Complementary Omics Strategies to Dissect p53 Signaling Networks Under Nutrient Stress

Signaling trough p53 is a major cellular stress response mechanism and increases upon nutrient stresses such as starvation. Here, we show in a human hepatoma cell line that starvation leads to robust nuclear p53 stabilization. Using BioID, we determine the cytoplasmic p53 interaction network within the immediate-early starvation response and show that p53 is dissociated from several metabolic enzymes and the kinase PAK2 for which direct binding with the p53 DNA-binding domain was confirmed with NMR studies. Furthermore, proteomics after p53 immunoprecipitation (RIME) uncovered the nuclear interactome under prolonged starvation, where we confirmed the novel p53 interactors SORBS1 (insulin receptor signaling) and UGP2 (glycogen synthesis). Finally, transcriptomics after p53 re-expression revealed a distinct starvation-specific transcriptome response and suggested previously unknown nutrient-dependent p53 target genes. Together, our complementary approaches delineate several nodes of the p53 signaling cascade upon starvation, shedding new light on the mechanisms of p53 as nutrient stress sensor. Given the central role of p53 in cancer biology and the beneficial effects of fasting in cancer treatment, the identified interaction partners and networks could pinpoint novel pharmacologic targets to fine-tune p53 activity.

Unraveling the Mechanism of Platelet Aggregation Suppression by Monoterpenoids

Platelet aggregation causes various diseases and therefore challenges the development of novel antiaggregatory drugs. In this study, we report the possible mechanism of platelet aggregation suppression by newly synthesized myrtenol-derived monoterpenoids carrying different heteroatoms (sulphur, oxygen, or nitrogen). Despite all tested compounds suppressed the platelet aggregation in vitro, the most significant effect was observed for the S-containing compounds. The molecular docking confirmed the putative interaction of all tested compounds with the platelet’s P2Y12 receptor suggesting that the anti-aggregation properties of monoterpenoids are implemented by blocking the P2Y12 function. The calculated binding force depended on heteroatom in monoterpenoids and significantly decreased with the exchanging of the sulphur atom with oxygen or nitrogen. On the other hand, in NMR studies on dodecyl phosphocholine (DPC) as a membrane model, only S-containing compound was found to be bound with DPC micelles surface. Meanwhile, no stable complexes between DPC micelles with either O- or N-containing compounds were observed. The binding of S-containing compound with cellular membrane reinforces the mechanical properties of the latter, thereby preventing its destabilization and subsequent clot formation on the phospholipid surface. Taken together, our data demonstrate that S-containing myrtenol-derived monoterpenoid suppresses the platelet aggregation in vitro via both membrane stabilization and blocking the P2Y12 receptor and, thus, appears as a promising agent for hemostasis control.

Exploring the Bioactive Potentials of C60-AgNPs Nano-Composites against Malignancies and Microbial Infections

At present, the potential role of the AgNPs/endo-fullerene molecule metal nano-composite has been evaluated over the biosystems in-vitro. The intra-atomic configuration of the fullerene molecule (C60) has been studied in-vitro for the anti-proliferative activity of human breast adenocarcinoma (MDA-MB-231) cell lines and antimicrobial activity against a few human pathogens that have been augmented with the pristine surface plasmonic electrons and antibiotic activity of AgNPs. Furthermore, FTIR revealed the basic vibrational signatures at ~3300 cm−1, 1023 cm−1, 1400 cm−1 for O-H, C-O, and C-H groups, respectively, for the carbon and oxygen atoms of the C60 molecule. NMR studies exhibited the different footprints and magnetic moments at ~7.285 ppm, explaining the unique underlying electrochemical attributes of the fullerene molecule. Such unique electronic and physico-chemical properties of the caged carbon structure raise hope for applications into the drug delivery domain. The in-vitro dose-dependent application of C60 elicits a toxic response against both the breast adenocarcinoma cell lines and pathogenic microbes. That enables the use of AgNPs decorated C60 endo fullerene molecules to design an effective anti-cancerous drug delivery and antimicrobial agent in the future, bringing a revolutionary change in the perspective of a treatment regime.

