Tetrameric Form Research Articles

A morpheein equilibrium regulates catalysis in phosphoserine phosphatase SerB2 from Mycobacterium tuberculosis

Mycobacterium tuberculosis phosphoserine phosphatase MtSerB2 is of interest as a new antituberculosis target due to its essential metabolic role in L-serine biosynthesis and effector functions in infected cells. Previous works indicated that MtSerB2 is regulated through an oligomeric transition induced by L-Ser that could serve as a basis for the design of selective allosteric inhibitors. However, the mechanism underlying this transition remains highly elusive due to the lack of experimental structural data. Here we describe a structural, biophysical, and enzymological characterisation of MtSerB2 oligomerisation in the presence and absence of L-Ser. We show that MtSerB2 coexists in dimeric, trimeric, and tetrameric forms of different activity levels interconverting through a conformationally flexible monomeric state, which is not observed in two near-identical mycobacterial orthologs. This morpheein behaviour exhibited by MtSerB2 lays the foundation for future allosteric drug discovery and provides a starting point to the understanding of its peculiar multifunctional moonlighting properties.

FRI162 Ligand-dependent Photocrosslinking of Mineralocorticoid Receptors Using Site-directed Incorporation of Unnatural Amino Acids

Abstract Disclosure: B. Almeida-Prieto: None. A.J. Gonzalez-Hernandez: None. T. Giraldez: None. D. Alvarez de la Rosa: None. Steroid receptors dimerize prior DNA binding as a key step for translational activation of target genes. It appears that several domains of the receptors play a role in the process. In addition, current evidence suggests that the active conformation of steroid receptors may not always be a dimer, but involves different quaternary structures, characteristic for each receptor. Recent data obtained by our group suggests that agonist-bound mineralocorticoid receptors (MR) adopt a tetrameric form in the nucleoplasm, forming higher order oligomers when bound to hormone response elements in the chromatin. The formation of such complexes would likely requires more than one oligomerization interface. To generate a tool for investigating inter-subunit contacts, we incorporated photoactivatable crosslinkers into specific sites in MR using a genetically-encoded unnatural amino acid (p-Benzoyl-L-phenylalanine, BzF). We created a library of 26 amber stop codon mutants substituting conserved A, F, W and Y residues distributed along the N-terminal domain (NTD), DNA-binding domain (DBD) and ligand-binding domain (LBD) of the mouse MR coding sequence. Site-directed incorporation of BzF into MR was performed by co-expressing an orthogonal pair of BzF-specific aminoacyl-tRNA synthetase and an amber-suppressor tRNA with the mutant receptors. Characterization of the library showed that 18 mutants produced functional receptors after BzF incorporation. Using an in silico approach we identified position W914 in helix 10 of the LBD as a potential candidate for photocrosslinking. Treatment of cells expressing W914BzF with UV light selectively increased aldosterone-bound, but not cortisol-bound, MR transcriptional activity by approximately 50%. Increased activity was still apparent after hormone washout, suggesting that BzF-induced photocrosslinking at position 914 contributes to stabilizing an active conformation and identifying H10 as a putative region for ligand-specific oligomerization interface. Our results support the strong potential of this methodology for studying this new organization model proposed for MR and other steroid receptors, along with the characterization of functional protein-protein interactions. Presentation Date: Friday, June 16, 2023

Quantum Chemical Investigation of the Interaction of Thalidomide Monomeric, Dimeric, Trimeric, and Tetrameric Forms with Guanine DNA Nucleotide Basis in DMSO and Water Solution: A Thermodynamic and NMR Spectroscopy Analysis.

