorders. Biochemical constituents of Withania somnifera like withanolideA, withanolide D, withaferin A and withaniamides play an important role in its pharmacological properties. Proteins like Withania somnifera glycoprotein and withania lectin like-protein possess potent therapeutic properties like antimicrobial, anti-snake venom poison and antimicrobial. In this review, we have tried to present different pharmacological properties associated with different extract preparations, phytochemical constituents and protein component of Withania somnifera. Future insights in this direction have also been highlighted.

Ignored avenues in alpha-synuclein associated proteopathy.

Alpha-Synuclein (αSyn) is a 14 kDa pre-synaptic protein predominantly expressed in various regions of brain comprising neocortex, hippocampus, striatum, thalamus and cerebellum. αSyn aggregates have special neuropathologic relevance for comprehending Parkinson's disease (PD) and other synucleopathies due to the presence of αSyn aggregates in brain of patients suffering from these diseases. Direct relationship between PD and various single nuclear polymorphisms of αSyn further displays an inherent significance of mutated αSyn in increasing the risk for developing PD. So far, various theories have been emerged to explain αSyn mediated neuronal cell toxicity seen in patients with PD, including interaction of αSyn aggregates with biomolecules, vesicle dystrafficking, augmented oxidative stress, mitochondrial dysfunction, and disruption of synaptic function. Despite the advances in understanding of PD pathophysiology, current available treatments are still aiming at giving symptomatic relief. Lately, PD vaccines against αSyn aggregates are also being considered. However, various other avenues for e.g. post-translational and conformational modifications of αSyn, effect of cellular small molecules such as polyamines and osmolytes on αSyn aggregation, still remain unexplored and we believe that therapeutics directed at these ignored targets will surface as a successful combinational therapy for PD. Additionally, understanding mechanisms behind the interplay between PD and other health conditions, such as Gaucher's disease, Cardiovascular disorders, Hypertension, Homocystinuria, Type-II Diabetes, and Cancer are also speculated to provide great insight for novel therapeutic interventions. In the current review, we have precisely discussed all these ignored avenues with their possible clinical implications. Link between PD and other associated diseases has also been extensively reviewed.

Molecular linkages between diabetes and Alzheimer's disease: current scenario and future prospects.

After the revolutionary Rotterdam study that suggested there was an increased risk of developing Alzheimer's disease (AD) in patients with type-2 diabetes mellitus (T2DM), a number of studies have provided direct evidence for the linkage between AD and T2DM. In recent years, AD is considered as a neuroendocrine disorder, also referred as type-3 diabetes. There is a growing list of evidence to suggest that, in addition to impaired insulin signaling, there are a number of additional factors that may act as mechanistic links between AD and T2DM. These factors mainly include hypercholesterolemia, dyslipidemia, hypercystinemia, inflammation, impaired insulin signaling and impaired central nervous response to the adipose tissue-derived hormone leptin. Increased cholesterol plays a crucial role in the abnormal metabolism of the amyloid precursor protein, leading to the accumulation of ß-amyloid. In addition to impaired insulin signaling, diabetes has been found to accelerate the appearance of cerebrovascular inflammation and ß-amyloid peptide (Aß) deposition. Increased oxidative stress and production of advanced glycation end products are other probable marker linkages. However, the details of many of these molecular links still require extensive investigation. It is possible that a number of common molecular linkages exist between T2DM and AD. Understanding and analyzing the various molecular linkages between AD and T2DM may shed light on new tools that can be used for the early diagnosis and treatment of AD and also accelerate the identification of T2DM patients who are at high risk of AD.

An overview on potential neuroprotective compounds for management of Alzheimer's disease.

Alzheimer's disease (AD) is one of the major neurodegenerative diseases affecting almost 28 million people around the globe. It consistently remains one of the major health concerns of present world. Due to the clinical limitations like severe side effects of some synthesized drugs, alternative forms of treatments are gaining global acceptance in the treatment of AD. Neuroprotective compounds of natural origin and their synthetic derivatives exhibit promising results with minimal side effects and some of them are in their different phases of clinical trials. Alkaloids and their synthetic derivatives form one of the groups which have been used in treatment of neurodegenerative diseases like AD. We have further grouped these alkaloids into different sub groups like Indoles, piperdine and isoquinolines. Polyphenols form another important class of natural compounds used in AD management.

Manifestations of native topology in the denatured state ensemble of Rhodopseudomonas palustris cytochrome c'.

To provide insight into the role of local sequence in the nonrandom coil behavior of the denatured state, we have extended our measurements of histidine-heme loop formation equilibria for cytochrome c' to 6 M guanidine hydrochloride. We observe that there is some reduction in the scatter about the best fit line of loop stability versus loop size data in 6 M versus 3 M guanidine hydrochloride, but the scatter is not eliminated. The scaling exponent, ν(3), of 2.5 ± 0.2 is also similar to that found previously in 3 M guanidine hydrochloride (2.6 ± 0.3). Rates of histidine-heme loop breakage in the denatured state of cytochrome c' show that some histidine-heme loops are significantly more persistent than others at both 3 and 6 M guanidine hydrochloride. Rates of histidine-heme loop formation more closely approximate random coil behavior. This observation indicates that heterogeneity in the denatured state ensemble results mainly from contact persistence. When mapped onto the structure of cytochrome c', the histidine-heme loops with slow breakage rates coincide with chain reversals between helices 1 and 2 and between helices 2 and 3. Molecular dynamics simulations of the unfolding of cytochrome c' at 498 K show that these reverse turns persist in the unfolded state. Thus, these portions of the primary structure of cytochrome c' set up the topology of cytochrome c' in the denatured state, predisposing the protein to fold efficiently to its native structure.

Relationship between functional activity and protein stability in the presence of all classes of stabilizing osmolytes.

We report the effects of stabilizing osmolytes (low molecular mass organic compounds that raise the midpoint of thermal denaturation) on the stability and function of RNase-A under physiological conditions (pH 6.0 and 25 degrees C). Measurements of Gibbs free energy change at 25 degrees C (DeltaG(D) degrees ) and kinetic parameters, Michaelis constant (K(m)) and catalytic constant (k(cat)) of the enzyme mediated hydrolysis of cytidine monophosphate, enabled us to classify stabilizing osmolytes into three different classes based on their effects on kinetic parameters and protein stability. (a) Polyhydric alcohols and amino acids and their derivatives do not have significant effects on DeltaG(D) degrees and functional activity (K(m) and k(cat)). (b) Methylamines increase DeltaG(D) degrees and k(cat), but decrease K(m). (c) Sugars increase DeltaG(D) degrees , but decrease both K(m) and k(cat). These findings suggest that, among the stabilizing osmolytes, (a) polyols, amino acids and amino acid derivatives are compatible solutes in terms of both stability and function, (b) methylamines are the best refolders (stabilizers), and (c) sugar osmolytes stabilize the protein, but they apparently do not yield functionally active folded molecules.