Lipoate protein ligase B primarily recognizes the C8-phosphopantetheine arm of its donor substrate and weakly binds the acyl carrier protein

Lipoic acid is a sulfur containing cofactor, indispensable for the function of several metabolic enzymes. In microorganisms, lipoic acid can be salvaged from the surroundings by Lipoate protein ligase A (LplA), an ATP-dependent enzyme. Alternatively, it can be synthesized by the sequential action of Lipoate protein ligase B (LipB) and Lipoyl synthase (LipA). LipB uptakes octanoyl- chain from C8-acyl carrier protein (C8-ACP), a byproduct of the type II fatty acid synthesis pathway and transfers it to a conserved lysine of the lipoyl domain of a dehydrogenase. The molecular basis of substrate recognition by LipB is still not fully understood. Using E. coli LipB as a model enzyme, we show that an octanoyl-transferase mainly recognizes the 4-phosphopantetheine tethered acyl-chain of its donor substrate and weakly binds the apo-acyl carrier protein. LipB can accept octanoate- from its own ACP, noncognate ACPs, as well as C8-CoA. Further, our NMR studies demonstrate the presence of an adenine and phosphate binding site in LipB, akin to LplA. A loop containing 71RGG73 sequence, analogous to the lipoate binding loop of LplA is also conserved in LipB. Collectively, our studies highlight commonalities between LipB and LplA in their mechanism of substrate recognition. This knowledge might be of significance in the treatment of mitochondrial fatty acid synthesis related disorders.

Identification and characterization of two drug-like fragments that bind to the same cryptic binding pocket of Burkholderia pseudomallei DsbA

Disulfide-bond-forming proteins (Dsbs) play a crucial role in the pathogenicity of many Gram-negative bacteria. Disulfide-bond-forming protein A (DsbA) catalyzes the formation of the disulfide bonds necessary for the activity and stability of multiple substrate proteins, including many virulence factors. Hence, DsbA is an attractive target for the development of new drugs to combat bacterial infections. Here, two fragments, bromophenoxy propanamide (1) and 4-methoxy-N-phenylbenzenesulfonamide (2), were identified that bind to DsbA from the pathogenic bacterium Burkholderia pseudomallei, the causative agent of melioidosis. The crystal structures of oxidized B. pseudomallei DsbA (termed BpsDsbA) co-crystallized with 1 or 2 show that both fragments bind to a hydrophobic pocket that is formed by a change in the side-chain orientation of Tyr110. This conformational change opens a `cryptic' pocket that is not evident in the apoprotein structure. This binding location was supported by 2D-NMR studies, which identified a chemical shift perturbation of the Tyr110 backbone amide resonance of more than 0.05 p.p.m. upon the addition of 2 mM fragment 1 and of more than 0.04 p.p.m. upon the addition of 1 mM fragment 2. Although binding was detected by both X-ray crystallography and NMR, the binding affinity (K d) for both fragments was low (above 2 mM), suggesting weak interactions with BpsDsbA. This conclusion is also supported by the crystal structure models, which ascribe partial occupancy to the ligands in the cryptic binding pocket. Small fragments such as 1 and 2 are not expected to have a high energetic binding affinity due to their relatively small surface area and the few functional groups that are available for intermolecular interactions. However, their simplicity makes them ideal for functionalization and optimization. The identification of the binding sites of 1 and 2 to BpsDsbA could provide a starting point for the development of more potent novel antimicrobial compounds that target DsbA and bacterial virulence.

Synthesis of a hydrophilic derivative of ecdysterone and development of its water-soluble form

The article presents materials on the isolation of ecdysterone substance from medicinal plant raw materials Silene wolgensis (Hornem.) Bess. ex. Spreng (Volga smolyovka). For the first time, the optimization of the method for ecdysterone substance obtaining from the aboveground part of the superconcentrator of phytoecdysteroids of the Silene wolgensis was carried out and based on it a pilot industrial regulation for the isolation of ecdysterone and an encapsulated water-soluble form were developed. It was found, that the interaction of the substrate molecule and the clathrate forms a substance that can dissolve in water and other more polar solvents, thereby solving the problem of bioavailability of the main hydrophobic drug. The method developed for producing the substance ecdysterone and its water-soluble encapsulated with β-cyclodextrin form was implemented into production at the Karaganda pharmaceutical plant. NMR studies of changes in the chemical shifts of protons of substrates and receptors illustrated that ecdysterone interacts with β-cyclodextrin to form supramolecular inclusion complexes with stoichiometric composition of 1:1

Two distinct binding modes provide the RNA binding protein RbFox with extraordinary sequence specificity

