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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.
Exploring the SARS-CoV-2 structural proteins for multi-epitope vaccine development: an in-silico approach.
INTRODUCTION: The ongoing life-threatening pandemic of coronavirus disease 2019 (COVID-19) has extensively affected the world. During this global health crisis, it is fundamentally crucial to find strategies to combat SARS-CoV-2. Despite several efforts in this direction and continuing clinical trials, no vaccine has been approved for it yet. METHODS: To find a preventive measure, we have computationally designed a multi-epitopic subunit vaccine using immuno-informatic approaches. RESULTS: The structural proteins of SARS-CoV-2 involved in its survival and pathogenicity were used to predict antigenic epitopes. The antigenic epitopes were capable of eliciting a strong humoral as well as cell-mediated immune response, our predictions suggest. The final vaccine was constructed by joining the all epitopes with specific linkers and to enhance their stability and immunogenicity. The physicochemical property of the vaccine was assessed. The vaccine 3D structure prediction and validation were done and docked with the human TLR-3 receptor. Furthermore, molecular dynamics simulations of the vaccine-TLR-3 receptor complex are employed to assess its dynamic motions and binding stability in-silico. CONCLUSION: Based on this study, we strongly suggest synthesizing this vaccine, which further can be tested in-vitro and in-vivo to check its potency in a cure for COVID-19.
Japanese encephalitis virus - exploring the dark proteome and disorder-function paradigm.
Japanese encephalitis virus (JEV) is one of the major causes of viral encephalitis all around the globe. Approximately 3 billion people in endemic areas are at risk of Japanese encephalitis. To develop a wholistic understanding of the viral proteome, it is important to investigate both its ordered and disordered proteins. However, the functional and structural significance of disordered regions in the JEV proteome has not been systematically investigated as of yet. To fill this gap, we used here a set of bioinformatics tools to analyze the JEV proteome for the predisposition of its proteins for intrinsic disorder and for the presence of the disorder-based binding regions (also known as molecular recognition features, MoRFs). We also analyzed all JEV proteins for the presence of the probable nucleic acid-binding (DNA and RNA) sites. The results of these computational studies are experimentally validated using JEV capsid protein as an illustrative example. In agreement with bioinformatic analysis, we found that the N-terminal region of the JEV capsid (residues 1-30) is intrinsically disordered. We showed that this region is characterized by the temperature response typical for highly disordered proteins. Furthermore, we have experimentally shown that this disordered N-terminal domain of a capsid protein has a noticeable 'gain-of-structure' potential. In addition, using DOPS liposomes, we demonstrated the presence of pronounced membrane-mediated conformational changes in the N-terminal region of JEV capsid. In our view, this disorder-centric analysis would be helpful for a better understanding of the JEV pathogenesis.
Salvianolic acid B noncovalently interacts with disordered c-Myc: a computational and spectroscopic-based study.
Aims: c-Myc, along with its partner MAX, regulates the expression of several genes, leading to an oncogenic phenotype. The MAX interacting interface of c-Myc is disordered and uncharacterized for small molecule binding. Salvianolic acid B possesses numerous therapeutic properties, including anticancer activity. The current study was designed to elucidate the interaction of the Sal_Ac_B with the disordered bHLH domain of c-Myc using computational and biophysical techniques. Materials & methods: The binding of Sal_Ac_B with Myc was studied using computational and biophysical techniques, including molecular docking and simulation, fluorescence lifetime, circular dichroism and anisotropy. Results & conclusions: The study demonstrated a high binding potential of Sal_Ac_B against the disordered Myc peptide. The binding of the compounds leads to an overall conformational change in Myc. Moreover, an extensive simulation study showed a stable Sal_Ac_B/Myc binding.
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.
Reprofiling of approved drugs against SARS-CoV-2 main protease: an in-silico study.
