Structural impairment of p53 C-terminal due to the effect of phosphorylation and acetylation: a study on the interdependence of PTM.

The C-terminal of tumor suppressor protein p53 is intrinsically disordered while unbound. This particular segment often shows structural plasticity when bound to other binding partners. The disordered component undergoes a disordered to ordered transition upon recognition. Post-translational modifications (PTMs), namely phosphorylation and acetylation, significantly alter the structural motifs of the segment. Among the various types of PTMs, phosphorylation, and acetylation of p53 at both N- and C- terminals lead to stabilization and activation. It has been noted experimentally that phosphorylation often regulates (enhances or reduces) the acetylation at specific sites. The phosphorylation of Thr377 and Ser378 reduces the acetylation of Lys373 and Lys382. Mutations of Thr377 and Ser378 to neutral Ala enhance and phospho mimic Asp reduce the acetylation of Lys373 and Lys382. Simulations of several single-point and pair-wise mutated systems have been generated to compare how the presence or absence of phosphorylation favors or disfavors the acetylation by thermodynamic and conformational analysis. We are using implicit solvent replica exchange molecular dynamics simulations to get 200 ns well-converged conformational ensembles of each system. Different sets of systems having both single and double PTMs are simulated. The results admit the appreciable change in the secondary structural level upon specific PTM. Also, the residual structure of the unbound p53 with single-point PTM varies significantly with pair-wise modifications. These observations further shed light on the relationship between the interdependencies of the specific PTM sites and the secondary structural levels.Communicated by Ramaswamy H. Sarma.

2D Metal-Organic Frameworks: Properties, Synthesis, and Applications in Electrochemical and Optical Biosensors.

Two-dimensional (2D) nanomaterials like graphene, layered double hydroxides, etc., have received increasing attention owing to their unique properties imparted by their 2D structure. The newest member in this family is based on metal-organic frameworks (MOFs), which have been long known for their exceptional physicochemical properties-high surface area, tunable pore size, catalytic properties, etc., to list a few. 2D MOFs are promising materials for various applications as they combine the exciting properties of 2D materials and MOFs. Recently, they have been extensively used in biosensors by virtue of their enormous surface area and abundant, accessible active sites. In this review, we provide a synopsis of the recent progress in the field of 2D MOFs for sensor applications. Initially, the properties and synthesis techniques of 2D MOFs are briefly outlined with examples. Further, electrochemical and optical biosensors based on 2D MOFs are summarized, and the associated challenges are outlined.

Structural modulation of p53TAD1-TAZ2 complex upon mutations and post-translational modification.

The tumour suppressing p53 is a target for genetic alterations in human cancer. Native p53, found in latent state in cells, gets activated following various intracellular or extracellular responses. It plays imperative role in cell-cycle control, via growth-arrest, DNA repair and apoptosis, mainly regulated by post-translational modifications (PTM). However, the influence of PTMs on the activity of p53 is still under extensive experimental and computational study. There are numerous PTM sites in p53, which are reported to regulate its binding affinities with other proteins. Of the many, Thr18 at transactivational domain (TAD) of p53 is reported to amplify p53 activity upon phosphorylation. To understand the molecular basis of p53 recognition by its binding partner upon mutations and PTMs, we have exploited all atom molecular dynamic (MD) simulation of p53TAD1 bound to TAZ2 domain of p300. The MD simulation inferred that phosphorylated and mutated Thr18, as a phospho-mimic, bound with TAZ2, redistributed the charge environment of the interface, thereby modulating the stronger interactions with TAZ2 to enhance the binding efficiency. The electrostatic interactions due to different charge environment together with H-bonding and hydrophobic interaction dictate diverse binding approach between the two. The results of this computational study further explain the importance of the Thr18 as a PTM site in atomistic detail, hence shedding further light to the understanding of how PTMs are imperative for p53 activity to protect the cellular world.Communicated by Ramaswamy H. Sarma.

Single Amino-Acid Based Self-Assembled Biomaterials with Potent Antimicrobial Activity.

