Role of MicroRNA-21 in Prostate Cancer Progression and Metastasis: Molecular Mechanisms to Therapeutic Targets.

The role of noncoding RNA has made remarkable progress in understanding progression, metastasis, and metastatic castration-resistant prostate cancer (mCRPC). A better understanding of the miRNAs has enhanced our knowledge of their targeting mainly at the therapy level in solid tumors, such as prostate cancer (PCa). microRNAs (miRNAs) belong to a class of endogenous RNA that deficit encoded proteins. Therefore, the role of miRNAs has been well-coined in the progression and development of PCa. miR-21 has a dual nature in its work both as a tumor suppressor and oncogenic role, but most of the recent studies showed that miR-21 is a tumor promoter and also is involved in castration-resistant prostate cancer (CRPC). Upregulation of miR-21 suppresses programmed cell death and inducing metastasis and castration resistant in PCa. miR-21 is involved in the different stages, such as proliferation, angiogenesis, migration, and invasion, and plays an important role in the progression, metastasis, and advanced stages of PCa. Recently, various studies directly linked the role of high levels of miR-21 with a poor therapeutic response in the patient of PCa. In the present review, we have explained the molecular mechanisms/pathways of miR-21 in PCa progression, metastasis, and castration resistant and summarized the role of miR-21 in diagnosis and therapeutic levels in PCa. In addition, we have spotlighted the recent therapeutic strategies for targeting different stages of PCa.

Functional inhibition of c-Myc using novel inhibitors identified through "hot spot" targeting.

Protein-protein interactions drive various biological processes in healthy as well as disease states. The transcription factor c-Myc plays a crucial role in maintaining cellular homeostasis, and its deregulated expression is linked to various human cancers; therefore, it can be considered a viable target for cancer therapeutics. However, the structural heterogeneity of c-Myc due to its disordered nature poses a major challenge to drug discovery. In the present study, we used an in silico alanine scanning mutagenesis approach to identify "hot spot" residues within the c-Myc/Myc-associated factor X interface, which is highly disordered and has not yet been systematically analyzed for potential small molecule binding sites. We then used the information gained from this analysis to screen potential inhibitors using a conformation ensemble approach. The fluorescence-based biophysical experiments showed that the identified hit molecules displayed noncovalent interactions with these hot spot residues, and further cell-based experiments showed substantial in vitro potency against diverse c-Myc-expressing cancer/stem cells by deregulating c-Myc activity. These biophysical and computational studies demonstrated stable binding of the hit compounds with the disordered c-Myc protein. Collectively, our data indicated effective drug targeting of the disordered c-Myc protein via the determination of hot spot residues in the c-Myc/Myc-associated factor X heterodimer.

Platelet-derived microvesicles activate human platelets via intracellular calcium mediated reactive oxygen species release.

Platelet-derived microvesicles (PMVs) are the most abundant microvesicles in circulation, originating from blood platelets via membrane blebbing. PMVs act as biological cargo carrying key molecules from platelets, including immunomodulatory molecules, growth factors, clotting molecules, and miRNAs that can regulate recipient cellular functions. Formation and release of PMVs play an essential role in the pathophysiology of vascular diseases such as hemostasis, inflammation, and thrombosis. Platelet activation is considered the critical event in thrombosis, and a growing number of evidence suggests that oxidative stress-mediated signaling plays a significant role in platelet activation. Ca2+ is a notable player in the generation of ROS in platelets. Reports have established that microvesicles exhibit dual nature in redox mechanisms as they possess both pro-oxidant and antioxidant machinery. However, the impact of PMVs and their ROS machinery on platelets is still a limited explored area. Here, we have demonstrated that PMVs mediate platelet activation via intracellular ROS generation. PMVs interacted with platelets and induced calcium-mediated intracellular ROS production via NADPH oxidase (NOX), leading to platelet activation. Our findings will open up new insights into the tangible relationship of PMVs with platelets and will further contribute to the therapeutic aspects of PMVs in vascular injury and tissue remodeling.