Given the COVID-19 pandemic, currently, there are many drugs in clinical trials against this virus. Among the excellent drug targets of SARS-CoV-2 are its proteases (Nsp3 and Nsp5) that plays vital role in polyprotein processing giving rise to functional nonstructural proteins, essential for viral replication and survival. Nsp5 (also known as Mpro) hydrolyzes replicase polyprotein (1ab) at eleven different sites. For targeting Mpro, we have employed drug repurposing approach to identify potential inhibitors of SARS-CoV-2 in a shorter time span. Screening of approved drugs through docking reveals Hyaluronic acid and Acarbose among the top hits which are showing strong interactions with catalytic site residues of Mpro. We have also performed docking of drugs Lopinavir, Ribavirin, and Azithromycin on SARS-CoV-2 Mpro. Further, binding of these compounds (Hyaluronic acid, Acarbose, and Lopinavir) is validated by extensive molecular dynamics simulation of 500 ns where these drugs show stable binding with Mpro. We believe that the high-affinity binding of these compounds will help in designing novel strategies for structure-based drug discovery against SARS-CoV-2.Communicated by Ramaswamy H. Sarma.
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.
Microsecond simulations and CD spectroscopy reveals the intrinsically disordered nature of SARS-CoV-2 spike-C-terminal cytoplasmic tail (residues 1242-1273) in isolation.
All available SARS-CoV-2 spike protein crystal and cryo-EM structures have shown missing electron densities for cytosolic C-terminal regions (CTR). Generally, the missing electron densities point towards the intrinsically disordered nature of the protein region (IDPR). This curiosity has led us to investigate the cytosolic CTR of the spike glycoprotein of SARS-CoV-2 in isolation. The spike CTR is supposed to be from 1235 to 1273 residues or 1242-1273 residues based on our used prediction. Therefore, we have demonstrated the structural conformation of cytosolic region and its dynamics through computer simulations up to microsecond timescale using OPLS and CHARMM forcefields. The simulations have revealed the unstructured conformation of cytosolic region. Further, we have validated our computational observations with circular dichroism (CD) spectroscopy-based experiments and found its signature spectra at 198 nm. We believe that our findings will surely help in understanding the structure-function relationship of the spike protein's cytosolic region.
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.
Mitoxantrone dihydrochloride, an FDA approved drug, binds with SARS-CoV-2 NSP1 C-terminal.
One of the major virulence factors of SARS-CoV-2, NSP1, is a vital drug target due to its role in host immune evasion through multiple pathways. NSP1 protein is associated with inhibiting host mRNA translation by binding to the small subunit of ribosome through its C-terminal region. Previously, we have shown the structural dynamics of the NSP1 C-terminal region (NSP1-CTR) in different physiological environments. So, it would be very interesting to investigate the druggable compounds that could bind with NSP1-CTR. Here, in this article, we have performed different spectroscopic technique-based binding assays of an anticancer drug mitoxantrone dihydrochloride (MTX) against the NSP1-CTR. We have also performed molecular dynamics simulations of the docked complex with two different force fields up to one microsecond. Overall, our results have suggested good binding between NSP1-CTR and MTX and may have implications in developing therapeutic strategies targeting the NSP1 protein of SARS-CoV-2.