Thalidomide (TLD) was used worldwide as a sedative, but it was revealed to cause teratogenicity when taken during early pregnancy. It has been stated that the (R) enantiomer of TLD has therapeutic effects, while the (S) form is teratogenic. Clinical studies, however, demonstrated the therapeutic efficacy of thalidomide in several intractable diseases, so TLD and its derivatives have played an important role in the development and therapy of anticancer drugs. Therefore, it is important to know the molecular mechanism of action of the TLD, although this is still not clear. In what molecular interactions are concerned, it is known that drug molecules can interact with DNA in different ways, for example, by intercalation between base pairs. Furthermore, the ability of the TLD to interact with DNA has been confirmed experimentally. In this work, we report a theoretical investigation of the interaction of the R and S enantiomers of TLD, in its monomeric, dimeric, trimeric, and tetrameric forms, with guanine (GUA) DNA nucleotide basis in solution using density functional theory (DFT). Our initial objective was to evaluate the interaction of TLD-R/S with GUA through thermodynamic and spectroscopic study in dimethyl sulfoxide (DMSO) solvent and an aqueous solution. Comparison of the experimental 1H nuclear magnetic resonance (NMR) spectrum in DMSO-d6 solution with calculated DFT-PCM-DMSO chemical shifts revealed that TLD can undergo molecular association in solution, and interaction of its dimeric form with a DNA base ((TLD)2-GUA and (TLD)2-2GUA, for example) through H-bond formation is likely to take place. Our results strongly indicated that we must consider the plausibility of the existence of TLD associations in solution when modeling the complexation of the TLD with biological targets. This is new information that may provide further insight into our understanding of drug binding to biological targets at the molecular level.

Dimeric ACE2-FC Is Equivalent to Monomeric ACE2 in the Surrogate Virus Neutralization Test.

Angiotensin-converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of protective humoral immune response to a viral infection or vaccine using the surrogate virus neutralization test (sVNT). Soluble ACE2 is also considered as an option for antiviral therapy potentially insensitive to the changes in the SARS-CoV-2 spike protein. Extensive testing of the samples of patient's serum by the sVNT method requires using preparations of ACE2 or ACE2 conjugates with constant properties. We have previously obtained a cell line that is a producer of a soluble monomeric ACE2 and showed that this ACE2 variant can be used in sVNT, preferably as a conjugate with horseradish peroxidase. A cell line that generates an ACE2-Fc fusion protein with high productivity, more than 150 mg/liter of the target protein when cultured in a stirred flask, was obtained for producing a stable and universally applicable form of soluble ACE2. The affinity-purified ACE2-Fc fusion contains a mixture of dimeric and tetrameric forms, but allows obtaining linear response curves for inhibition of binding with the receptor-binding domain of the SARS-CoV-2 spike protein by antibodies. The ACE2-Fc-HRP-based sVNT testing system can be used for practical measurements of the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

Studies of Benzotriazole on and into the Copper Electrodeposited Layer by Cyclic Voltammetry, Time-of-Flight Secondary-Ion Mass Spectrometry, Atomic Force Microscopy, and Surface Enhanced Raman Spectroscopy.

Benzotriazole (BTA) is an important compound that demonstrates the strongest anticorrosion properties of copper and plays a role as a leveler and an additive to the electroplating bath for control of the roughness and corrosion resistance of the electrodeposited copper layer. In this paper, we combined cyclic voltammetry (CV), time-of-flight secondary-ion mass spectrometry (TOF-SIMS), surface enhanced Raman spectroscopy (SERS), and atomic force microscopy (AFM) to study the interaction of BTA with copper surfaces at varied concentrations with and without the presence of chloride ions. We identified the most relevant molecular copper and its complex forms with BTA on the copper electrodeposited layer. BTA is adsorbed and incorporated into the copper surface in monomeric, dimeric, trimeric, tetrameric, and pentameric forms, inhibiting the copper electrodeposition. The addition of chloride ions diminishes the inhibiting properties of BTA. The Cu-BTA-Cl complexes were identified in the forms C12H8N6Cu2Cl- and C6H4N3CuCl-. Coadsorption of chloride ions and BTA molecules depends on their concentration and applied potential. Chloride ions are replaced by BTA molecules. BTA and chloride ions, depending on their concentration and applied potential, control the copper nucleation processes at the micro- and nanoscales. We compared the abilities and limitations of TOF-SIMS and SERS for studies of the interactions of benzotriazole with copper and chloride ions at the molecular level.