Structure-based virtual screening against multiple Plasmodium falciparum kinases reveals antimalarial compounds.

The continuous emergence of resistance against most frontline antimalarial drugs has led to countless deaths in malaria-endemic countries, counting 619,000 deaths in 2021, with mutation in drug targets being the sole cause. As mutation is correlated frequently with fitness cost, the likelihood of mutation emergence in multiple targets at a time is extremely low. Hence, multitargeting compounds may seem promising to address drug resistance issues with additional benefits like increased efficacy, improved safety profile, and the requirement of fewer pills compared to traditional single and combinational drugs. In this study, we attempted to use the High Throughput Virtual Screening approach to predict multitarget inhibitors against six chemically validated Plasmodium falciparum (Pf) kinases (PfPKG, PfMAP2, PfCDPK4, PfTMK, PfPK5, PfPI4K), resulting in 21 multitargeting hits. The molecular dynamic simulation of the top six complexes (Myricetin-MAP2, Quercetin-CDPK4, Myricetin-TMK, Quercetin-PKG, Salidroside-PK5, and Salidroside-PI4K) showed stable interactions. Moreover, hierarchical clustering reveals the structural divergence of the compounds from the existing antimalarials, indicating less chance of cross-resistance. Additionally, the top three hits were validated through parasite growth inhibition assays, with quercetin and myricetin exhibiting an IC50 value of 1.84 and 3.93 µM, respectively.

Intrinsic disorder in proteins associated with oxidative stress-induced JNK signaling.

The c-Jun N-terminal kinase (JNK) signaling cascade is a mitogen-activated protein kinase (MAPK) signaling pathway that can be activated in response to a wide range of environmental stimuli. Based on the type, degree, and duration of the stimulus, the JNK signaling cascade dictates the fate of the cell by influencing gene expression through its substrate transcription factors. Oxidative stress is a result of a disturbance in the pro-oxidant/antioxidant homeostasis of the cell and is associated with a large number of diseases, such as neurodegenerative disorders, cancer, diabetes, cardiovascular diseases, and disorders of the immune system, where it activates the JNK signaling pathway. Among different biological roles ascribed to the intrinsically disordered proteins (IDPs) and hybrid proteins containing ordered domains and intrinsically disordered protein regions (IDPRs) are signaling hub functions, as intrinsic disorder allows proteins to undertake multiple interactions, each with a different consequence. In order to ensure precise signaling, the cellular abundance of IDPs is highly regulated, and mutations or changes in abundance of IDPs/IDPRs are often associated with disease. In this study, we have used a combination of six disorder predictors to evaluate the presence of intrinsic disorder in proteins of the oxidative stress-induced JNK signaling cascade, and as per our findings, none of the 18 proteins involved in this pathway are ordered. The highest level of intrinsic disorder was observed in the scaffold proteins, JIP1, JIP2, JIP3; dual specificity phosphatases, MKP5, MKP7; 14-3-3ζ and transcription factor c-Jun. The MAP3Ks, MAP2Ks, MAPKs, TRAFs, and thioredoxin were the proteins that were predicted to be moderately disordered. Furthermore, to characterize the predicted IDPs/IDPRs in the proteins of the JNK signaling cascade, we identified the molecular recognition features (MoRFs), posttranslational modification (PTM) sites, and short linear motifs (SLiMs) associated with the disordered regions. These findings will serve as a foundation for experimental characterization of disordered regions in these proteins, which represents a crucial step for a better understanding of the roles of IDPRs in diseases associated with this important pathway.

