Distinct Binding Research Articles

Enhanced antidiabetic and antioxidant properties of gold nanoparticles green synthesized using blossom extract of Azadirachta indica: In vitro studies

This study describes the in situ biosynthesis of gold nanoparticles (AuNPs) using neem blossom (Azadirachta indica) extract with enhanced in vitro anti-diabetic and antioxidant properties. The formation of AuNPs was confirmed by the characteristic optical response through the surface plasmon resonance (SPR) band observed around 530 nm. The effect of phytochemical in the formation of AuNPs was investigated using Fourier-transform infrared spectroscopy (FTIR). The effect of reaction parameters such as temperature, pH and precursor concentration on the size and surface morphology of AuNPs was optimized using the SPR band. The metallic form of phytosynthesised AuNPs was confirmed by X-ray photoelectron spectra (XPS) results with a distinctive binding energy difference of 4f shells ΔE = 3.8 eV. Transmission electron microscopy (TEM) and X-ray diffraction (XRD) studies confirmed the crystalline nature of the particles formed. The antioxidant property of AuNPs demonstrated the influence of phytochemical surfactants coated on the surface of AuNPs and the results revealed the higher antioxidant (90 %) efficiency compared to standard ascorbic acid. phytosynthesised AuNPs demonstrated 79 % enzyme inhibition efficiency in α-amylase assays. The antimicrobial evaluation results revealed the exceptional microbicidal efficacy of AuNPs against both gram-positive and gram-negative bacteria. Phytochemically activated AuNPs showed improved antioxidant, antimicrobial and antidiabetic properties and it could be a promising candidate for biological applications in medicine.

C/EBPα Acts As an RNA Binding Protein Essential for, and Promotes End-Stage Macrophage Differentiation

The transcription factor C/EBPα is a well characterized DNA binding protein with essential functions in controlling and regulating myeloid differentiation and lipid metabolism. Herein, we describe C/EBPα's separate and distinct RNA binding characteristics. Using Chromatin RNA Immunoprecipitation, we identified that C/EBPα interacts primarily with RNA introns in both HL-60 and THP-1 cells, with a preference for a palindromic GC-rich binding motif. Structural prediction in conjunction with RNA electrophoretic mobility shift assays show that C/EBPα interacts with RNA through two previously undescribed domains located towards the proteins N terminus. These domains are distinct from C/EBPα's DNA binding b-ZIP domain which is instead located on the proteins C terminus. Mouse bone marrow transplantation and in vitro cytokine assays reveal that C/EBPα RNA binding appears to be essential for macrophage maturation but not neutrophil differentiation. To better understand these phenotypic differences, we expanded our observations into the transcriptomic space by utilizing single-cell CITE-Seq. In line with biochemical data, notable mature cell populations were absent when the C/EBPα's RNA binding domains are excluded from the protein. Intriguingly, differential gene expression revealed strong, selective upregulation of Lipoprotein Lipase (Lpl) in monocyte, but not neutrophil populations (Panel 1). It has been previously reported that dysregulation of Lpl affects bone marrow monocyte progenitor differentiation and results in dysregulated cellular mobilization from the bone marrow, a phenotype that is commonly seen in cancers such as AML. The increased abundance of monocyte and neutrophil populations in the C/EBPα RNA deletion samples is therefore suggestive that C/EBPα RNA binding is critical in regulating terminal differentiation and mobilization circuits which may become dysregulated or perturbed in diseases such as AML. On-going analyses are investigating the RNA velocity trajectory of captured cell populations in an attempt to better understand at which stage C/EBPα RNA binding becomes essential for terminal maturation and differentiation of select cell populations. Taken together, we demonstrate that C/EBPα is also a bona fida RNA binding protein with unique functions distinct from its DNA binding activity that are essential for monocyte maturation, differentiation and mobilization. Panel 1 - Identified Cell Populations Following scCITE-Seq with C/EBPα Full-Length (WT) and C/EBPα RNA Binding Deletion (DD). Each circle represents a single cell presented in UMAP space. The left panel shows populations identified with the full-length C/EBPα construct. The right panel shows the cell populations identified with the RNA domain deletion construct. Notable differences in cell populations and numbers are evident. Overlayed onto the cells is the expression of Lipoprotein Lipase (Lpl). Expression is shown as a gradient from black up to green, representing a log2(4) expression. Lpl expression is shown to be expressed uniquely in the monocyte population of cells almost exclusively in the RNA deletion sample.

