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Thermophoresis and diffusiophoresis or the directional motion of particles in liquids and gases driven by a temperature and concentration gradients. These phenomena are of both fundamental interest and practical use. So far, thermophoresis, diffusiophoresis along with their dynamics have been investigated and applied. In this paper, a type of thermodynamic force is studied and applied to explain the thermophoresis processes in gases. This force is known as the general thermophoretic force driven by both the temperature and the concentration gradients. It is caused by elastic collision between gas molecules and particles suspended in the gases. The results show that: (i) Based on the kinetic theory of gases, the equation describes the general thermophoretic force can be derived. (ii) Both the thermophoretic force (which is driven solely by a temperature gradient) and the diffusiophoretic force (which is driven solely by a concentration gradient) are special cases of the general thermophoretic force. (iii) Both the thermophoretic and the diffusiophoretic forces depend on the size of gas molecules and particles. (iv) The general thermophoretic force can be applied to explain the thermophoresis: the negative thermophoresis (negative Soret effect) can occur when the diffusiophoretic force opposes the thermophoretic force. (v) The Soret coefficient can be determined by the balanced state of the general thermophoretic force. (vi) The thermophoretic force and diffusiophoretic force of the gases exerted on gas molecules are not depended on the size of molecules. (vii) Under the normal condition (temperature is 20 °C and the pressure is 1 atm), the thermophoretic force and the diffusiophoretic force exerted on gas molecules are approximately 2.45 × 10–24 N and 1.74 × 10–24 N respectively. [ABSTRACT FROM AUTHOR]

Oncolytic virotherapy is a promising approach for cancer treatment. However, when introduced into the body, the virus provokes the production of virus-neutralizing antibodies, which can reduce its antitumor effect. To shield viruses from the immune system, aptamers that can cover the membrane of the viral particle are used. Aptamers that specifically bind to the JX-594 strain of the vaccinia virus were developed earlier. However, the parameters for binding to the recombinant virus VV-GMCSF-Lact, developed based on the LIVP strain of the vaccinia virus, may differ due its different repertoire of antigenic determinants on its membrane compared to JX-594. In this work, the spatial atomic structures of aptamers to JX-594 and bifunctional aptamers were determined using molecular modeling. The efficiency of viral particles binding to the aptamers (EC50), as well as the cytotoxicity and stability of the aptamers were studied. The synergistic effect of the VV-GMCSF-Lact combination with the aptamers in the presence of serum was investigated using human glioblastoma cells. This proposed approach allowed us to conduct a preliminary screening of sequences using in silico modeling and experimental methods, and identified potential candidates that are capable of shielding VV-GMCSF-Lact from virus-neutralizing antibodies. [ABSTRACT FROM AUTHOR]

The potency of frontline antimalarial drug artemisinin (ART) derivatives is triggered by heme-induced cleavage of the endoperoxide bond to form reactive heme-ART alkoxy radicals and covalent heme-ART adducts, which are highly toxic to the parasite. ART-resistant (ART-R) parasites with mutations in the Plasmodium falciparum Kelch-containing protein Kelch13 (PfKekch13) exhibit impaired hemoglobin uptake, reduced yield of hemoglobin-derived heme, and thus decreased ART activation. However, any direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that the purified recombinant PfKelch13 wild-type (WT) protein displays measurable binding affinity for iron and heme, the main effectors for ART activation. The heme-binding property is also exhibited by the native PfKelch13 protein from parasite culture. The two ART-R recombinant PfKelch13 mutants (C580Y and R539T) display weaker heme binding affinities compared to the ART-sensitive WT and A578S mutant proteins, which further translates into reduced yield of heme-ART derivatives when ART is incubated with the heme molecules bound to the mutant PfKelch13 proteins. In conclusion, this study provides the first evidence for ART activation via the heme-binding propensity of PfKelch13. This mechanism may contribute to the modulation of ART-R levels in malaria parasites through a novel function of PfKelch13. Elucidation of heme binding affinity and artemisinin activation by the Plasmodium falciparum Kelch13 protein variants. [ABSTRACT FROM AUTHOR]

Objective: Isoscopoletin is one of the primary metabolites of natural product scoparone, which was reported to against tumor proliferation. The aim of this study was to explore the mechanism of isoscopoletin against hepatocellular carcinoma (HCC). Methods: Transcriptomics was used to reveal the possible pathways of isoscopoletin against HCC in vitro. The potential targets of isoscopoletin against HCC through affecting glycolysis were analyzed by network pharmacology, then the potential binding abilities of isoscopoletin to glycolysis-related proteins were initially verified by high throughput virtual molecular docking. The affinities of isoscopoletin for glycolysis-related proteins were assayed using microscale thermophoresis (MST), which was reverse-validated by inhibiting the binding ability of isoscopoletin to GPD2. Glucose consumption and lactate production were examined to evaluate the effects of isoscopoletin on intracellular glycolysis, and the regulation of glycolysis-related targets by isoscopoletin was detected using RT-qPCR and ELISA kits. Results: The results of transcriptomics showed that the differentially expressed genes (DEGs) were mainly enriched in glycolysis and other metabolic-related pathways. Network pharmacology and molecular docking revealed that GPD2, GPI, HSP90AA1 and PGK2 were the core targets in the glycolysis process of isoscopoletin against HCC. MST results showed that there was a strong affinity between isoscopoletin and GPD2, GPI, Hsp90α and PGK2. In vitro results showed that isoscopoletin inhibited glucose consumption and lactate production, while regulating the levels of glycolysis-related proteins. Conclusion: This study suggests that isoscopoletin may exist an anti-tumor effect by regulating the glycolysis-related proteins GPD2, GPI, Hsp90α and PGK2, inhibiting the glycolysis process in HCC cells, then blocking the energy supply of tumor cells. [ABSTRACT FROM AUTHOR]