Detection of neuronal OFF periods as low amplitude neural activity segments
Abstract Background During non-rapid eye movement sleep (NREM), alternating periods of synchronised high (ON period) and low (OFF period) neuronal activity are associated with high amplitude delta band (0.5–4 Hz) oscillations in neocortical electrophysiological signals termed slow waves. As this oscillation is dependent crucially on hyperpolarisation of cortical cells, there is an interest in understanding how neuronal silencing during OFF periods leads to the generation of slow waves and whether this relationship changes between cortical layers. A formal, widely adopted definition of OFF periods is absent, complicating their detection. Here, we grouped segments of high frequency neural activity containing spikes, recorded as multiunit activity from the neocortex of freely behaving mice, on the basis of amplitude and asked whether the population of low amplitude (LA) segments displayed the expected characteristics of OFF periods. Results Average LA segment length was comparable to previous reports for OFF periods but varied considerably, from as short as 8 ms to > 1 s. LA segments were longer and occurred more frequently in NREM but shorter LA segments also occurred in half of rapid eye movement sleep (REM) epochs and occasionally during wakefulness. LA segments in all states were associated with a local field potential (LFP) slow wave that increased in amplitude with LA segment duration. We found that LA segments > 50 ms displayed a homeostatic rebound in incidence following sleep deprivation whereas short LA segments (< 50 ms) did not. The temporal organisation of LA segments was more coherent between channels located at a similar cortical depth. Conclusion We corroborate previous studies showing neural activity signals contain uniquely identifiable periods of low amplitude with distinct characteristics from the surrounding signal known as OFF periods and attribute the new characteristics of vigilance-state-dependent duration and duration-dependent homeostatic response to this phenomenon. This suggests that ON/OFF periods are currently underdefined and that their appearance is less binary than previously considered, instead representing a continuum.
Autophagy preserves hematopoietic stem cells by restraining mTORC1-mediated cellular anabolism
Adult stem cells are long-lived and quiescent with unique metabolic requirements. Macroautophagy/autophagy is a fundamental survival mechanism that allows cells to adapt to metabolic changes by degrading and recycling intracellular components. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained mTOR (mammalian target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. mTOR inhibition by rapamycin treatment in vivo was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting mTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation.
Explaining paranoia: cognitive and social processes in the occurrence of extreme mistrust.
BACKGROUND: Paranoia-incorrectly thinking that others are deliberating trying to harm you-causes distress, undermines social interactions and leads to withdrawal. It presents across multiple psychiatric diagnoses. OBJECTIVE: The primary aim was to determine the extent that cognitive and social processes may explain paranoia. The secondary aim was to identify explanatory factors that distinguished paranoia and social anxiety. METHODS: 10 382 UK adults, quota sampled to match the population for age, gender, ethnicity, income and region, participated in a non-probability survey. All participants completed a paranoia measure and assessments of cognitive and social processes. Structural equation modelling was conducted. FINDINGS: 2586 (24.9%) participants described being mistrustful of other people. 1756 (16.9%) participants wanted help to trust more. 66.7% of variance in paranoia was explained by a model comprising (in descending order of importance): within-situation defence behaviours, negative images, negative self-beliefs, discrimination, dissociation, aberrant salience, anxiety sensitivity, agoraphobic distress, worry, less social support, agoraphobic avoidance, less analytical reasoning and alcohol use. All explanatory factors were associated with paranoia and social anxiety. Ten factors were more closely associated with paranoia than social anxiety, including discrimination, hallucinations, negative images, aberrant salience and alcohol use. Nine factors were more closely associated with social anxiety, including less positive self-belief, an external locus of control, worry and less analytical reasoning. CONCLUSIONS: Multiple causes are likely to be involved in paranoia. Cognitive and social processes may explain a high degree of paranoia. CLINICAL IMPLICATIONS: Multiple clear targets for intervention to reduce paranoia are identified.
The signal peptide of the amyloid precursor protein forms amyloid-like aggregates and enhances Aβ42 aggregation
Signal sequences are short peptides at the N terminus of proteins destined for the secretion pathway. Typically, after cleavage by peptidases, signal peptides are degraded by intra-membrane proteases. In some cases, however, signal peptides can be processed further and released into the endoplasmic reticulum, secretion pathways, or cytoplasm. The consequences of these processes remain unclear, in particular considering that dysregulated signal peptides could potentially aggregate and induce cytotoxicity. To investigate this problem, we study the signal peptide of the amyloid precursor protein (APP), which originates the Alzheimer's β-amyloid (Aβ) peptide. Our results show that this signal peptide (residues 1–17 of APP [APP1–17SP]) can form amyloid-like cytotoxic aggregates. We further demonstrate that APP1–17SP seeds promote aggregation of Aβ, which raises the intriguing possibility of an interplay between APP1–17SP and Aβ aggregation in disease processes.