The design and development of soft biomaterials based on amino acid and short-peptide have gained much attention due to their potent biomedical applications. A slight alteration in the side-chain of single amino acid in a peptide or protein sequence has a huge impact on the structure and function. Phenylalanine is one of the most studied amino acids, which contains an aromatic phenyl group connected through a flexible -CH2 - unit. In this work, we have examined whether flexibility and aromatic functionality of phenylalanine (Phe) are important in gel formation of model gelator Fmoc-Phe-OH or not. To examine this hypothesis, we synthesized Fmoc-derivatives of three analogues unnatural amino acids including cyclohexylalanine, phenylglycine, and homophenylalanine; which are slightly varied from Phe. Interestingly, all these three new analogues formed hydrogels in phosphate buffer at pH 7.0 having different gelation efficacy and kinetics. This study suggests that the presence of aromatic side-chain and flexibility are not mandatory for the gelation of this model gelator. Newly synthesized unnatural amino acid derivatives have also exhibited promising antimicrobial activity towards gram-positive bacteria by inhibiting cellular oxygen consumption. We further determined the biocompatibility of these amino acid derivatives by using a hemolysis assay on human blood cells. Overall studies described the development of single amino acid-based new injectable biomaterials with improved antimicrobial activity by the slight alteration in the side-chain of amino acid.

Spike protein mutational landscape in India during the complete lockdown phase: Could Muller's ratchet be a future game-changer for COVID-19?

The dire need of effective preventive measures and treatment approaches against SARS-CoV-2 virus, causing COVID-19 pandemic, calls for an in-depth understanding of its evolutionary dynamics with attention to specific geographic locations, since lockdown and social distancing to prevent the virus spread could lead to distinct localized dynamics of virus evolution within and between countries owing to different environmental and host-specific selection pressures. To decipher any correlation between SARS-CoV-2 evolution and its epidemiology in India, we studied the mutational diversity of spike glycoprotein, the key player for the attachment, fusion and entry of virus to the host cell. For this, we analyzed the sequences of 630 Indian isolates as available in GISAID database till June 07, 2020 (during the time-period before the start of Unlock 1.0 in India on and from June 08, 2020), and detected the spike protein variants to emerge from two major ancestors - Wuhan-Hu-1/2019 and its D614G variant. Average stability of the docked spike protein - host receptor (S-R) complexes for these variants correlated strongly (R2 = 0.96) with the fatality rates across Indian states. However, while more than half of the variants were found unique to India, 67% of all variants showed lower stability of S-R complex than the respective ancestral variants, indicating a possible fitness loss in recently emerged variants, despite a continuous increase in mutation rate. These results conform to the sharply declining fatality rate countrywide (>7-fold during April 11 - June 28, 2020). Altogether, while we propose the potential of S-R complex stability to track disease severity, we urge an immediate need to explore if SARS-CoV-2 is approaching mutational meltdown in India.

Magnetic field assisted high capacity durable Li-ion battery using magnetic α-Fe2O3 nanoparticles decorated expired drug derived N-doped carbon anode.

We have synthesized a novel ferromagnetic material by coating α-Fe2O3 nanoparticles with N-doped carbon matrix using a simple combustion method. Expired paracetamol drugs are used as nitrogen and carbon source. This α-Fe2O3/NC shows ferromagnetic property due to the incorporation of oxygen defects. When used as the Li-ion battery anode, α-Fe2O3/NC shows higher capacity compared to commercial α-Fe2O3 due to the occurrence of both intercalation and conversion reaction. Further, application of magnetic field at the anode of the freshly assembled cell at the first charge-discharge cycle, results in ~two-fold enhancement in specific capacity. For the cycled cell also, increase in the capacity from 80 mAh. g-1 to 150 mAh. g-1 at 5 A. g-1 is observed during the application of magnetic field at the 501st charging cycle. This improved performance is attributed to the field-dependent enhancement of diffusion and convection due to the magnetohydrodynamic effect. Further, application of the magnetic field at 1001st, 1501st and 1751st charging cycles shows improved LIB performance. We can show that not only the magnetic field, magnetic properties of the anode α-Fe2O3/NC also play a crucial role in influencing the battery performance. Moreover, utilization of expired drug helps in dramatically reducing pollution caused by its disposal.