Distinct receptor binding domain IgG thresholds predict protective host immunity across SARS-CoV-2 variants and time

SARS-CoV-2 neutralising antibodies provide protection against COVID-19. Evidence from early vaccine trials suggested binding antibody thresholds could serve as surrogate markers of neutralising capacity, but whether these thresholds predict sufficient neutralising capacity against variants of concern (VOCs), and whether this is impacted by vaccine or infection history remains unclear. Here we analyse individuals recovered from, vaccinated or with hybrid immunity against SARS-CoV-2. An NT50 ≥ 100 IU confers protection in vaccine trials, however, as VOC induce a reduction in NT50, we use NT50 ≥ 1000 IU as a cut off for WT NT50 that would retain neutralisation against VOC. In unvaccinated convalescent participants, a receptor binding domain (RBD) IgG of 456 BAU/mL predicts an NT50 against WT of 1000 IU with an accuracy of 80% (95%CI 73–86%). This threshold maintains accuracy in determining loss of protective immunity against VOC in two vaccinated cohorts. It predicts an NT50 < 100 IU against Beta with an accuracy of 80% (95%CI 67–89%) in 2 vaccine dose recipients. In booster vaccine recipients with a history of COVID-19 (hybrid immunity), accuracy is 87% (95%CI 77–94%) in determining an NT50 of <100 IU against BA.5. This analysis provides a discrete threshold that could be used in future clinical studies.

Implications of albumin in cell culture media on the biological action of vanadates(V)

In this article, the implications of binding competition of vanadates(V) with dodecyl sulfates for bovine serum albumin on cytotoxicity of vanadium(V) species against prostate cancer cells have been investigated. The pH- and SDS-dependent vanadate(V)-BSA interactions were observed. At pH 5, there is only one site capable of binding ten vanadates(V) ions (logK(ITC)1 = 4.96 ± 0.06; ΔH(ITC)1 = −1.04 ± 0.03 kcal mol−1), whereas at pH 7 two distinctive binding sites on protein were found, saturated with two and seven V(V) ions, respectively (logK(ITC)1 = 6.11 ± 0.06; ΔH(ITC)1 = 0.78 ± 0.12 kcal mol−1; logK(ITC)2 = 4.80 ± 0.02; ΔH(ITC)2 = − 4.95 ± 0.14 kcal mol−1). SDS influences the stoichiometry and the stability of the resulting V(V)-BSA complexes. Finally, the cytotoxicity of vanadates(V) against prostate cancer cells (PC3 line) was examined in the presence and absence of SDS in the culture medium. In the case of a 24-h incubation with 100 μM vanadate(V), a ca. 20 % reduction in viability of PC3 cells was observed in the presence of SDS. However, in other considered cases (various concentrations and time of incubation) SDS does not affect the dose-dependent action of vanadates(V) on the investigated prostate cancer cells.

Dissecting the intricacies of human antibody responses to SARS-CoV-1 and SARS-CoV-2 infection

The 2003 severe acute respiratory syndrome coronavirus (SARS-CoV-1) causes more severe disease than SARS-CoV-2, which is responsible for COVID-19. However, our understanding of antibody response to SARS-CoV-1 infection remains incomplete. Herein, we studied the antibody responses in 25 SARS-CoV-1 convalescent patients. Plasma neutralization was higher and lasted longer in SARS-CoV-1 patients than in severe SARS-CoV-2 patients. Among 77 monoclonal antibodies (mAbs) isolated, 60 targeted the receptor-binding domain (RBD) and formed 7 groups (RBD-1 to RBD-7) based on their distinct binding and structural profiles. Notably, RBD-7 antibodies bound to a unique RBD region interfaced with the N-terminal domain of the neighboring protomer (NTD proximal) and were more prevalent in SARS-CoV-1 patients. Broadly neutralizing antibodies for SARS-CoV-1, SARS-CoV-2, and bat and pangolin coronaviruses were also identified. These results provide further insights into the antibody response to SARS-CoV-1 and inform the design of more effective strategies against diverse human and animal coronaviruses.