Background: Huashi Baidu Formula (HBF) is a clinical formula known for its efficacy against coronavirus disease 2019 (COVID-19). HBF may reduce the number of patients with abnormal serum creatinine while improving respiratory symptoms, suggesting that this formula may have potential for treating acute kidney injury (AKI). However, the protective effect of HBF on AKI has not been definitively confirmed, and the mechanism remains unclear. Therefore, the present study explored the renoprotective effects and molecular mechanisms of HBF and screened for its active ingredients to identify new potential applications of renoprotection by HBF. Methods: The present study first assessed the protective effects of HBF on AKI in a DOX-induced mouse model. Then, RNA-seq and bioinformatics analyses were used to explore the related pathological processes and potential molecular mechanisms, which were subsequently validated using qRT-PCR and Western blotting. Furthermore, candidate compounds with potential binding affinity to two pivotal targets, sphingosine kinase 1 (SphK1) and plasminogen activator inhibitor-1 (PAI-1), were screened from the 29 constituents present in the blood using Microscale Thermophoresis (MST). Finally, to identify the active ingredients, the candidate components were re-screened using the SphK1 kinase activity detection system or the uPA/PAI-1 substrate colorimetric assay system. Results: In the DOX-induced AKI mouse model, therapeutic administration of HBF significantly reduced the levels of CRE, BUN, TNF-α, IL-1β, IL-6, and UA in plasma and the levels of MDA, T-CHO, and TG in kidney tissue. Additionally, the levels of TP and Alb in plasma and SOD and CAT in the kidney tissue were significantly increased. Histopathological assessment revealed that HBF reduced tubular vacuolation, renal interstitial inflammatory cell infiltration, tubular atrophy, and positive staining of renal interstitial collagen. RNA-seq and bioinformatics analyses showed that oxidative stress, the immune-inflammatory response, and extracellular matrix (ECM) formation could be the pathological processes that HBF targets to exerts its renoprotective effects. Furthermore, HBF regulated the APJ/SPHK1/NF-κB and APJ/PAI-1/TGFβ signaling axes and reduced the phosphorylation levels of NF-κB p65 and SMAD2 and the expression of cytokines and the ECM downstream of the axis. Finally, six SphK1 inhibitors (paeoniflorin, astragalin, emodin, glycyrrhisoflavone, quercetin, and liquiritigenin) and three PAI-1 inhibitors (glycyrrhisoflavone, licochalcone B, and isoliquiritigenin) were identified as potentially active ingredients in HBF. Conclusion: In brief, our investigation underscores the renoprotective effect of HBF in a DOX-induced AKI model mice, elucidating its mechanisms through distinct pathological processes and identifying key bioactive compounds. These findings offer new insights for broadening the clinical applications of HBF and unravelling its molecular mode of action. [ABSTRACT FROM AUTHOR]

Seed size, a key determinant of rice yield, is regulated by brassinosteroid (BR); however, the BR pathway in rice has not been fully elucidated. Here, we report the cloning and characterization of the quantitative trait locus Rice Big Grain 1 (qRBG1) from single-segment substitution line Z499. Our data show that qRBG1Z is an unselected rare promoter variation that reduces qRBG1 expression to increase cell number and size, resulting in larger grains, whereas qRBG1 overexpression causes smaller grains in recipient Nipponbare. We demonstrate that qRBG1 encodes a non-canonical BES1 (Bri1-EMS-Suppressor1)/BZR1(Brassinazole-Resistant1) family member, OsBZR5, that regulates grain size upon phosphorylation by OsGSK2 (GSK3-like Kinase2) and binding to D2 (DWARF2) and OFP1 (Ovate-Family-Protein1) promoters. qRBG1 interacts with OsBZR1 to synergistically repress D2, and to antagonistically mediate OFP1 for grain size. Our results reveal a regulatory network controlling grain size via OsGSK2–qRBG1–OsBZR1–D2–OFP1 module, providing a target for improving rice yield. A rare variation in the promoter of qRBG1/OsBZR5 is associated with larger rice grains. OsBZR5 is found to negatively regulate grain size in rice, a biological role opposite to other OsBZR members. A regulatory module of OsGSK2–qRBG1–OsBZR1–D2–OFP1 is further revealed. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for virion budding. In addition to enveloped virions, HBV-replicating cells nonlytically release non-enveloped (naked) capsids independent of the integral ESCRT machinery, but the exact secretory mechanism remains elusive. Here, we provide more detailed information about the existence and characteristics of naked capsid, as well as the viral and host regulations of naked capsid egress. HBV capsid/core protein has two highly conserved Lysine residues (K7/K96) that potentially undergo various types of posttranslational modifications for subsequent biological events. Mutagenesis study revealed that the K96 residue is critical for naked capsid egress, and the intracellular egress-competent capsids are associated with ubiquitinated host proteins. Consistent with a previous report, the ESCRT-III-binding protein Alix and its Bro1 domain are required for naked capsid secretion through binding to intracellular capsid, and we further found that the ubiquitinated Alix binds to wild type capsid but not K96R mutant. Moreover, screening of NEDD4 E3 ubiquitin ligase family members revealed that AIP4 stimulates the release of naked capsid, which relies on AIP4 protein integrity and E3 ligase activity. We further demonstrated that AIP4 interacts with Alix and promotes its ubiquitination, and AIP4 is essential for Alix-mediated naked capsid secretion. However, the Bro1 domain of Alix is non-ubiquitinated, indicating that Alix ubiquitination is not absolutely required for AIP4-induced naked capsid secretion. Taken together, our study sheds new light on the mechanism of HBV naked capsid egress in viral life cycle. Author summary: HBV-infected cells can produce various viral particles with different sizes and morphologies. Virions are complete particles with both nucleocapsids and surface antigen (HBsAg) envelope, which exploits the endosomal sorting complexes required for transport (ESCRT)/multivesicular body (MVB) pathway for budding. In addition to virion, HBV-infected cells also secrete non-enveloped (or naked) capsid particles. It remains unclear how HBV naked capsid is released from the infected cells. In this study, we further characterized the naked capsid and identified the Lysine 96 of HBV core protein as an important contact point for cellular protein Alix to mediate naked capsid secretion. We also determined NEDD4 E3 ubiquitin ligase family member AIP4 stimulates the release of naked capsid, regardless of their genome contents. AIP4 interacts with Alix and promotes its ubiquitination. However, the AIP4 protein per se rather than its E3 ubiquitin ligase activity is sufficient for Alix-induced naked capsid secretion. In summary, these findings provide new insights into a better understanding of HBV naked capsid egress. [ABSTRACT FROM AUTHOR]