The specificity of RNA-binding proteins for their target sequences varies considerably. Yet, it is not understood how certain proteins achieve markedly higher sequence specificity than most others. Here we show that the RNA Recognition Motif of RbFox accomplishes extraordinary sequence specificity by employing functionally and structurally distinct binding modes. Affinity measurements of RbFox for all binding site variants reveal the existence of two different binding modes. The first exclusively binds the cognate and a closely related RNA variant with high affinity. The second mode accommodates all other RNAs with greatly reduced affinity, thereby imposing large thermodynamic penalties on even near-cognate sequences. NMR studies indicate marked structural differences between the two binding modes, including large conformational rearrangements distant from the RNA binding site. Distinct binding modes by a single RNA binding module explain extraordinary sequence selectivity and reveal an unknown layer of functional diversity, cross talk and regulation for RNA-protein interactions.

An Easily Synthesized Covalent Nanocage that Hosts Fullerenes in Multiple Charge States and Selectively Binds C70

Discrete nanocages provide a way to solubilize, separate, and tune the properties of molecular guests, including fullerenes and other aromatics. However, few such nanocages can be synthesized efficiently from inexpensive starting materials, limiting their practical utility. To address this limitation, we developed a new pyridinium-linked cofacial porphyrin nanocage (Cage4+) that can be prepared efficiently on a gram scale. NMR studies in CD3CN reveal that Cage4+ binds C60 and C70 with association constants >108 M-1 and complete selectivity for extracting C70 from mixtures of both fullerenes. The solubility of Cage4+ in polar solvents enabled electrochemical characterization of the host-guest complexes C60@Cage4+ and C70@Cage4+, finding that the redox properties of the encapsulated fullerenes are minimally affected despite the positive charge of the host. Complexes of the −1 and −2 charge states of the fullerenes bound in Cage4+ were subsequently characterized by UV-vis-NIR and NMR spectroscopies. The relatively easy preparation of Cage4+ and its ability to bind fullerenes without substantially affecting their redox properties suggests that C60@Cage4+ and C70@Cage4+ may be directly useful as solubilized fullerene derivatives.

The Impact of Hydration and Dehydration on the Mobility and Location of Ibuprofen Molecules in the Voids of Ultra-Stable Zeolite Y

Mesoporous dealuminated zeolites are used as hosts for ibuprofen. This drug experiences high mobility when confined in mesopores, which is largely dependent on the water content. Zeolites are materials that are naturally hydrated under ambient conditions. Nitrogen adsorption and X-ray diffraction (XRD) show that the samples with the content of ibuprofen up to 38% have the guest phase residing only in mesopores. 1H and 13C MAS NMR studies of samples in ambient conditions, after dehydration, and in hydration prove the impact of water for increased mobility of ibuprofen. Increased mobility of the introduced phase was also detected for samples with no water content. It was ascribed to ibuprofen located outside mesopores, which experiences a prolonged time of cooling to room temperature. This phenomenon is important for all the future uses of the melting method in guest–host systems and the future use of zeolites for biomedical applications.

1H, 13C and 15N resonance assignment of the SARS-CoV-2 full-length nsp1 protein and its mutants reveals its unique secondary structure features in solution

Structural characterization of the SARS-CoV-2 full length nsp1 protein will be an essential tool for developing new target-directed antiviral drugs against SARS-CoV-2 and for further understanding of intra- and intermolecular interactions of this protein. As a first step in the NMR studies of the protein, we report the 1H, 13C and 15N resonance backbone assignment as well as the Cβ of the apo form of the full-lengthSARS-CoV-2 nsp1 including the folded domain together with the flaking N- and C- terminal intrinsically disordered fragments. The 19.8 kD protein was characterized by high-resolution NMR. Validation of assignment have been done by using two different mutants, H81P and K129E/D48E as well as by amino acid specific experiments. According to the obtained assignment, the secondary structure of the folded domain in solution was almost identical to its previously published X-ray structure as well as another published secondary structure obtained by NMR, but some discrepancies have been detected. In the solution SARS-CoV-2 nsp1 exhibited disordered, flexible N- and C-termini with different dynamic characteristics. The short peptide in the beginning of the disordered C-terminal domain adopted two different conformations distinguishable on the NMR time scale. We propose that the disordered and folded nsp1 domains are not fully independent units but are rather involved in intramolecular interactions. Studies of the structure and dynamics of the SARS-CoV-2 mutant in solution are on-going and will provide important insights into the molecular mechanisms underlying these interactions.