The molecular basis of drug selectivity for α5 subunit-containing GABAA receptors

α5 subunit-containing γ-aminobutyric acid type A (GABAA) receptors represent a promising drug target for neurological and neuropsychiatric disorders. Altered expression and function contributes to neurodevelopmental disorders such as Dup15q and Angelman syndromes, developmental epilepsy and autism. Effective drug action without side effects is dependent on both α5-subtype selectivity and the strength of the positive or negative allosteric modulation (PAM or NAM). Here we solve structures of drugs bound to the α5 subunit. These define the molecular basis of binding and α5 selectivity of the β-carboline, methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), type II benzodiazepine NAMs, and a series of isoxazole NAMs and PAMs. For the isoxazole series, each molecule appears as an ‘upper’ and ‘lower’ moiety in the pocket. Structural data and radioligand binding data reveal a positional displacement of the upper moiety containing the isoxazole between the NAMs and PAMs. Using a hybrid molecule we directly measure the functional contribution of the upper moiety to NAM versus PAM activity. Overall, these structures provide a framework by which to understand distinct modulator binding modes and their basis of α5-subtype selectivity, appreciate structure–activity relationships, and empower future structure-based drug design campaigns.

3site Multisubstrate-Bound State of Cytochrome P450cam.

We identified a multisubstrate-bound state, hereby referred as a 3site state, in cytochrome P450cam via integrating molecular dynamics simulation with nuclear magnetic resonance (NMR) pseudocontact shift measurements. The 3site state is a result of simultaneous binding of three camphor molecules in three locations around P450cam: (a) in a well-established "catalytic" site near heme, (b) in a kink-separated "waiting" site along channel-1, and (c) in a previously reported "allosteric" site at E, F, G, and H helical junctions. These three spatially distinct binding modes in the 3site state mutually communicate with each other via homotropic allostery and act cooperatively to render P450cam functional. The 3site state shows a significantly superior fit with NMR pseudo contact shift (PCS) data with a Q-score of 0.045 than previously known bound states and consists of D251 free of salt-bridges with K178 and R186, rendering the enzyme functionally primed. To date, none of the reported cocomplex of P450cam with its redox partner putidaredoxin (pdx) has been able to match solution NMR data and controversial pdx-induced opening of P450cam's channel-1 remains a matter of recurrent discourse. In this regard, inclusion of pdx to the 3site state is able to perfectly fit the NMR PCS measurement with a Q-score of 0.08 and disfavors the pdx-induced opening of channel-1, reconciling previously unexplained remarkably fast hydroxylation kinetics with a koff of 10.2 s-1. Together, our findings hint that previous experimental observations may have inadvertently captured the 3site state as an in vitro solution state, instead of the catalytic state alone, and provided a distinct departure from the conventional understanding of cytochrome P450.

Probing a distinct druggable tubulin binding site with gatorbulins 1–7, their metabolic and physicochemical properties, and pharmacological consequences

Microtubules, consisting of α/β-tubulin heterodimers, are prime targets for anticancer drug discovery. Gatorbulin-1 (GB1, 1a) is a recently described marine natural product that targets tubulin at a new, seventh pharmacological site at the tubulin intradimer interface. Using our previously developed robust route towards GB1 (1a), we synthesized simplified, first-generation gatorbulins, GB2–7 (1b–1g) of this highly modified cyclodepsipeptide (GB1) that does not contain any proteinogenic amino acid. We systematically investigated the structure-activity relationship at the biochemical and cellular level using GB1-susceptible ovarian and cervical cancer cells. We validated that the hydroxamate moiety in the N-methyl-alanine residue is critical for activity. All other structural modifications present in GB1, including C-hydroxylation of asparagine, methylation at C-4 of proline, and sp2 hybridization in dehydro-alanine, were proven to be functionally relevant. Replacement of the primary amide with a methyl ester also resulted in reduced activity, indicating the intricate scaffold optimization by the GB1-producing cyanobacterium. Inhibition of tubulin polymerization in vitro and binding affinities correlated very well, translating into differentials in cellular efficacy. We used docking and molecular dynamics to evaluate the effects of the chemical simplification at the structural level, indicating that each modification resulted in loss of target interactions, although energetically modest. Similar to cevipabulin that targets two different sites on the tubulin dimer, GB1 promotes proteasome-mediated tubulin degradation but by an unknown mechanism, presumably distinct from that of cevipabulin. Comparison with cevipabulin indicated that this compound binds to the same tubulin region as GB1 (1a), although the binding mode is distinct. Cevipabulin almost exclusively interacts with α-tubulin, including nonexchangeable GTP. In contrast, GB1 (1a) makes extensive contact and hydrogen bonds with both α- and β-chains of tubulin. GB1-7 showed excellent solubility and much higher than that of paclitaxel. Hepatic microsome stability was excellent, human cytochrome P450s were not inhibited and plasma binding was minimal with high free fractions. Passive permeability was predicted to be high based on PAMPA. Parent compound GB1 (1a) was further evaluated using a cellular model with MDCK cells stably transduced with the human efflux transporter MDR1/P-gp, showing similar permeability with and against transporter gradient, indicating that GB1 (1a) is a poor P-gp substrate.