Genome sequencing has revealed frequent mutations in Ras homolog family member A (RHOA) among various cancers with unique aberrant profiles and pathogenic effects, especially in peripheral T-cell lymphoma (PTCL). The discrete positional distribution and types of RHOA amino acid substitutions vary according to the tumor type, thereby leading to different functional and biological properties, which provide new insight into the molecular pathogenesis and potential targeted therapies for various tumors. However, the similarities and discrepancies in characteristics of RHOA mutations among various histologic subtypes of PTCL have not been fully elucidated. Herein we highlight the inconsistencies and complexities of the type and location of RHOA mutations and demonstrate the contribution of RHOA variants to the pathogenesis of PTCL by combining epigenetic abnormalities and activating multiple downstream pathways. The promising potential of targeting RHOA as a therapeutic modality is also outlined. This review provides new insight in the field of personalized medicine to improve the clinical outcomes for patients. [ABSTRACT FROM AUTHOR]

Protein methylation is a functionally important post-translational modification that occurs on diverse amino acid residues. The current proteomics approaches are inefficient to discover the methylation on residues other than Arg and Lys, which hinders the deep understanding of the functional role of rare protein methylation. Herein, we present a methyl-specific metabolic labeling approach for global methylome mapping, which enable the acquisition of methylome dataset covering diverse methylation types. Interestingly, of the identified methylation events, His methylation is found to be preferably occurred in C3H1 zinc fingers (ZFs). These His methylation events are determined to be Nπ specific and catalyzed by CARNMT1. The His methylation is found to stabilize the structure of ZFs. U2AF1 is used as a proof-of-concept to highlight the functional importance of His methylation in ZFs in RNA binding and RNA metabolism. The results of this study enable novel understanding of how protein methylation regulates cellular processes. Here the authors developed a methyl-specific metabolic labeling approach for global analysis covering diverse methylation types. His methylation is found in C3H1 zinc fingers. U2AF1 is used to highlight the role of His methylation in ZFs in RNA metabolism. [ABSTRACT FROM AUTHOR]

Modular nanotransporters (MNTs) are drug delivery systems for targeted cancer treatment. As MNTs are composed of several modules, they offer the advantage of high specificity and biocompatibility in delivering drugs to the target compartment of cancer cells. The large carrier module brings together functioning MNT modules and serves as a platform for drug attachment. The development of smaller-sized MNTs via truncation of the carrier module appears advantageous in facilitating tissue penetration. In this study, two new MNTs with a truncated carrier module containing either an N-terminal (MNTN) or a C-terminal (MNTC) part were developed by genetic engineering. Both new MNTs demonstrated a high affinity for target receptors, as revealed by fluorescent-labeled ligand-competitive binding. The liposome leakage assay proved the endosomolytic activity of MNTs. Binding to the importin heterodimer of each truncated MNT was revealed by a thermophoresis assay, while only MNTN possessed binding to Keap1. Finally, the photodynamic efficacy of the photosensitizer attached to MNTN was significantly higher than when attached to either MNTC or the original MNTs. Thus, this work reveals that MNT's carrier module can be truncated without losing MNT functionality, favoring the N-terminal part of the carrier module due to its ability to bind Keap1. [ABSTRACT FROM AUTHOR]

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-d-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3–7. Here we show that glutamate and its structural analogues, including NMDAR antagonist l-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4–7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.Glutamate functions as a positive allosteric modulator for acid-sensing ion channels to exacerbate ischaemic neurotoxicity. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance is an ongoing "one health" challenge of global concern. The acyl-ACP synthetase (termed AasS) of the zoonotic pathogen Vibrio harveyi recycles exogenous fatty acid (eFA), bypassing the requirement of type II fatty acid synthesis (FAS II), a druggable pathway. A growing body of bacterial AasS-type isoenzymes compromises the clinical efficacy of FAS II-directed antimicrobials, like cerulenin. Very recently, an acyl adenylate mimic, C10-AMS, was proposed as a lead compound against AasS activity. However, the underlying mechanism remains poorly understood. Here we present two high-resolution cryo-EM structures of AasS liganded with C10-AMS inhibitor (2.33 Å) and C10-AMP intermediate (2.19 Å) in addition to its apo form (2.53 Å). Apart from our measurements for C10-AMS' Ki value of around 0.6 μM, structural and functional analyses explained how this inhibitor interacts with AasS enzyme. Unlike an open state of AasS, ready for C10-AMP formation, a closed conformation is trapped by the C10-AMS inhibitor. Tight binding of C10-AMS blocks fatty acyl substrate entry, and therefore inhibits AasS action. Additionally, this intermediate analog C10-AMS appears to be a mixed-type AasS inhibitor. In summary, our results provide proof of principle that inhibiting salvage of eFA by AasS reverses the FAS II bypass. This facilitates the development of next-generation of anti-bacterial therapeutics, esp. the dual therapy consisting of C10-AMS scaffold derivatives combined with certain FAS II inhibitors. Author summary: An AasS-aided eFA recycling compromises the clinical efficacy of FAS II-directed antimicrobials. The C10-AMS compound is an effective inhibitor for AasS with the Ki value of ~0.6 μM. Efficient binding of AasS by C10-AMS blocks the entry of fatty acyl substrates of various length. The C10-AMS inhibitor mimics an acyl-AMP adenylate to trap AasS in a closed conformation rather than an open state. Collectively, inhibiting a bacterial eFA scavenger AasS enables the re-sensitization of FAS II-targeted antimicrobials. This provides proof of concept that a combination of C10-AMS scaffold derivatives with appropriate FAS II inhibitors constitutes a next-generation anti-virulence biotherapy. [ABSTRACT FROM AUTHOR]