Delineating the cascade of molecular events in protein aggregation triggered by Glyphosate, aminomethylphosphonic acid, and Roundup in serum albumins

The molecular basis of protein unfolding on exposure to the widely used herbicide, Glyphosate (GLY), its metabolite aminomethylphosphonic acid (AMPA), and the commercial formulation Roundup have been probed using human and bovine serum albumins (HSA and BSA). Protein solutions were exposed to chemical stress at set experimental conditions. The study proceeds with spectroscopic and imaging tools. Steady-state and time-resolved fluorescence (TRF) measurements indicated polarity changes with the possibility of forming a ground-state complex. Atomic force microscopy imaging results revealed the formation of fibrils from BSA and dimer, trimer, and tetramer forms of oligomers from HSA under the chemical stress of GLY. In the presence of AMPA, serum albumins (SAs) form a compact network of oligomers. The compact network of oligomers was transformed into fibrils for HSA with increasing concentrations of AMPA. In contrast, Roundup triggered the formation of amorphous aggregates from SAs. Analysis of the Raman amide I band of all aggregates showed a significant increase in antiparallel β-sheet fractions at the expense of α-helix. The highest percentage, 24.6%, of antiparallel β-sheet fractions was present in amorphous aggregate formed from HSA under the influence of Roundup. These results demonstrated protein unfolding, which led to the formation of oligomers and fibrils.

Chemokine Heteromers and Their Impact on Cellular Function—A Conceptual Framework

Chemoattractant cytokines or chemokines are proteins involved in numerous biological activities. Their essential role consists of the formation of gradient and (immune) cell recruitment. Chemokine biology and its related signaling system is more complex than simple ligand–receptor interactions. Beside interactions with their cognate and/or atypical chemokine receptors, and glycosaminoglycans (GAGs), chemokines form complexes with themselves as homo-oligomers, heteromers and also with other soluble effector proteins, including the atypical chemokine MIF, carbohydrate-binding proteins (galectins), damage-associated molecular patterns (DAMPs) or with chemokine-binding proteins such as evasins. Likewise, nucleic acids have been described as binding targets for the tetrameric form of CXCL4. The dynamic balance between monomeric and dimeric structures, as well as interactions with GAGs, modulate the concentrations of free chemokines available along with the nature of the gradient. Dimerization of chemokines changes the canonical monomeric fold into two main dimeric structures, namely CC- and CXC-type dimers. Recent studies highlighted that chemokine dimer formation is a frequent event that could occur under pathophysiological conditions. The structural changes dictated by chemokine dimerization confer additional biological activities, e.g., biased signaling. The present review will provide a short overview of the known functionality of chemokines together with the consequences of the interactions engaged by the chemokines with other proteins. Finally, we will present potential therapeutic tools targeting the chemokine multimeric structures that could modulate their biological functions.

Base pairs with 5-chloroorotic acid and comparison with the natural nucleobase. Structural and spectroscopic study, and three suggested antiviral modified nucleosides

A structural and spectroscopic study of 5-chloroorotic acid (5-ClOA) biomolecule was carried out by IR and FT-Raman and the results obtained were compared to those achieved in 5-fluoroorotic acid and 5-aminoorotic acid compounds. The structures of all possible tautomeric forms were determined using DFT and MP2 methods. To know the tautomer form present in the solid state, the crystal unit cell was optimized through dimer and tetramer forms in several tautomeric forms. The keto form was confirmed through an accurate assignment of all the bands. For this purpose, an additional improvement in the theoretical spectra was carried out using linear scaling equations (LSE) and polynomic equations (PSE) deduced from uracil molecule. Base pairs with uracil, thymine and cytosine nucleobases were optimized and compared to the natural Watson-Crick (WC) pairs. The counterpoise (CP) corrected interaction energies of the base pairs were also calculated. Three nucleosides were optimized based on 5-ClOA as nucleobase, and their corresponding WC pairs with adenosine. These modified nucleosides were inserted in DNA:DNA and RNA:RNA microhelices, which were optimized. The position of the -COOH group in the uracil ring of these microhelices interrupts the DNA/RNA helix formation. Because of the special characteristic of these molecules they can be used as antiviral drugs. Communicated by Ramaswamy H. Sarma

Structural and functional analyses of Pcal_0917, an α-glucosidase from hyperthermophilic archaeon Pyrobaculum calidifontis

Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry.