Atypical homodimerization revealed by the structure of the (S)-enantioselective haloalkane dehalogenase DmmarA from Mycobacterium marinum.

Haloalkane dehalogenases (HLDs) are a family of α/β-hydrolase fold enzymes that employ SN2 nucleophilic substitution to cleave the carbon-halogen bond in diverse chemical structures, the biological role of which is still poorly understood. Atomic-level knowledge of both the inner organization and supramolecular complexation of HLDs is thus crucial to understand their catalytic and noncatalytic functions. Here, crystallographic structures of the (S)-enantioselective haloalkane dehalogenase DmmarA from the waterborne pathogenic microbe Mycobacterium marinum were determined at 1.6 and 1.85 Å resolution. The structures show a canonical αβα-sandwich HLD fold with several unusual structural features. Mechanistically, the atypical composition of the proton-relay catalytic triad (aspartate-histidine-aspartate) and uncommon active-site pocket reveal the molecular specificities of a catalytic apparatus that exhibits a rare (S)-enantiopreference. Additionally, the structures reveal a previously unobserved mode of symmetric homodimerization, which is predominantly mediated through unusual L5-to-L5 loop interactions. This homodimeric association in solution is confirmed experimentally by data obtained from small-angle X-ray scattering. Utilizing the newly determined structures of DmmarA, molecular modelling techniques were employed to elucidate the underlying mechanism behind its uncommon enantioselectivity. The (S)-preference can be attributed to the presence of a distinct binding pocket and variance in the activation barrier for nucleophilic substitution.

Tailoring stimuli-responsive PVDF-based copolymer membrane with engineered pore structure for efficient antibody purification

Membrane chromatography is considered as a sustainable alternative for downstream purification to overcome the production bottleneck of biopharmaceuticals. However, two of the major factors impeding its industrial adoption are the lack of controllable functionalization and competitive binding capacity. This work aimed to develop a one-pot synthesis strategy for fabricating a new class of pH-responsive poly(vinylidene fluoride)-graft-poly(dimethylaminoethyl methacrylate) (PVDF-g-PDMAEMA) membranes with anion exchangers, to achieve fast and accurate removal of albumin impurities from antibody streams. By utilizing a pre-functionalized copolymer with controlled ligand density, PVDF-g-PDMAEMA membranes with advantageous bicontinuous pore network structure were obtained via an unconventional nonsolvent thermally induced phase separation method. The membrane showed a competitive water permeance of > 6000 L m-2h−1 bar−1. Combining with dedicated static binding and isotherm studies, the membranes demonstrated distinct pH-responsive binding behaviour to model impurity bovine serum albumin (BSA) with maximal binding of 318.0 mg/g at pH 5.5. The dynamic flow-through binding experiments indicated that the membrane could effectively capture BSA from immunoglobulin G (IgG) (model antibody)/BSA mixed solution with a competitive binding capacity of 210.5 mg/g, producing IgG with purity > 90 % and nearly full recovery. Also, comprehensive analysis demonstrated the robustness of the membrane performance through five consecutive operation cycles, with or without harsh alkaline regeneration. The biocompatibility analysis further proved the membranes’ suitability in biopharmaceutical applications. This study provided a new route for designing future chromatographic membranes to promote its industrial uptake, pushing for energy-saving and effective downstream bioprocessing.

Mechanistic insights into the enzymatic activity of E3 ligase HOIL-1L and its regulation by the linear ubiquitin chain binding.