Microscale thermophoresis (MST) is a well-established method to quantify protein-RNA interactions. In this study, we employed MST to analyze the RNA binding properties of glycine-rich RNA binding protein 7 (GRP7), which is known to have multiple biological functions related to its ability to bind different types of RNA. However, the exact mechanism of GRP7's RNA binding is not fully understood. While the RNA-recognition motif of GRP7 is known to be involved in RNA binding, the glycine-rich region (known as arginine-glycine-glycine-domain or RGG-domain) also influences this interaction. To investigate to which extend the RGG-domain of GRP7 is involved in RNA binding, mutation studies on putative RNA interacting or modulating sites were performed. In addition to MST experiments, we examined liquid–liquid phase separation of GRP7 and its mutants, both with and without RNA. Furthermore, we systemically investigated factors that might affect RNA binding selectivity of GRP7 by testing RNAs of different sizes, structures, and modifications. Consequently, our study revealed that GRP7 exhibits a high affinity for a variety of RNAs, indicating a lack of pronounced selectivity. Moreover, we established that the RGG-domain plays a crucial role in binding longer RNAs and promoting phase separation. [ABSTRACT FROM AUTHOR]

The sigma-1 receptor (σ1R) is a non-opioid membrane receptor, which responds to a diverse array of synthetic ligands to exert various pharmacological effects. Meanwhile, candidates for endogenous ligands of σ1R have also been identified. However, how endogenous ligands bind to σ1R remains unknown. Here, we present crystal structures of σ1R from Xenopus laevis (xlσ1R) bound to two endogenous neurosteroid ligands, progesterone (a putative antagonist) and dehydroepiandrosterone sulfate (DHEAS) (a putative agonist), at 2.15-3.09 Å resolutions. Both neurosteroids bind to a similar location in xlσ1R mainly through hydrophobic interactions, but surprisingly, with opposite binding orientations. DHEAS also forms hydrogen bonds with xlσ1R, whereas progesterone interacts indirectly with the receptor through water molecules near the binding site. Binding analyses are consistent with the xlσ1R-neurosteroid complex structures. Furthermore, molecular dynamics simulations and structural data reveal a potential water entry pathway. Our results provide insight into binding of two endogenous neurosteroid ligands to σ1R. How endogenous ligands bind to the sigma-1 receptor (σ1R) remains elusive. Here, the authors report crystal structures of σ1R from Xenopus laevis bound to two neurosteroid ligands, providing insight into their binding mechanism to σ1R. [ABSTRACT FROM AUTHOR]

Dermatan sulfate (DS) is a glycosaminoglycan characterized by having a variable structure and wide distribution in animal tissues. We previously demonstrated that some structural variants of DS were able to rapidly induce moderate necroptosis in luminal breast cancer cells when used at a high concentration. We have now investigated the mechanisms underlying the DS-mediated activation of the necroptotic executor MLKL using immunofluorescence, Western blotting and pharmacological inhibition. The two main processes, by which DS influences the phosphorylation of MLKL, are the activation of NFκB, which demonstrates a suppressive impact, and the induction of oxidative stress, which has a stimulatory effect. Moreover, the triggering of the redox imbalance by DS occurs via the modulatory influence of this glycosaminoglycan on the rearrangement of the actin cytoskeleton, requiring alterations in the activity of small Rho GTP-ase Rac1. All of these processes that were elicited by DS in luminal breast cancer cells showed a dependence on the structure of this glycan and the type of cancer cells. Furthermore, our results suggest that a major mechanism that is involved in the stimulation of necroptosis in luminal breast cancer cells by high doses of DS is mediated via the effect of this glycan on the activity of adhesion molecules. [ABSTRACT FROM AUTHOR]

The majority of rod-shaped and some filamentous plant viruses encode a cysteine-rich protein (CRP) that functions in viral virulence; however, the roles of these CRPs in viral infection remain largely unknown. Here, we used barley stripe mosaic virus (BSMV) as a model to investigate the essential role of its CRP in virus morphogenesis. The CRP protein γb directly interacts with BSMV coat protein (CP), the mutations either on the His-85 site in γb predicted to generate a potential CCCH motif or on the His-13 site in CP exposed to the surface of the virions abolish the zinc-binding activity and their interaction. Immunogold-labeling assays show that γb binds to the surface of rod-shaped BSMV virions in a Zn2+-dependent manner, which enhances the RNA binding activity of CP and facilitates virion assembly and stability, suggesting that the Zn2+-dependent physical association of γb with the virion is crucial for BSMV morphogenesis. Intriguingly, the tightly binding of diverse CRPs to their rod-shaped virions is a general feature employed by the members in the families Virgaviridae (excluding the genus Tobamovirus) and Benyviridae. Together, these results reveal a hitherto unknown role of CRPs in the assembly and stability of virus particles, and expand our understanding of the molecular mechanism underlying virus morphogenesis. Author summary: The symmetrical structures of plant RNA viruses are usually composed of numerous identical coat protein (CP) subunits encapsidated one molecule of viral genomic RNA. Interestingly, a large number of rod-shaped and filamentous plant viruses encode a cysteine-rich protein (CRP), which usually function as viral suppressors of RNA silencing and pathogenicity determinants during viral infection. However, their roles in viral life cycle remain poorly understood. Here, our study reveals the essential role of CRPs in virus morphogenesis. The cysteine-rich γb protein binds to the surface of rod-shaped barley stripe mosaic virus (BSMV) virion by interacting with CP in a Zn2+-dependent manner, thereby facilitating virion assembly and stability. Both His-85 site in γb predicted to generate a potential CCCH motif and His-13 site in CP exposed to the surface of the virions are key amino acids in γb-CP interaction and the zinc-binding activity. Attractively, the phenomenon of CRPs being tightly bound to their rod-shaped virions occurs in a broad range of distantly related viruses, implying a conserved role for the CRPs in orchestrating virus morphogenesis. [ABSTRACT FROM AUTHOR]