A novel inhibitor L755507 efficiently blocks c-Myc-MAX heterodimerization and induces apoptosis in cancer cells.

c-Myc is a transcription factor that plays a crucial role in cellular homeostasis, and its deregulation is associated with highly aggressive and chemotherapy-resistant cancers. After binding with partner MAX, the c-Myc-MAX heterodimer regulates the expression of several genes, leading to an oncogenic phenotype. Although considered a crucial therapeutic target, no clinically approved c-Myc-targeted therapy has yet been discovered. Here, we report the discovery via computer-aided drug discovery of a small molecule, L755507, which functions as a c-Myc inhibitor to efficiently restrict the growth of diverse Myc-expressing cells with low micromolar IC50 values. L755507 successfully disrupts the c-Myc-MAX heterodimer, resulting in decreased expression of c-Myc target genes. Spectroscopic and computational experiments demonstrated that L755507 binds to the c-Myc peptide and thereby stabilizes the helix-loop-helix conformation of the c-Myc transcription factor. Taken together, this study suggests that L755507 effectively inhibits the c-Myc-MAX heterodimerization and may be used for further optimization to develop a c-Myc-targeted antineoplastic drug.

Role of structural disorder in the multi-functionality of flavivirus proteins.

INTRODUCTION: The life cycle of a virus involves interacting with the host cell, entry, hijacking host machinery for viral replication, evading the host's immune system, and releasing mature virions. However, viruses, being small in size, can only harbor a genome large enough to code for the minimal number of proteins required for the replication and maturation of the virions. As a result, many viral proteins are multifunctional machines that do not directly obey the classic structure-function paradigm. Often, such multifunctionality is rooted in intrinsic disorder that allows viral proteins to interact with various cellular factors and remain functional in the hostile environment of different cellular compartments. AREAS COVERED: This report covers the classification of flaviviruses, their proteome organization, and the prevalence of intrinsic disorder in the proteomes of different flaviviruses. Further, we have summarized the speculations made about the apparent roles of intrinsic disorder in the observed multifunctionality of flaviviral proteins. EXPERT OPINION: Small sizes of viral genomes impose multifunctionality on their proteins, which is dependent on the excessive usage of intrinsic disorder. In fact, intrinsic disorder serves as a universal functional tool, weapon, and armor of viruses and clearly plays an important role in their functionality and evolution.

Understanding COVID-19 via comparative analysis of dark proteomes of SARS-CoV-2, human SARS and bat SARS-like coronaviruses.

The recently emerged coronavirus designated as SARS-CoV-2 (also known as 2019 novel coronavirus (2019-nCoV) or Wuhan coronavirus) is a causative agent of coronavirus disease 2019 (COVID-19), which is rapidly spreading throughout the world now. More than 1.21 million cases of SARS-CoV-2 infection and more than 67,000 COVID-19-associated mortalities have been reported worldwide till the writing of this article, and these numbers are increasing every passing hour. The World Health Organization (WHO) has declared the SARS-CoV-2 spread as a global public health emergency and admitted COVID-19 as a pandemic now. Multiple sequence alignment data correlated with the already published reports on SARS-CoV-2 evolution indicated that this virus is closely related to the bat severe acute respiratory syndrome-like coronavirus (bat SARS-like CoV) and the well-studied human SARS coronavirus (SARS-CoV). The disordered regions in viral proteins are associated with the viral infectivity and pathogenicity. Therefore, in this study, we have exploited a set of complementary computational approaches to examine the dark proteomes of SARS-CoV-2, bat SARS-like, and human SARS CoVs by analysing the prevalence of intrinsic disorder in their proteins. According to our findings, SARS-CoV-2 proteome contains very significant levels of structural order. In fact, except for nucleocapsid, Nsp8, and ORF6, the vast majority of SARS-CoV-2 proteins are mostly ordered proteins containing less intrinsically disordered protein regions (IDPRs). However, IDPRs found in SARS-CoV-2 proteins are functionally important. For example, cleavage sites in its replicase 1ab polyprotein are found to be highly disordered, and almost all SARS-CoV-2 proteins contains molecular recognition features (MoRFs), which are intrinsic disorder-based protein-protein interaction sites that are commonly utilized by proteins for interaction with specific partners. The results of our extensive investigation of the dark side of SARS-CoV-2 proteome will have important implications in understanding the structural and non-structural biology of SARS or SARS-like coronaviruses.