Heme-oxidized IRP2 ubiquitin ligase 1 (HOIL-1L) serves as a unique E3 ligase to catalyze the mono-ubiquitination of relevant protein or sugar substrates and plays vital roles in numerous cellular processes in mammals. However, the molecular mechanism underpinning the E3 activity of HOIL-1L and the related regulatory mechanism remain elusive. Here, we report the crystal structure of the catalytic core region of HOIL-1L and unveil the key catalytic triad residues of HOIL-1L. Moreover, we discover that HOIL-1L contains two distinct linear di-ubiquitin binding sites that can synergistically bind to linear tetra-ubiquitin, and the binding of HOIL-1L with linear tetra-ubiquitin can promote its E3 activity. The determined HOIL-1L/linear tetra-ubiquitin complex structure not only elucidates the detailed binding mechanism of HOIL-1L with linear tetra-ubiquitin but also uncovers a unique allosteric ubiquitin-binding site for the activation of HOIL-1L. In all, our findings provide mechanistic insights into the E3 activity of HOIL-1L and its regulation by the linear ubiquitin chain binding.

The PWWP domain and the evolution of unique DNA methylation toolkits in Hymenoptera

DNMT3 in Hymenoptera has a unique duplication of the essential PWWP domain. Using GST-tagged PWWP fusion proteins and histone arrays we show that these domains have gained new properties and represent the first case of PWWP domains binding to H3K27 chromatin modifications, including H3K27me3, a key modification that is important during development. Phylogenetic analyses of 107 genomes indicate that the duplicated PWWP domains separated into two sister clades, and their distinct binding capacities are supported by 3D modeling. Other features of this unique DNA methylation system include variable copies, losses, and duplications of DNMT1 and DNMT3, and combinatorial generations of DNMT3 isoforms including variants missing the catalytic domain. Some of these losses and duplications of are found only in parasitic wasps. We discuss our findings in the context of the crosstalk between DNA methylation and histone methylation, and the expanded potential of epigenomic modifications in Hymenoptera to drive evolutionary novelties.

Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the currentbest performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method thatnot only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 μM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

Dynamically switchable porphyrin-based molecular tweezer for on−off fullerene recognition

Herein, a novel molecular tweezer based on 2,2′-bipyridine-bridged porphyrin subunits was constructed for efficient fullerenes recognition. The syn conformation of the molecule, which was obtained by Zn(Ⅱ) coordination, gives rise to a proper cavity to interact with fullerene guests to form a stable 1:1 complex in toluene solution. It exhibits distinct binding selectivity towards C60 over C70. Moreover, the fullerene recognition capacity can be adequately suppressed by importing H2PO4− to competitively capture Zn(Ⅱ) along with syn-anti conformational conversion. Subsequently, the molecular tweezer regenerated to bind the fullerene by introducing the Ca2+ into the system. Significantly, the association-disassociation process can be switched reversibly and repeatedly.

Multidimensional quantitative phenotypic and molecular analysis reveals neomorphic behaviors of p53 missense mutants

Mutations in the TP53 tumor suppressor gene occur in >80% of the triple-negative or basal-like breast cancer. To test whether neomorphic functions of specific TP53 missense mutations contribute to phenotypic heterogeneity, we characterized phenotypes of non-transformed MCF10A-derived cell lines expressing the ten most common missense mutant p53 proteins and observed a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and 3D mammosphere architecture. The p53 mutants R248W, R273C, R248Q, and Y220C are the most aggressive while G245S and Y234C are the least, which correlates with survival rates of basal-like breast cancer patients. Interestingly, a crucial amino acid difference at one position—R273C vs. R273H—has drastic changes on cellular phenotype. RNA-Seq and ChIP-Seq analyses show distinct DNA binding properties of different p53 mutants, yielding heterogeneous transcriptomics profiles, and MD simulation provided structural basis of differential DNA binding of different p53 mutants. Integrative statistical and machine-learning-based pathway analysis on gene expression profiles with phenotype vectors across the mutant cell lines identifies quantitative association of multiple pathways including the Hippo/YAP/TAZ pathway with phenotypic aggressiveness. Further, comparative analyses of large transcriptomics datasets on breast cancer cell lines and tumors suggest that dysregulation of the Hippo/YAP/TAZ pathway plays a key role in driving the cellular phenotypes towards basal-like in the presence of more aggressive p53 mutants. Overall, our study describes distinct gain-of-function impacts on protein functions, transcriptional profiles, and cellular behaviors of different p53 missense mutants, which contribute to clinical phenotypic heterogeneity of triple-negative breast tumors.