Receptors of cytokines are major regulators of the immune response. In this work, we have discovered two new ligands that can activate the TNFR1 (tumor necrosis factor receptor 1) receptor. Earlier, we found that the peptide of the Tag (PGLYRP1) protein designated 17.1 can interact with the TNFR1 receptor. Here, we have found that the Mts1 (S100A4) protein interacts with this peptide with a high affinity (Kd = 1.28 × 10−8 M), and that this complex is cytotoxic to cancer cells that have the TNFR1 receptor on their surface. This complex induces both apoptosis and necroptosis in cancer cells with the involvement of mitochondria and lysosomes in cell death signal transduction. Moreover, we have succeeded in locating the Mts1 fragment that is responsible for protein–peptide interaction, which highly specifically interacts with the Tag7 protein (Kd = 2.96 nM). The isolated Mts1 peptide M7 also forms a complex with 17.1, and this peptide–peptide complex also induces the TNFR1 receptor-dependent cell death. Molecular docking and molecular dynamics experiments show the amino acids involved in peptide binding and that may be used for peptidomimetics' development. Thus, two new cytotoxic complexes were created that were able to induce the death of tumor cells via the TNFR1 receptor. These results may be used in therapy for both cancer and autoimmune diseases. [ABSTRACT FROM AUTHOR]

Transglutaminase (EC 2.3.2.13, TGase), an enzyme that catalyzes the formation of covalent cross-links between protein or peptide molecules, plays a critical role in commercial food processing, medicine, and textiles. TGase from Streptomyces is the sole commercial enzyme preparation for cross-linking proteins. In this study, we revealed that the SOS response repressor protein LexA in Streptomyces mobaraensis not only triggers morphological development but also enhances TGase synthesis. The absence of lexA significantly diminished TGase production and sporulation. Although LexA does not bind directly to the promoter region of the TGase gene, it indirectly stimulates transcription of the tga gene, which encodes TGase. Furthermore, LexA directly enhances the expression of genes associated with protein synthesis and transcription factors, thus favorably influencing TGase synthesis at both the transcriptional and posttranscriptional levels. Moreover, LexA activates four crucial genes involved in morphological differentiation, promoting spore maturation. Overall, our findings suggest that LexA plays a dual role as a master regulator of the SOS response and a significant contributor to TGase regulation and certain aspects of secondary metabolism, offering insights into the cellular functions of LexA and facilitating the strategic engineering of TGase overproducers. [ABSTRACT FROM AUTHOR]

Small GTPases are monomeric (approximately 21 kDa) proteins that regulate a number of biological processes, such as vesicle transport, cell division cycle, cell migration, invasion, adhesion, proliferation, and DNA repair. They are also involved in carcinogenesis and neurodegenerative diseases. Some of these proteins, such as the Rho family proteins, are essential regulators of actin cytoskeleton, controlling cell adhesion and motility. This review examines both normal and pathological processes in human cells that are regulated by small GTPases of the Rho family. Particular attention is paid to small GTPase inhibitors and their use in the treatment of various diseases. [ABSTRACT FROM AUTHOR]

This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [ N 1112 (OH) ][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [ N 1112 (OH) ][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs' biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity. [ABSTRACT FROM AUTHOR]

The affinity constant, also known as the equilibrium constant, binding constant, equilibrium association constant, or the reciprocal value, the equilibrium dissociation constant (Kd), can be considered as one of the most important characteristics for any antibody–antigen pair. Many methods based on different technologies have been proposed and used to determine this value. However, since a very large number of publications and commercial datasheets do not include this information, significant obstacles in performing such measurements seem to exist. In other cases where such data are reported, the results have often proved to be unreliable. This situation may indicate that most of the technologies available today require a high level of expertise and effort that does not seem to be available in many laboratories. In this paper, we present a simple approach based on standard immunoassay technology that is easy and quick to perform. It relies on the effect that the molar IC50 approaches the Kd value in the case of infinitely small concentrations of the reagent concentrations. A two-dimensional dilution of the reagents leads to an asymptotic convergence to Kd. The approach has some similarity to the well-known checkerboard titration used for the optimization of immunoassays. A well-known antibody against the FLAG peptide, clone M2, was used as a model system and the results were compared with other methods. This approach could be used in any case where a competitive assay is available or can be developed. The determination of an affinity constant should belong to the crucial parameters in any quality control of antibody-related products and assays and should be mandatory in papers using immunochemical protocols. [ABSTRACT FROM AUTHOR]

ATP-binding cassette transporter B6 (ABCB6), a protein essential for heme biosynthesis in mitochondria, also functions as a heavy metal efflux pump. Here, we present cryo-electron microscopy structures of human ABCB6 bound to a cadmium Cd(II) ion in the presence of antioxidant thiol peptides glutathione (GSH) and phytochelatin 2 (PC2) at resolutions of 3.2 and 3.1 Å, respectively. The overall folding of the two structures resembles the inward-facing apo state but with less separation between the two halves of the transporter. Two GSH molecules are symmetrically bound to the Cd(II) ion in a bent conformation, with the central cysteine protruding towards the metal. The N-terminal glutamate and C-terminal glycine of GSH do not directly interact with Cd(II) but contribute to neutralizing positive charges of the binding cavity by forming hydrogen bonds and van der Waals interactions with nearby residues. In the presence of PC2, Cd(II) binding to ABCB6 is similar to that observed with GSH, except that two cysteine residues of each PC2 molecule participate in Cd(II) coordination to form a tetrathiolate. Structural comparison of human ABCB6 and its homologous Atm-type transporters indicate that their distinct substrate specificity might be attributed to variations in the capping residues situated at the top of the substrate-binding cavity. Cryo-EM structure of human ATP-binding cassette transporter 6 (ABCB6) in complex with Cd(II) and glutathione (GSH) provides molecular insight into its dual role, essential for heme biosynthesis and as a heavy metal efflux pump. [ABSTRACT FROM AUTHOR]

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure. NEW & NOTEWORTHY: SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components. [ABSTRACT FROM AUTHOR]