A multitude of signaling pathways associated with Alzheimer's disease and their roles in AD pathogenesis and therapy.

The exact molecular mechanisms associated with Alzheimer's disease (AD) pathology continue to represent a mystery. In the past decades, comprehensive data were generated on the involvement of different signaling pathways in the AD pathogenesis. However, the utilization of signaling pathways as potential targets for the development of drugs against AD is rather limited due to the immense complexity of the brain and intricate molecular links between these pathways. Therefore, finding a correlation and cross-talk between these signaling pathways and establishing different therapeutic targets within and between those pathways are needed for better understanding of the biological events responsible for the AD-related neurodegeneration. For example, autophagy is a conservative cellular process that shows link with many other AD-related pathways and is crucial for maintenance of the correct cellular balance by degrading AD-associated pathogenic proteins. Considering the central role of autophagy in AD and its interplay with many other pathways, the finest therapeutic strategy to fight against AD is the use of autophagy as a target. As an essential step in this direction, this comprehensive review represents recent findings on the individual AD-related signaling pathways, describes key features of these pathways and their cross-talk with autophagy, represents current drug development, and introduces some of the multitarget beneficial approaches and strategies for the therapeutic intervention of AD.

Conformational dynamics of 13 amino acids long NSP11 of SARS-CoV-2 under membrane mimetics and different solvent conditions.

The intrinsically disordered proteins/regions (IDPs/IDPRs) are known to be responsible for multiple cellular processes and are associated with many chronic diseases. In viruses, the existence of a disordered proteome is also proven and is related to its conformational dynamics inside the host. The SARS-CoV-2 has a large proteome, in which, structure and functions of all proteins are not known yet, along with non-structural protein 11 (nsp11). In this study, we have performed extensive experimentation on nsp11. Our results based on the CD spectroscopy gives characteristic disordered spectrum for IDPs. Further, we investigated the conformational behavior of nsp11 in the presence of membrane mimetic environment, α-helix inducer, and natural osmolyte. In the presence of negatively charged and neutral liposomes, nsp11 remains disordered. However, with SDS micelle, it adopted an α-helical conformation, suggesting the helical propensity of nsp11. Finally, we again confirmed the IDP behavior of nsp11 using MD simulations. In future, this conformational dynamic study could help to clarify its functional importance in SARS-CoV-2 infection.

Investigating the conformational dynamics of SARS-CoV-2 NSP6 protein with emphasis on non-transmembrane 91-112 & 231-290 regions.

The NSP6 protein of SARS-CoV-2 is a transmembrane protein, with some regions lying outside the membrane. Besides a brief role of NSP6 in autophagosome formation, this is not studied significantly. Also, there is no structural information available to date. Based on the prediction by TMHMM server for transmembrane prediction, it is found that the N-terminal residues (1-11), middle region residues (91-112), and C-terminal residues (231-290) lies outside the membrane. Molecular Dynamics (MD) simulations showed that NSP6 consists of helical structures. In contrast, the membrane outside lying region (91-112) showed partial helicity, which was further used as a model and obtained disordered type conformation during 1.5 µs. Additionally, a 200ns simulation study of residues 231-290 have shown significant conformational changes. As compared to helical and beta-sheet conformations in its structure model, the 200ns simulation resulted in the loss of beta-sheet structures while helical regions remained intact. Further, we have experimentally characterized the residue 91-112 by using reductionist approaches. CD spectroscopy suggests that the NSP6 (91-112) is disordered-like region in isolation, which gains helical conformation in different biological mimic environmental conditions. These studies can be helpful to study NSP6 (91-112) interactions with host proteins, where different protein conformations might play a significant role. The present study adds up more information about the NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis.

The dark side of Alzheimer's disease: unstructured biology of proteins from the amyloid cascade signaling pathway.