Single-molecule-binding studies of antivirals targeting the hepatitis C virus core protein.

The hepatitis C virus is associated with nearly 300,000 deaths annually. At the core of the virus is an RNA-protein complex called the nucleocapsid, which consists of the viral genome and many copies of the core protein. Because the assembly of the nucleocapsid is a critical step in viral replication, a considerable amount of effort has been devoted to identifying antiviral therapeutics that can bind to the core protein and disrupt assembly. Although several candidates have been identified, little is known about how they interact with the core protein or how those interactions alter the structure and thus the function of this viral protein. Our work biochemically characterizes several of these binding interactions, highlighting both similarities and differences as well as strengths and weaknesses. These insights bolster the notion that this viral protein is a viable target for novel therapeutics and will help to guide future developments of these candidate antivirals.

Synthesis of Upper-Rim Sulfanylpropyl- and p-Methoxyphenylazo-Substituted Calix[4]arenes as Chromogenic Sensors for Hg2+ and Ag+ Ions.

A series of calix[4]arenes with upper-rim sulfanylpropyl and p-methoxyphenylazo groups (compounds 8-10) were synthesized and found to be effective chromogenic sensors for selectively detecting Hg2+, Hg+, and Ag+ ions among 18 screened metal perchlorates. In comparison to previously reported diallyl- and dithioacetoxypropyl-substituted calix[4]arenes (5, 6, 14, 15, and 16) and the newly synthesized compound 7, the distal (5,17)-disulfanylpropyl-substituted di-p-methoxyphenylazocalix[4]arene 9 demonstrated superior performance with a limit of detection of 0.028 μM for Hg2+ ions in a chloroform/methanol (v/v = 399/1) cosolvent. Job's plot revealed 1:1 binding stoichiometry for all these upper-rim sulfanylpropyl- and p-methoxyphenylazo-substituted calix[4]arenes 8-10 with Hg2+ ions, and Benesi-Hildebrand plots from ultraviolet/visible (UV-vis) titration spectra were used for the determination of their association constants. Our findings indicated that the distal orientation of two p-methoxyphenylazo and two sulfanylpropyl groups in calix[4]arenes 8-10 is more favorable for binding Hg2+ ions than the proximal (5,11-) orientation; moreover, the adjacent sulfanylpropyl groups exhibited superior coordination as ligands compared to the allyl and thioacetoxypropyl groups. Notably, compounds 8-10 displayed a comparable trend in their association with Ag+ ions, albeit with 1 order of magnitude lower binding constants and a distinct binding mode compared to Hg2+ ions. UV-vis spectroscopy, Job's plots, high-resolution mass spectrometry, and 1H nuclear magnetic resonance titration studies are presented and discussed.

Effects of spike proteins on angiotensin converting enzyme 2 (ACE2)

The Coronavirus Disease 2019 (COVID-19) pandemic was caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which enters host cells through interactions of its spike protein to Angiotensin-Converting Enzyme 2 (ACE2). ACE2 is a peptidase that cleaves Angiotensin II, a critical pathological mediator. This study investigated if the spike protein binding to ACE2 compromises its peptidase activity. Spike/ACE2 Binding Assays suggested that spike proteins of SARS-CoV-2, SARS-CoV and MERS-CoV, but not HKU1, bind to ACE2. S1 and receptor-binding domain (RBD), but not S2, extracellular domain (ECD) or CendR domain, bind to ACE2. While glycosylated spike proteins prepared in HEK293 cells bind to ACE2, non-glycosylated proteins produced in E. coli do not. Cysteine residues of the spike protein expressed in HEK293 cells are fully oxidized, while those of the protein expressed in E. coli are reduced. The deglycosylation of HEK cell-produced protein attenuates the ACE2 binding, while the oxidation of the E. coli protein does not promote the binding. The S1 protein of SARS-CoV-2 enhances the ACE2 peptidase activity, while SARS-CoV, MERS-CoV or HKU1 does not. The ACE2 activity is enhanced by RBD, but not ECD or CendR. In contrast to distinct ACE2 binding capacities of proteins expressed in HEK293 cells and in E. coli, spike proteins expressed in both systems enhance the ACE2 activity. Thus, the spike protein of SARS-CoV-2, but not other coronaviruses, enhances the ACE2 peptidase activity through its RBD in a glycosylation-independent manner.