Introduction: B-cell activation triggers the release of endoplasmic reticulum calcium stores through the store-operated calcium entry (SOCE) pathway resulting in calcium influx by calcium release-activated calcium (CRAC) channels on the plasma membrane. B-cell-specific murine knockouts of SOCE do not impact humoral immunity suggesting that alternative channels may be important. Methods: We identified a member of the calcium-permeable transient receptor potential (TRP) ion channel family, TRPV5, as a candidate channel expressed in B cells by a quantitative polymerase chain reaction (qPCR) screen. To further investigate the role of TRPV5 in B-cell responses, we generated a murine TRPV5 knockout (KO) by CRISPR-Cas9. Results: We found TRPV5 polarized to B-cell receptor (BCR) clusters upon stimulation in a PI3K-RhoA-dependent manner. TRPV5 KO mice have normal B-cell development and mature B-cell numbers. Surprisingly, calcium influx upon BCR stimulation in primary TRPV5 KO B cells was not impaired; however, differential expression of other calcium-regulating proteins, such as ORAI1, may contribute to a compensatory mechanism for calcium signaling in these cells. We demonstrate that TRPV5 KOB cells have impaired spreading and contraction in response to membranebound antigen. Consistent with this, TRPV5 KO B cells have reduced BCR signaling measured through phospho-tyrosine residues. Lastly, we also found that TRPV5 is important for early T-dependent antigen specific responses post-immunization. Discussion: Thus, our findings identify a role for TRPV5 in BCR signaling and Bcell activation. [ABSTRACT FROM AUTHOR]

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity. [ABSTRACT FROM AUTHOR]

The emergence of biopolymer building blocks is a crucial step during the origins of life1–6. However, all known formation pathways rely on rare pure feedstocks and demand successive purification and mixing steps to suppress unwanted side reactions and enable high product yields. Here we show that heat flows through thin, crack-like geo-compartments could have provided a widely available yet selective mechanism that separates more than 50 prebiotically relevant building blocks from complex mixtures of amino acids, nucleobases, nucleotides, polyphosphates and 2-aminoazoles. Using measured thermophoretic properties7,8, we numerically model and experimentally prove the advantageous effect of geological networks of interconnected cracks9,10 that purify the previously mixed compounds, boosting their concentration ratios by up to three orders of magnitude. The importance for prebiotic chemistry is shown by the dimerization of glycine11,12, in which the selective purification of trimetaphosphate (TMP)13,14 increased reaction yields by five orders of magnitude. The observed effect is robust under various crack sizes, pH values, solvents and temperatures. Our results demonstrate how geologically driven non-equilibria could have explored highly parallelized reaction conditions to foster prebiotic chemistry.Heat flows through thin, crack-like geo-compartments are shown to purify previously mixed compounds and enhance their reactivity, providing a selective mechanism for separating molecules relevant to the chemical origins of life. [ABSTRACT FROM AUTHOR]

Protein–protein interactions (PPIs) offer great opportunities to expand the druggable proteome and therapeutically tackle various diseases, but remain challenging targets for drug discovery. Here, we provide a comprehensive pipeline that combines experimental and computational tools to identify and validate PPI targets and perform early-stage drug discovery. We have developed a machine learning approach that prioritizes interactions by analyzing quantitative data from binary PPI assays or AlphaFold-Multimer predictions. Using the quantitative assay LuTHy together with our machine learning algorithm, we identified high-confidence interactions among SARS-CoV-2 proteins for which we predicted three-dimensional structures using AlphaFold-Multimer. We employed VirtualFlow to target the contact interface of the NSP10-NSP16 SARS-CoV-2 methyltransferase complex by ultra-large virtual drug screening. Thereby, we identified a compound that binds to NSP10 and inhibits its interaction with NSP16, while also disrupting the methyltransferase activity of the complex, and SARS-CoV-2 replication. Overall, this pipeline will help to prioritize PPI targets to accelerate the discovery of early-stage drug candidates targeting protein complexes and pathways. Synopsis: A new pipeline for prioritizing protein-protein interactions (PPIs) for drug discovery, combines machine learning-based scoring of quantitative PPI data, protein complex structure prediction and virtual drug screening. A multi-adaptive support vector machine (maSVM) classifier is used for scoring PPIs from quantitative interaction and structure prediction data. The machine learning-based classifier is applicable to PPI datasets from various assays and AlphaFold-Multimer predictions improving comparability between different methods. Interaction mapping with LuTHy and maSVM-based scoring identified high-confidence SARS-CoV-2 PPIs. Subsequent AlphaFold-Multimer predictions revealed key interaction residues within the NSP10-NSP16 methyltransferase complex. Targeting the complex with virtual compound screening identified an early-stage small molecule inhibitor that disrupts the NSP10-NSP16 interaction and SARS-CoV-2 replication. A new pipeline for prioritizing protein-protein interactions (PPIs) for drug discovery, combines machine learning-based scoring of quantitative PPI data, protein complex structure prediction and virtual drug screening. [ABSTRACT FROM AUTHOR]

Naphthylisoquinoline (NIQ) alkaloids are rising as a promising class of secondary metabolites with pharmaceutical potential. NF-κB has already been recognized as a significant modulator of cancer proliferation and drug resistance. We have previously reported the mechanisms behind the cytotoxic effect of dioncophylline A, an NIQ monomer, in leukemia cells. In the current study, we have investigated the cytotoxic effect of jozimine A2, an NIQ dimer, on leukemia cells in comparison to a second, structurally unsymmetric dimer, michellamine B. To this end, molecular docking was applied to predict the binding affinity of the dimers towards NF-κB, which was then validated through microscale thermophoresis. Next, cytotoxicity assays were performed on CCRF-CEM cells and multidrug-resistant CEM/ADR5000 cells following treatment. Transcriptome analysis uncovered the molecular networks affected by jozimine A2 and identified the cell cycle as one of the major affected processes. Cell death modes were evaluated through flow cytometry, while angiogenesis was measured with the endothelial cell tube formation assay on human umbilical vein endothelial cells (HUVECs). The results indicated that jozimine A2 bound to NF-κB, inhibited its activity and prevented its translocation to the nucleus. In addition, jozimine A2 induced cell death through apoptosis and prevented angiogenesis. Our study describes the cytotoxic effect of jozimine A2 on leukemia cells and explains the interactions with the NF-κB signaling pathway and the anticancer activity. [ABSTRACT FROM AUTHOR]