Studying the structural organization of non-membranous protein hemoglobin in a lipid environment after reconstitution

In our current work we have developed a supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer with embedded hemoglobin, reconstituted via detergent-mediated method. Microscopic studies revealed that the hemoglobin molecules could be visualized without any labelling agents. The reconstituted proteins assemble themselves as supramolecular structures to adapt to lipid bilayer environment. The nonionic detergent, n-octyl-β-d-glucoside (NOG) used for insertion of hemoglobin played an important role in formation of these structures. When concentrations of lipid, protein and detergent were raised by four folds, we observed phase separation by protein molecules within bilayer via protein-protein assembly. This phase separation process exhibited extremely slow kinetics to form large stable domains with correlation times in the order of minutes. Confocal Z-scanning images showed that these supramolecular structures generated membrane deformities. UV–Vis, Fluorescence and Circular Dichroism (CD) measurement indicated minor structural change to expose the hydrophobic regions of the protein to adjust the hydrophobic stress of the lipid environment whilst Small Angle Neutron Scattering (SANS) results indicated that the hemoglobin molecules retained their overall tetrameric form in the system. In conclusion, we state that this investigation allowed us to closely inspect some rare but noteworthy phenomena like the formation of supramolecular structures, large domain formation and membrane deformation etc.

Phase 1 trial of GD2-SADA:177Lu-DOTA drug complex in patients with recurrent or refractory metastatic solid tumors known to express GD2 including small cell lung cancer (SCLC), sarcoma, and malignant melanoma.

TPS3162 Background: GD2 is a disialoganglioside with high expression in small cell lung cancers (SCLC), sarcomas and melanomas and limited expression in normal tissues. The SADA platform represents a 2-step pretargeted radioimmunotherapy approach developed to optimize tumor targeting and reduce normal tissue irradiation. The GD2-SADA molecule is composed of a non-radioactive, bispecific antibody (anti-GD2 x anti-DOTA) linked to a tetramerization sequence from the p53 protein. In the first step, the GD2-SADA molecule is administered in a tetrameric form with a high affinity to the GD2 tumor target. In preclinical research, any unbound GD2-SADA molecule will disassemble to monomers and has shown to clear via the kidneys. In the second step, the radioactive payload 177Lu-DOTA is administered, which binds to the anti-DOTA part of the GD2-SADA and leads to radiation-induced damage at the target and has shown limited exposure to non-target tissues in preclinical research. This pre-targeting approach supports the concept of highly focused irradiation of the tumor target with the potential of administering higher radioactivity doses. Methods: Trial 1001 (NCT05130255) is a first-in-human, dose-escalation, single-arm, open-label, non-randomized, multicenter phase 1 trial with the purpose of evaluating safety and tolerability of GD2-SADA:177Lu-DOTA. The trial is composed of 3 parts: Part A is dose escalation of the GD2-SADA dose and testing of different intervals between GD2-SADA and 177Lu-DOTA. Part B is dose escalation of the 177Lu-DOTA radioactivity dose to establish its maximum tolerated dose (MTD). Part C involves repeated dosing to assess the safety profile and determine the Recommended Phase 2 Dose (RP2D). Parts B and C may only begin upon completion of dose-limiting toxicity (DLT) observation period of Parts A and B, respectively. Escalation is based on classical 3+3 design. Parts A, B, and C will include 18, 12, and 32 patients, respectively. Patients can only be assigned to one part of the trial. The trial will include adult/adolescent patients with either SCLC, sarcoma, or melanoma with radiographical relapse/progression on standard therapies. The patients must be in ECOG performance status 0-1 and have adequate hematological, renal, and liver function. The patients must have measurable disease according to RECIST 1.1. Patients with active CNS metastases are ineligible. Safety endpoints (DLTs and adverse events) will be summarized by trial part and cohort. Objective response according to RECIST 1.1 and disease control rates at 6, 12 and 24 months will be presented with exact 95% CI. PFS, OS, and duration of response will be estimated using Kaplan-Meier methods. Additional objectives include dosimetry, PK, and immunogenicity data. The study is actively enrolling at US sites. Clinical trial information: NCT05130255 .

Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes.

Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Deoxyguanosine-Linked Bifunctional Inhibitor of SAMHD1 dNTPase Activity and Nucleic Acid Binding.