Differentiating the roles of Mycobacterium tuberculosis substrate binding proteins, FecB and FecB2, in iron uptake.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, poses a great threat to human health. With the emergence of drug resistant Mtb strains, new therapeutics are desperately needed. As iron is critical to the growth and survival of Mtb, mechanisms through which Mtb acquires host iron represent attractive therapeutic targets. Mtb scavenges host iron via Mtb siderophore-dependent and heme iron uptake pathways. While multiple studies describe the import of heme and ferric-siderophores and the export of apo-siderophores across the inner membrane, little is known about their transport across the periplasm and cell-wall environments. Mtb FecB and FecB2 are predicted periplasmic binding proteins implicated in host iron acquisition; however, their precise roles are not well understood. This study sought to differentiate the roles FecB and FecB2 play in Mtb iron acquisition. The crystallographic structures of Mtb FecB and FecB2 were determined to 2.0 Å and 2.2 Å resolution, respectively, and show distinct ligand binding pockets. In vitro ligand binding experiments for FecB and FecB2 were performed with heme and bacterial siderophores from Mtb and other species, revealing that both FecB and FecB2 bind heme, while only FecB binds the Mtb sideophore ferric-carboxymycobactin (Fe-cMB). Subsequent structure-guided mutagenesis of FecB identified a single glutamate residue-Glu339-that significantly contributes to Fe-cMB binding. A role for FecB in the Mtb siderophore-mediated iron acquisition pathway was corroborated by Mycobacterium smegmatis and Mtb pull-down assays, which revealed interactions between FecB and members of the mycobacterial siderophore export and import machinery. Similarly, pull-down assays with FecB2 confirms its role in heme uptake revealing interactions with a potential inner membrane heme importer. Due to ligand preference and protein partners, our data suggest that Mtb FecB plays a role in siderophore-dependent iron and heme acquisition pathways; in addition, we confirm that Mtb FecB2 is involved in heme uptake.

2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation.

Fluorescent glucose derivatives are valuable tools as glucose analogs in plant research to explore metabolic pathways, study enzyme activity, and investigate cellular processes related to glucose metabolism and sugar transport. They allow visualization and tracking of glucose uptake, its utilization, and distribution within plant cells and tissues. This study investigates the phenotypic and metabolic impact of the exogenously fed glucose derivative, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose) (2-NBDG) on the fibers of Gossypium hirsutum (Upland cotton) ovule in vitro cultures. The presence of 2-NBDG in the culture medium did not lead to macroscopic morphological alterations in ovule and fiber development or to the acquisition of fluorescence or yellow coloration. Confocal laser scanning microscope imaging and chromatographic analysis of cotton ovules' outer rim cross-sections showed that the 2-NBDG is transported from the extracellular space and accumulated inside some outer integument cells, epidermal cells, and fertilized epidermal cells (fibers), but is not incorporated into the cell walls. Untargeted metabolic profiling of the fibers revealed significant changes in the relative levels of metabolites involved in glycolysis and upregulation of alternative energy-related pathways. To provide biochemical and structural evidence for the observed downregulation of glycolysis pathways in the fibers containing 2-NBDG, kinetics analysis and docking simulations were performed on hexokinase from G. hirsutum (GhHxk). Notably, the catalytic activity of heterologously expressed recombinant active GhHxk exhibited a five-fold decrease in reaction rates compared to D-glucose. Furthermore, GhHxk exhibited a linear kinetic behavior in the presence of 2-NBDG instead of the Michaelis-Menten kinetics found for D-glucose. Docking simulations suggested that 2-NBDG interacts with a distinct binding site of GhHxk9, possibly inducing a conformational change. These results highlight the importance of considering fluorescent glucose derivatives as ready-to-use analogs for tracking glucose-related biological processes. However, a direct comparison between their mode of action and its extrapolation into biochemical considerations should go beyond microscopic inspection and include complementary analytical techniques.