SMADs are the canonical intracellular effector proteins of the TGF-β (transforming growth factor-β). SMADs translocate from plasma membrane receptors to the nucleus regulated by many SMAD-interacting proteins through phosphorylation and other post-translational modifications that govern their nucleocytoplasmic shuttling and subsequent transcriptional activity. The signaling pathway of TGF-β/SMAD exhibits both tumor-suppressing and tumor-promoting phenotypes in epithelial-derived solid tumors. Collectively, the pleiotropic nature of TGF-β/SMAD signaling presents significant challenges for the development of effective cancer therapies. Here, we review preclinical studies that evaluate the efficacy of inhibitors targeting major SMAD-regulating and/or -interacting proteins, particularly enzymes that may play important roles in epithelial or mesenchymal compartments within solid tumors. [ABSTRACT FROM AUTHOR]

Background and Objectives: 4-Methylumbelliferone (4-MU) is a coumarin compound that can be extracted from the medicinal plant with anti-cancer properties, Smilax china L. In recent years, studies have revealed its potential as an anti-tumor and anti-metastasis drug with promising effects in cancer treatment. Despite an increase in research on the metabolic patterns of tumor cells, no prior research has suggested that 4-MU inhibits tumor proliferation by blocking glycolysis. This thesis presents evidence that 4-MU binds to proteins involved in glycolysis, thus mediating its anti-tumor effects. Materials and Methods: Network pharmacology, transcriptomics, and molecular docking were utilized to forecast the potential targets and probable pathways of 4-MU's anti-cancer activity, and the affinity of 4-MU towards potential targets was discovered using microscale thermophoresis (MST) detection. Results: The results of transcriptome analysis brought to light that the genes with differential expressions were primarily enriched in metabolic pathways, including glycolysis-related proteins. Using network pharmacology and molecular docking, our study identified Heat Shock Protein 90 Alpha Family Class A Member 1 (Hsp90AA1), mitochondria, phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2), and glucose-6-phosphate isomerase (GPI) as potential targets of 4-MU. The strong binding affinity between 4-MU and these proteins was confirmed by the MST assay. Conclusion: The findings indicate that 4-MU can hinder glycolysis by binding to glycolysis-associated proteins such as Hsp90AA1, PGK2, GPD2, and GPI. This results in the prevention of the energy supply to the tumor tissue, which ultimately curbs tumor growth, thereby demonstrating its anti-tumor properties. These results conclude that 4-MU has the capacity to be a novel glycolysis inhibitor for cancer treatment. Moreover, the identification of these glycolysis-associated proteins as possible targets for cancer therapy offers new avenues for research in the field of cancer treatment, thus providing further valuable evidence for the anti-cancer mechanism of 4-MU. [ABSTRACT FROM AUTHOR]

Interferon alpha-2b (IFN- α 2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN- α 2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C ​ 2 C ​ 1 Im][C ​ 4 F ​ 9 SO ​ 3 ] and [N ​ 1112 (OH) ][C ​ 4 F ​ 9 SO ​ 3 ]) vs. mere fluoro-containing IL ([C ​ 4 C ​ 1 Im][CF ​ 3 SO ​ 3 ]), in combination with sucrose or [N ​ 1112 (OH) ][H ​ 2 PO ​ 4 ] (well-known globular protein stabilizers), or high-charge-density salt K ​ 3 PO ​ 4 were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN- α 2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN- α 2b high in the enriched IFN- α 2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN- α 2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN- α 2b and BSA with the ABS phase-forming components were assessed by MST, showing the strong interaction between FILs aggregates and both proteins. In view of their biocompatibility, customizable properties, and selectivity, FIL-based ABSs are suggested as an improved purification step that could facilitate the development of biologics. [ABSTRACT FROM AUTHOR]

Proteins are large biomolecules with a specific structure that is composed of one or more long amino acid chains. Correct protein structures are directly linked to their correct function, and many environmental factors can have either positive or negative effects on this structure. Thus, there is a clear need for methods enabling the study of proteins, their correct folding, and components affecting protein stability. There is a significant number of label-free methods to study protein stability. In this review, we provide a general overview of these methods, but the main focus is on fluorescence-based low-instrument and -expertise-demand techniques. Different aspects related to thermal shift assays (TSAs), also called differential scanning fluorimetry (DSF) or ThermoFluor, are introduced and compared to isothermal chemical denaturation (ICD). Finally, we discuss the challenges and comparative aspects related to these methods, as well as future opportunities and assay development directions. [ABSTRACT FROM AUTHOR]

Breast-milk αS1-casein is a Toll-like receptor 4 (TLR4) agonist, whereas phosphorylated αS1-casein does not bind TLR4. The objective of this study was to analyse the structural requirements for these effects. In silico analysis of αS1-casein indicated high α-helical content with coiled-coil characteristics. This was confirmed by CD-spectroscopy, showing the α-helical conformation to be stable between pH 2 and 7.4. After in vitro phosphorylation, the α-helical content was significantly reduced, similar to what it was after incubation at 80 °C. This conformation showed no in vitro induction of IL-8 secretion via TLR4. A synthetic peptide corresponding to V77-E92 of αS1-casein induced an IL-8 secretion of 0.95 ng/mL via TLR4. Our results indicate that αS1-casein appears in two distinct conformations, an α-helical TLR4-agonistic and a less α-helical TLR4 non-agonistic conformation induced by phosphorylation. This is to indicate that the immunomodulatory role of αS1-casein, as described before, could be regulated by conformational changes induced by phosphorylation. [ABSTRACT FROM AUTHOR]

The aims of this study were to determine whether it is possible to use peptide microarrays obtained using the SPOT technique (immobilized on cellulose) and specific polyclonal antibodies to select fragments that reconstruct the outer sphere of proteins and to ascertain whether the selected peptide fragments can be useful in the study of their protein–protein and/or peptide–protein interactions. Using this approach, epidermal growth factor (EGF) fragments responsible for the interaction with the EGF receptor were searched. A library of EGF fragments immobilized on cellulose was obtained using triazine condensing reagents. Experiments on the interactions with EGFR confirmed the high affinity of the selected peptide fragments. Biological tests on cells showed the lack of cytotoxicity of the EGF fragments. Selected EGF fragments can be used in various areas of medicine. [ABSTRACT FROM AUTHOR]