Sterile alpha motif histidine-aspartate domain protein 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase that exists in monomeric, dimeric, and tetrameric forms. It is activated by GTP binding to an A1 allosteric site on each monomer subunit, which induces dimerization, a prerequisite for dNTP-induced tetramerization. SAMHD1 is a validated drug target stemming from its inactivation of many anticancer nucleoside drugs leading to drug resistance. The enzyme also possesses a single-strand nucleic acid binding function that promotes RNA and DNA homeostasis by several mechanisms. To discover small molecule inhibitors of SAMHD1, we screened a custom ∼69 000-compound library for dNTPase inhibitors. Surprisingly, this effort yielded no viable hits and indicated that exceptional barriers for discovery of small molecule inhibitors existed. We then took a rational fragment-based inhibitor design approach using a deoxyguanosine (dG) A1 site targeting fragment. A targeted chemical library was synthesized by coupling a 5'-phosphoryl propylamine dG fragment (dGpC3NH2) to 376 carboxylic acids (RCOOH). Direct screening of the products (dGpC3NHCO-R) yielded nine initial hits, one of which (R = 3-(3'-bromo-[1,1'-biphenyl]), 5a) was investigated extensively. Amide 5a is a competitive inhibitor against GTP binding to the A1 site and induces inactive dimers that are deficient in tetramerization. Surprisingly, 5a also prevented ssDNA and ssRNA binding, demonstrating that the dNTPase and nucleic acid binding functions of SAMHD1 can be disrupted by a single small molecule. A structure of the SAMHD1-5a complex indicates that the biphenyl fragment impedes a conformational change in the C-terminal lobe that is required for tetramerization.

Abnormal expression of lysosomal glycoproteins in patients with congenital disorders of glycosylation

ObjectiveThe study of the impact of some inherited defects in glycosylation on the biosynthesis of some lysosomal glycoproteins. Results description: Whole-exome sequencing revealed a homozygous variant; 428G > A; p. (R143K) in SRD5A3 in one patient and a heterozygous one c.46G > A p. (Gly16Arg) in SLC35A2 in the other patient. Both variants were predicted to be likely pathogenic. Lysosome-associated membrane glycoprotein 2 (LAMP2) immunodetection in both cases showed a truncated form of the protein. Cystinosin (CTN) protein appeared as normal and truncated forms in both patients in ratios of the mature to truncated forms of CTN were lower than the control. The levels of the truncated forms of both cellular proteins were higher in the SRD5A3-CDG case compared to the SLC35A2-CDG case. The tetrameric form of cathepsin C (CTSC) was expressed at low levels in both cases with congenital disorder of glycosylation (CDG). SLC35A2-CDG patient had one extra-unknown band while SRD5A3-CDG patient had a missing band of CTSC forms. The expression patterns of lysosomal glycoproteins could be different between different types of CDG.

Inhibition of ROS-Scavenging Enzyme System Is a Key Event in Tomato Genetic Resistance against Root-Knot Nematodes.

Genetic resistance in plants against incompatible pests is expressed by the activation of an immune system; however, the molecular mechanisms of pest recognition and expression of immunity, although long the object of investigation, are far from being fully understood. The immune response triggered by the infection of soil-borne parasites, such as root-knot nematodes (RKNs), to incompatible resistant tomato plants was studied and compared to the compatible response that occurred when RKNs attacked susceptible plants. In compatible interactions, the invading nematode juveniles were allowed to fully develop and reproduce, whilst that was impeded in incompatible interactions. In crude root extracts, a first assay of reactive oxygen species (ROS)-scavenging enzymatic activity was carried out at the earliest stages of tomato-RKN incompatible interaction. Membrane-bound and soluble CAT, which is the most active enzyme in hydrogen peroxide (H2O2) scavenging, was found to be specifically inhibited in roots of inoculated resistant plants until 5 days after inoculation, with respect to uninoculated plants. The expression of genes encoding for antioxidant enzymes, such as CAT and glutathione peroxidase (GPX), was not always inhibited in roots of nematode-infected resistant tomato. Therefore, the biochemical mechanisms of CAT inhibition were further investigated. Two CAT isozymes were characterized by size exclusion HPLC as a tetrameric form with a molecular weight of 220,000 dalton and its subunits (55,000 dalton). Fractions containing such isozymes were tested by their sensitivity to both salicylic acid (SA) and H2O2. It was evidenced that elevated concentrations of both chemicals led to a partial inactivation of CAT. Elevated concentrations of H2O2 in incompatible interactions have been suggested to be produced by membrane-bound superoxide anion generating, SOD, and isoperoxidase-enhanced activities. Such partial inactivation of CAT has been depicted as one of the earliest key metabolic events, which is specifically associated with tomato immunity to RKNs. Enhanced ROS production and the inhibition of ROS-scavenging systems have been considered to trigger all the metabolic events leading to cell death and tissue necrosis developed around the head of the invading juveniles by which this special type of plant resistance is exerted.