Alzheimer's disease (AD) is a leading cause of age-related dementia worldwide. Despite more than a century of intensive research, we are not anywhere near the discovery of a cure for this disease or a way to prevent its progression. Among the various molecular mechanisms proposed for the description of the pathogenesis and progression of AD, the amyloid cascade hypothesis, according to which accumulation of a product of amyloid precursor protein (APP) cleavage, amyloid ß (Aß) peptide, induces pathological changes in the brain observed in AD, occupies a unique niche. Although multiple proteins have been implicated in this amyloid cascade signaling pathway, their structure-function relationships are mostly unexplored. However, it is known that two major proteins related to AD pathology, Aß peptide, and microtubule-associated protein tau belong to the category of intrinsically disordered proteins (IDPs), which are the functionally important proteins characterized by a lack of fixed, ordered three-dimensional structure. IDPs and intrinsically disordered protein regions (IDPRs) play numerous vital roles in various cellular processes, such as signaling, cell cycle regulation, macromolecular recognition, and promiscuous binding. However, the deregulation and misfolding of IDPs may lead to disturbed signaling, interactions, and disease pathogenesis. Often, molecular recognition-related IDPs/IDPRs undergo disorder-to-order transition upon binding to their biological partners and contain specific disorder-based binding motifs, known as molecular recognition features (MoRFs). Knowing the intrinsic disorder status and disorder-based functionality of proteins associated with amyloid cascade signaling pathway may help to untangle the mechanisms of AD pathogenesis and help identify therapeutic targets. In this paper, we have used multiple computational tools to evaluate the presence of intrinsic disorder and MoRFs in 27 proteins potentially relevant to the amyloid cascade signaling pathway. Among these, BIN1, APP, APOE, PICALM, PSEN1 and CD33 were found to be highly disordered. Furthermore, their disorder-based binding regions and associated short linear motifs have also been identified. These findings represent important foundation for the future research, and experimental characterization of disordered regions in these proteins is required to better understand their roles in AD pathogenesis.

Amyloid formation by intrinsically disordered trans-activation domain of cMyb.

The cMyb trans-activation domain is one of the model systems to understand the folding and binding mechanisms in intrinsically disordered proteins. cMyb (291-315) TAD (cMyb TAD) upon interaction with KIX plays a crucial role in transcriptional regulation. However, nothing is known regarding its aggregation behaviour on change of buffer conditions or stressed environment. Notably, most of the disease-associated amyloid-forming proteins such as Aß, Tau, α-synuclein, and amylin are natively unstructured. Nevertheless, to date, very fewer evidence on aggregation behaviours on TAD domains are available. Therefore, this is necessary to investigate the aggregation propensity of intrinsically disordered cMyb TAD domain in isolation. As an essential step in that direction, we have extensively studied the aggregation behaviour of cMyb TAD using the standard approaches for aggregation studies and systematically probed the amyloid conformations. These aggregates are ThT and ANS-positive whose amyloid nature was also confirmed by Far-UV CD spectroscopic studies suggesting that cMyb TAD fibrils are rich in ß-sheet secondary structure, transmission electron microscopy revealed the formation of characteristic long branched amyloid fibrils of 6-16 nm diameter, and MTT assay in SH-SY5Y neuroblastoma cells suggest that these aggregates are cytotoxic. This amyloid nature of cMyb TAD may affect its binding with KIX and alter cMyb function (transcriptional regulation) under acidic/stressed conditions.

Zika virus NS4A N-Terminal region (1-48) acts as a cofactor for inducing NTPase activity of NS3 helicase but not NS3 protease.