Premature Termination of MexR Leads to Overexpression of MexAB-OprM Efflux Pump in Pseudomonas aeruginosa in a Tertiary Referral Hospital in India.

OBJECTIVES: The present study was undertaken to investigate the mutations that are present in mexR gene of multidrug resistant (MDR) isolates of Pseudomonas aeruginosa collected from a tertiary referral hospital of north east India. METHODS: 76 MDR clinical isolates of P. aeruginosa were obtained from the patients who were admitted to or attended the clinics of Silchar medical college and hospital. They were screened phenotypically for the presence of efflux pump activity by an inhibitor based method. Acquired resistance mechanisms were detected by multiplex PCR. Real time PCR was performed to study the expression of mexA gene of MexAB-OprM efflux pump in isolates with increase efflux pump activity. mexR gene of the isolates with overexpressed MexAB-OprM efflux pump was amplified, sequenced and analysed. RESULTS: Out of 76 MDR isolates, 24 were found to exhibit efflux pump activity phenotypically against ciprofloxacin and meropenem. Acquired resistance mechanisms were absent in 11 of them and among those isolates, 8 of them overexpressed MexAB-OprM. All the 8 isolates possessed mutation in mexR gene. 11 transversions, 4 transitions, 2 deletion mutations and 2 insertion mutations were found in all the isolates. However, the most significant observation was the formation of a termination codon at 35th position which resulted in the termination of the polypeptide and leads to overexpression of the MexAB-OprM efflux pump. CONCLUSIONS: This study highlighted emergence of a novel mutation which is probably associated with multi drug resistance. Therefore, further investigations and actions are needed to prevent or at least hold back the expansion and emergence of newer mutations in nosocomial pathogens which may compromise future treatment options.

A novel peptide interferes with Mycobacterium tuberculosis virulence and survival.

Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming rate, necessitating an urgent need for a new class of drug candidates. Here, we report that SL3, a novel 33-amino acid peptide, causes debilitating effects on mycobacterial morphology. Treatment with SL3 drastically inhibits the growth of Mycobacterium tuberculosis in vitro as well as in a pre-clinical mouse model for M.tb infection. Microarray analysis of SL3-expressing strain demonstrates wide-scale transcriptional disruption in M.tb. We therefore believe that SL3 and similar peptides may herald a new approach towards discovering new molecules for TB therapy.

A peptide fragment from the human COX3 protein disrupts association of Mycobacterium tuberculosis virulence proteins ESAT-6 and CFP10, inhibits mycobacterial growth and mounts protective immune response.

BACKGROUND: Tuberculosis (TB) is one of the most prevalent infectious diseases affecting millions worldwide. The currently available anti-TB drugs and vaccines have proved insufficient to contain this scourge, necessitating an urgent need for identification of novel drug targets and therapeutic strategies. The disruption of crucial protein-protein interactions, especially those that are responsible for virulence in Mycobacterium tuberculosis - for example the ESAT-6:CFP10 complex - are a worthy pursuit in this direction. METHODS: We therefore sought to improvise a method to attenuate M. tuberculosis while retaining the latter's antigenic properties. We screened peptide libraries for potent ESAT-6 binders capable of dissociating CFP10 from ESAT-6. We assessed the disruption by a peptide named HCL2, of the ESAT-6:CFP10 complex and studied its effects on mycobacterial survival and virulence. RESULTS: We found that HCL2, derived from the human cytochrome c oxidase subunit 3 (COX3) protein, disrupts ESAT-6:CFP10 complex, binds ESAT-6 potently, disintegrates bacterial cell wall and inhibits extracellular as well as intracellular mycobacterial growth. In addition, an HCL2 expressing M. tuberculosis strain induces both Th1 and Th17 host protective responses. CONCLUSIONS: Disruption of ESAT-6:CFP10 association could, therefore, be an alternate method for attenuating M. tuberculosis, and a possible route towards future vaccine generation.