Rhodospirillum rubrum L-Asparaginase Conjugates with Polyamines of Improved Biocatalytic Properties as a New Promising Drug for the Treatment of Leukemia

L-asparaginase Rhodospirillum rubrum (RrA) is an enzyme (amidohydrolases; EC 3.5.1.1) that catalyzes the L-asparagine hydrolysis reaction to form L-aspartic acid. Due to the shortcomings of existing L-asparaginases from Esherichia coli (EcA) and Erwinia chrysanthemi (ErA), RrA may turn out to be a new promising drug for the treatment of leukemia. RrA has a low homology with EcA and ErA, which makes the enzyme potentially less immunogenic. RrA has pronounced antitumor activity on a number of leukemia cells. However, there is a need to improve the biocatalytic properties of the enzyme. So, in this study, the RrA conjugates with polyamines with different molecular architectures were developed to regulate the catalytic properties of the enzyme. Linear polyethyleneimine (PEI), branched polyethyleneimine, modified with polyethylene glycol (PEI-PEG), and spermine (Spm) were used to obtain conjugates with RrA. It was discovered by gel permeation chromatography that Spm allows the most active tetrameric form of RrA to be obtained and stabilized. Molecular docking was used to study the binding of spermine to RrA subunits. The activity of the RrA conjugates with Spm and PEI-PEG was 23–30% higher than the native enzyme. The pH optimum of the conjugates shifted from 9.0 to 8.5. The conjugates had higher stability: Spm and PEI-PEG reduced the inactivation constant (kin) more than two-fold upon incubation at 53 °С. The conjugate RrA-PEI-PEG reduced the accessibility of trypsin to the protein surface and reduced kin by eight times. The modification of RrA with polyamines made it possible to obtain enzyme preparations with improved biocatalytic properties. These conjugates represent interest for further study as potential therapeutic agents.

Palmitoylation of the Parkinson's disease-associated protein synaptotagmin-11 links its turnover to α-synuclein homeostasis.

Synaptotagmin-11 (Syt11) is a vesicle-trafficking protein that is linked genetically to Parkinson's disease (PD). Likewise, the protein α-synuclein regulates vesicle trafficking, and its abnormal aggregation in neurons is the defining cytopathology of PD. Because of their functional similarities in the same disease context, we investigated whether the two proteins were connected. We found that Syt11 was palmitoylated in mouse and human brain tissue and in cultured cortical neurons and that this modification to Syt11 disrupted α-synuclein homeostasis in neurons. Palmitoylation of two cysteines adjacent to the transmembrane domain, Cys39 and Cys40, localized Syt11 to digitonin-insoluble portions of intracellular membranes and protected it from degradation by the endolysosomal system. In neurons, palmitoylation of Syt11 increased its abundance and enhanced the binding of α-synuclein to intracellular membranes. As a result, the abundance of the physiologic tetrameric form of α-synuclein was decreased, and that of its aggregation-prone monomeric form was increased. These effects were replicated by overexpression of wild-type Syt11 but not a palmitoylation-deficient mutant. These findings suggest that palmitoylation-mediated increases in Syt11 amounts may promote pathological α-synuclein aggregation in PD.

Structural and mechanistic insights into cancer promoting activities of pyruvate kinase muscle isoform-2.