Among Flaviviridae, in West Nile virus (WNV) and Hepatitis C virus (HCV), the non-structural protein NS4A modulates the NTPase activity of viral helicases during nucleic acid unwinding through its N-terminal disordered residues (1-50). In HCV, the acidic NS4A also serves as a cofactor for regulating the NS3 protease activity. However, in case of Zika virus (ZIKV), the role of NS4A and its impact on activities of NS3 helicase and protease is not known. In order to elucidate the role of NS4A, we checked the NTPase activity of NS3 helicase and protease activity of NS3 protease in presence of NS4A N-terminal region (residues 1-48) peptide. Our enzyme kinetics results together with binding experiment clearly demonstrate that NS3 helicase in presence of NS4A peptide increased the rate of ATP hydrolysis whereas the protease activity of NS3 protease was not affected. Therefore, like WNV and HCV, our results establish a role of ZIKV NS4A being a cofactor for modulating the NTPase activity of ZIKV NS3 helicase.

Conformational dynamics of p53 N-terminal TAD2 region under different solvent conditions.

Although the mystery molecule p53 has been studied extensively, still several unknown mechanisms need to be elucidated. Being a central hub of cellular signaling pathways, the function of p53 is precisely executed synergistically by its intrinsically disordered and structural domains. The disordered N-terminal region further modulates p53 function by undergoing promiscuous binding and folding with several partners with the help of TAD1 and TAD2 motifs. Among these regions, a significant contribution is made by TAD2 in terms of binding affinities. This heterogeneity in p53 TAD region motivates to employ a reductionist approach to understand the folding behavior of TAD2 region independently under a broad range of different pH, temperature and solvent conditions. Since the intracellular environment is complex and crowded with a variety of molecules providing different type of surfaces from polar to hydrophobic, it is mandatory to characterize the conformational heterogeneity of disordered proteins to completely understand their function. Different types of alcohols were used to estimate the structure forming capabilities of the TAD2 peptides using circular dichroism, fluorescence and lifetime spectroscopy. The alcohols ethanol, TFE and HFIP were previously known to induce increasing levels of hydrophobic environments in water-alcohol mixtures respectively. Our results have shown that TAD2 peptide undergoes a dehydration dependent induction of hydrophobic interactions leading towards structural transitions in presence of organic solvents. This study is highlighting the importance of hydrophobic surfaces playing a crucial role in TAD2 interaction and conformational transitions.

Folding and structural polymorphism of p53 C-terminal domain: One peptide with many conformations.

Proteins of the p53 family are best known for their role in the regulation of cell cycle. The p53 protein, as a model system, has been extensively explored in numerous cancer-related studies. The C-terminal domain (CTD) of p53 is an intrinsically disordered region that gains multiple different conformations at interaction with different binding partners. However, the impact of the surrounding environment on the structural preference of p53-CTD is not known. We investigated the impact of the surrounding environment on the conformational behavior and folding of p53-CTD. Although the entire CTD is predicted as a highly disordered region by several commonly used disorder predictors, based on the secondary structure prediction, we find that a part of the CTD sequence (residues 380-388) is "confused", being predicted to shuffle between the irregular, α-helical and ß-strand structures. First time, we are observing the effect of folding-induced organic solvents, trifluoroethanol and methanol, on the conformation of CTD. Water-miscible organic solvents exert hydrophobic interactions, which are major driving force to trigger structural changes in CTD. By lowering the solution dielectric constant, organic solvents can also strengthen electrostatic interactions. We have also performed Replica Exchange Molecular Dynamic (REMD) simulations for enhanced conformation sampling of the peptide. These simulation studies have also provided detailed insight into the peculiarities of this peptide, explaining its folding behavior in the presence of methanol. We consider that these hydrophobic interactions may have important roles for function-related structural changes of this disordered region.

Mammalian antimicrobial peptide protegrin-4 self assembles and forms amyloid-like aggregates: Assessment of its functional relevance.

Protegrin-4 (PG-4) is a member of the porcine leukocyte protegrins family of cysteine-rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the ß sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG-4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin-T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non-toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide-based antimicrobials with enhanced stability.

The mechanism of phosphatidylcholine-induced interference of PAP (248-286) aggregation.