Expression of the ARPC4 subunit of human Arp2/3 severely affects mycobacterium tuberculosis growth and suppresses immunogenic response in murine macrophages.

BACKGROUND: The search for molecules against Mycobacterium tuberculosis is urgent. The mechanisms facilitating the intra-macrophage survival of Mycobacterium tuberculosis are as yet not entirely understood. However, there is evidence showing the involvement of host cell cytoskeleton in every step of establishment and persistence of mycobacterial infection. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that expression of ARPC4, a subunit of the Actin related protein 2/3 (Arp2/3) protein complex, severely affects the pathogen's growth. TEM studies display shedding of the mycobacterial outer-coat. Furthermore, in infected macrophages, mycobacteria expressing ARPC4 were cleared off at a much faster rate, and were unable to mount a pro-inflammatory cytokine response. The translocation of ARPC4-expressing mycobacteria to the lysosome of the infected macrophage was also impaired. Additionally, the ARPC4 subunit was shown to interact with Rv1626, an essential secretory mycobacterial protein. Real-time PCR analysis showed that upon expression of ARPC4 in mycobacteria, Rv1626 expression is downregulated as much as six-fold. Rv1626 was found to also interact with mammalian cytoskeleton protein, Arp2/3, and enhance the rate of actin polymerization. CONCLUSIONS/SIGNIFICANCE: With crystal structures for Rv1626 and ARPC4 subunit already known, our finding lays out the effect of a novel molecule on mycobacteria, and represents a viable starting point for developing potent peptidomimetics.

A three-hybrid system to probe in vivo protein-protein interactions: application to the essential proteins of the RD1 complex of M. tuberculosis.

BACKGROUND: Protein-protein interactions play a crucial role in enabling a pathogen to survive within a host. In many cases the interactions involve a complex of proteins rather than just two given proteins. This is especially true for pathogens like M. tuberculosis that are able to successfully survive the inhospitable environment of the macrophage. Studying such interactions in detail may help in developing small molecules that either disrupt or augment the interactions. Here, we describe the development of an E. coli based bacterial three-hybrid system that can be used effectively to study ternary protein complexes. METHODOLOGY/PRINCIPAL FINDINGS: The protein-protein interactions involved in M. tuberculosis pathogenesis have been used as a model for the validation of the three-hybrid system. Using the M. tuberculosis RD1 encoded proteins CFP10, ESAT6 and Rv3871 for our proof-of-concept studies, we show that the interaction between the proteins CFP10 and Rv3871 is strengthened and stabilized in the presence of ESAT6, the known heterodimeric partner of CFP10. Isolating peptide candidates that can disrupt crucial protein-protein interactions is another application that the system offers. We demonstrate this by using CFP10 protein as a disruptor of a previously established interaction between ESAT6 and a small peptide HCL1; at the same time we also show that CFP10 is not able to disrupt the strong interaction between ESAT6 and another peptide SL3. CONCLUSIONS/SIGNIFICANCE: The validation of the three-hybrid system paves the way for finding new peptides that are stronger binders of ESAT6 compared even to its natural partner CFP10. Additionally, we believe that the system offers an opportunity to study tri-protein complexes and also perform a screening of protein/peptide binders to known interacting proteins so as to elucidate novel tri-protein complexes.

Phenylalanine-rich peptides potently bind ESAT6, a virulence determinant of Mycobacterium tuberculosis, and concurrently affect the pathogen's growth.

BACKGROUND: The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung. METHODOLOGY/PRINCIPAL FINDINGS: During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage. CONCLUSIONS/SIGNIFICANCE: While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide's binding to Esat6-as the latter is not an essential protein of M. tuberculosis-nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.