Pyruvate Kinase catalyzes the conversion of phosphoenolpyruvate (PEP) to pyruvate with the production of ATP in the final reaction of glycolysis. The M2 isoform of the PK gene is significantly upregulated in cancer tissues and is a critical regulator of Warburg effect. The tetrameric form of PKM2 has higher kinase activity than the dimer, which supports the growth of tumor by nuclear translocation. The study aims to determine how different conditions such as an alkaline pH (prevalent in cancer cells), and a hyperthermic environment (shown to kill tumor cells) can aid or alleviate cancer progression, by acting via PKM2 which is a central protein in cancer. An alkaline pH of 8.0 was shown to alter the oligomerization state of the protein, promoting the formation of the nuclear-translocating and cancer-promoting dimeric form. The kinase activity was also hampered supporting the higher oncoprotein function. On the other hand, a hyperthermic environment of 42°C was shown to induce changes in the overall structure and stability of PKM2 at secondary and tertiary level, ascertained via Circular Dichroism and Thermal Fluorimetry. As the protein was unable to retain its structure at the slightly higher temperature, the oncoprotein activity of the protein was also obliterated, which may hint towards a possible pathway through which the therapeutic hyperthermia works on cancer cells. Deeper analysis of these mechanisms was performed by testing these conditions with cancer patient-derived mutants of PKM2. The mutants were also crystallized and diffracted at <3Å resolution, which will be solved to gain in-depth structural insights on this protein. This study will unravel novel mechanisms and structural details of PKM2 which will aid in better understanding of the role of PKM2 in the manifestation of disease.

Molecular Dynamics-Based Comparative Analysis of Chondroitin and Dermatan Sulfates.

Glycosaminoglycans (GAGs) are a class of linear anionic periodic polysaccharides containing disaccharide repetitive units. These molecules interact with a variety of proteins in the extracellular matrix and so participate in biochemically crucial processes such as cell signalling affecting tissue regeneration as well as the onset of cancer, Alzheimer's or Parkinson's diseases. Due to their flexibility, periodicity and chemical heterogeneity, often termed "sulfation code", GAGs are challenging molecules both for experiments and computation. One of the key questions in the GAG research is the specificity of their intermolecular interactions. In this study, we make a step forward to deciphering the "sulfation code" of chondroitin sulfates-4,6 (CS4, CS6, where the numbers correspond to the position of sulfation in NAcGal residue) and dermatan sulfate (DS), which is different from CSs by the presence of IdoA acid instead of GlcA. We rigorously investigate two sets of these GAGs in dimeric, tetrameric and hexameric forms with molecular dynamics-based descriptors. Our data clearly suggest that CS4, CS6 and DS are substantially different in terms of their structural, conformational and dynamic properties, which contributes to the understanding of how these molecules can be different when they bind proteins, which could have practical implications for the GAG-based drug design strategies in the regenerative medicine.

Effects of Dimerization, Dendrimerization, and Chirality in p-BthTX-I Peptide Analogs on the Antibacterial Activity and Enzymatic Inhibition of the SARS-CoV-2 PLpro Protein

Recent studies have shown that the peptide [des-Cys11,Lys12,Lys13-(p-BthTX-I)2K] (p-Bth) is a p-BthTX-I analog that shows enhanced antimicrobial activity, stability and hemolytic activity, and is easy to obtain compared to the wild-type sequence. This molecule also inhibits SARS-CoV-2 viral infection in Vero cells, acting on SARS-CoV-2 PLpro enzymatic activity. Thus, the present study aimed to assess the effects of structural modifications to p-Bth, such as dimerization, dendrimerization and chirality, on the antibacterial activity and inhibitory properties of PLpro. The results showed that the dimerization or dendrimerization of p-Bth was essential for antibacterial activity, as the monomeric structure led to a total loss of, or significant reduction in, bacterial activities. The dimers and tetramers obtained using branched lysine proved to be prominent compounds with antibacterial activity against Gram-positive and Gram-negative bacteria. In addition, hemolysis rates were below 10% at the corresponding concentrations. Conversely, the inhibitory activity of the PLpro of SARS-CoV-2 was similar in the monomeric, dimeric and tetrameric forms of p-Bth. Our findings indicate the importance of the dimerization and dendrimerization of this important class of antimicrobial peptides, which shows great potential for antimicrobial and antiviral drug-discovery campaigns.