Seminal amyloids are well known for their role in enhancing HIV infection. Among all the amyloidogenic peptides identified in human semen, PAP248-286 was found to be the most active and was termed as semen-derived enhancer of viral infection (SEVI). Although amyloidogenic nature of the peptide is mainly linked with enhancement of the viral infection, the most active physiological conformation of the aggregated peptide remains inconclusive. Lipids are known to modulate aggregation pathway of a variety of proteins and peptides and constitute one of the most abundant biomolecules in human semen. PAP248-286 significantly differs from the other known amyloidogenic peptides, including Aß and IAPP, in terms of critical concentration, surface charge, fibril morphology, and structural transition during aggregation. Hence, in the present study, we aimed to assess the effect of a lipid, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), on PAP248-286 aggregation and the consequent conformational outcomes. Our initial observation suggested that the presence of the lipid considerably influenced the aggregation of PAP248-286 . Further, ZDOCK and MD simulation studies of peptide multimerization have suggested that the hydrophobic residues at C-terminus are crucial for PAP248-286 aggregation and are anticipated to be major DOPC-interacting partners. Therefore, we further assessed the aggregation behaviour of C-terminal (PAP273-286 ) fragment of PAP248-286 and observed that DOPC possesses the ability to interfere with the aggregation behaviour of both the peptides used in the current study. Mechanistically, we propose that the presence of DOPC causes considerable inhibition of the peptide aggregation by interfering with the peptide's disordered state to ß-sheet transition.

Role of the glutaredoxin domain and FAD in the stabilization of thioredoxin glutathione reductase.

Thioredoxin glutathione reductase (TGRsec) is a multi-domain flavoprotein that plays a principal role in redox homeostasis maintenance. We have previously demonstrated the role of selenocysteine in maintaining TGRsec structure-function, but the role of the glutaredoxin (Grx) domain and FAD is still unclear. In the present study, the urea-induced unfolding of recombinant Fasciola gigantica TGRsec (FgTGRsec) and its N-terminal truncated variant (ΔNTD-FgTGRsec) were examined to understand the role of the Grx domain and FAD in the stabilization of FgTGRsec and ΔNTD-FgTGRsec. Our results showed that both proteins underwent unfolding in a three state manner. First, the protein undergoes a conformational transition rendering a near-native state with no FAD bound, and then full unfolding of the apo-dimer occurs without dissociation. The Grx domain stabilized the global FgTGRsec structure and positively regulated FgTGRsec activity, and alteration in the FAD microenvironment was directly proportional to the loss of thioredoxin reductase (TrxR) and glutathione reductase activities. Based on these results, we concluded that the Grx domain stabilizes the full-length FgTGRsec protein for efficient catalysis. Thus, we suggest that in platyhelminth parasites, during evolution, the Grx domain merged with the TrxR domain to confer higher catalytic activity and provide additional structural stability to the full-length TGR.

Understanding the frustration arising from the competition between function, misfolding, and aggregation in a globular protein.

Folding and function may impose different requirements on the amino acid sequences of proteins, thus potentially giving rise to conflict. Such a conflict, or frustration, can result in the formation of partially misfolded intermediates that can compromise folding and promote aggregation. We investigate this phenomenon by studying frataxin, a protein whose normal function is to facilitate the formation of iron-sulfur clusters but whose mutations are associated with Friedreich's ataxia. To characterize the folding pathway of this protein we carry out a Φ-value analysis and use the resulting structural information to determine the structure of the folding transition state, which we then validate by a second round of rationally designed mutagenesis. The analysis of the transition-state structure reveals that the regions involved in the folding process are highly aggregation-prone. By contrast, the regions that are functionally important are partially misfolded in the transition state but highly resistant to aggregation. Taken together, these results indicate that in frataxin the competition between folding and function creates the possibility of misfolding, and that to prevent aggregation the amino acid sequence of this protein is optimized to be highly resistant to aggregation in the regions involved in misfolding.