Cytolethal distending toxins (CDTs) are intracellular-acting bacterial genotoxins generated by a diverse group of mucocutaneous human pathogens. CDTs must successfully bind to the plasma membrane of host cells in order to exert their modulatory effects. Maximal toxin activity requires all three toxin subunits, CdtA, CdtB, and CdtC, which, based primarily on high-resolution structural data, are believed to preassemble into a tripartite complex necessary for toxin activity. However, biologically active toxin has not been experimentally demonstrated to require assembly of the three subunits into a heterotrimer. Here, we experimentally compared concentration-dependent subunit interactions and toxin cellular activity of the Campylobacter jejuni CDT (Cj-CDT). Coimmunoprecipitation and dialysis retention experiments provided evidence for the presence of heterotrimeric toxin complexes, but only at concentrations of Cj-CdtA, Cj-CdtB, and Cj-CdtC several logs higher than required for Cj-CDTmediated arrest of the host cell cycle at the G2/M interface, which is triggered by the endonuclease activity associated with the catalytic Cj-CdtB subunit. Microscale thermophoresis confirmed that Cj-CDT subunit interactions occur with low affinity. Collectively, our data suggest that at the lowest concentrations of toxin sufficient for arrest of cell cycle progression, mixtures of Cj-CdtA, Cj-CdtB, and Cj-CdtC consist primarily of non-interacting, subunit monomers. The lack of congruence between toxin tripartite structure and cellular activity suggests that the widely accepted model that CDTs principally intoxicate host cells as preassembled heterotrimeric structures should be revisited. [ABSTRACT FROM AUTHOR]

The specific and sensitive detection of 17β-estradiol (E2) is critical for diagnosing and treating numerous diseases, and aptamers have emerged as promising recognition probes for developing detection platforms. However, traditional long-sequence E2 aptamers have demonstrated limited clinical performance due to redundant structures that can affect their stability and recognition ability. There is thus an urgent need to further optimize the structure of the aptamer to build an effective detection platform for E2. In this work, we have designed a novel short aptamer that retains the key binding structure of traditional aptamers to E2 while eliminating the redundant structures. The proposed aptamer was evaluated for its binding properties using microscale thermophoresis, a gold nanoparticle-based colorimetric method, and electrochemical assays. Our results demonstrate that the proposed aptamer has excellent specific recognition ability for E2 and a high affinity with a dissociation constant of 92 nM. Moreover, the aptamer shows great potential as a recognition probe for constructing a highly specific and sensitive clinical estradiol detection platform. The aptamer-based electrochemical sensor enabled the detection of E2 with a linear range between 5 pg mL–1 and 10 ng mL–1 (R2 = 0.973), and the detection capability of a definite low concentration level was 5 pg mL–1 (S/N = 3). Overall, this novel aptamer holds great promise as a valuable tool for future studies on the role of E2 in various physiological and pathological processes and for developing sensitive and specific diagnostic assays for E2 detection in clinical applications. [ABSTRACT FROM AUTHOR]

Developing a preventative vaccine for HIV-1 has been a global priority. The elicitation of broadly neutralizing antibodies (bNAbs) against a broad range of HIV-1 envelopes (Envs) from various strains appears to be a critical requirement for an efficacious HIV-1 vaccine. To understand their ability to neutralize HIV-1, it is important to characterize the binding characteristics of bNAbs. Our work is the first to utilize microscale thermophoresis (MST), a rapid, economical, and flexible in-solution temperature gradient method to quantitatively determine the binding affinities of bNAbs and non-neutralizing monoclonal antibodies (mAbs) to HIV-1 recombinant envelope monomer and trimer proteins of different subtypes, pseudoviruses (PVs), infectious molecular clones (IMCs), and cells expressing the trimer. Our results demonstrate that the binding affinities were subtype-dependent. The bNAbs exhibited a higher affinity to IMCs compared to PVs and recombinant proteins. The bNAbs and mAbs bound with high affinity to native-like gp160 trimers expressed on the surface of CEM cells compared to soluble recombinant proteins. Interesting differences were seen with V2-specific mAbs. Although they recognize linear epitopes, one of the antibodies also bound to the Envs on PVs, IMCs, and a recombinant trimer protein, suggesting that the epitope was not occluded. The identification of epitopes on the envelope surface that can bind to high affinity mAbs could be useful for designing HIV-1 vaccines and for down-selecting vaccine candidates that can induce high affinity antibodies to the HIV-1 envelope in their native conformation. [ABSTRACT FROM AUTHOR]

β-Lactams are the most widely prescribed class of antibiotics that inhibit penicillin-binding proteins (PBPs), particularly transpeptidases that function in peptidoglycan synthesis. A major mechanism of antibiotic resistance is the production of β-lactamase enzymes, which are capable of hydrolyzing β-lactam antibiotics. There have been many efforts to counter increasing bacterial resistance against β-lactams. These studies have mainly focused on three areas: discovering novel inhibitors against β-lactamases, developing new β-lactams less susceptible to existing resistance mechanisms, and identifying non-β-lactam inhibitors against cell wall transpeptidases. Drug discovery in the β-lactam field has afforded a range of research opportunities for academia. In this review, we summarize the recent new findings on both β-lactamases and cell wall transpeptidases because these two groups of enzymes are evolutionarily and functionally connected. Many efforts to develop new β-lactams have aimed to inhibit both transpeptidases and β-lactamases, while several promising novel β-lactamase inhibitors have shown the potential to be further developed into transpeptidase inhibitors. In addition, the drug discovery progress against each group of enzymes is presented in three aspects: understanding the targets, screening methodology, and new inhibitor chemotypes. This is to offer insights into not only the advancement in this field but also the challenges, opportunities, and resources for future research. In particular, cyclic boronate compounds are now capable of inhibiting all classes of β-lactamases, while the diazabicyclooctane (DBO) series of small molecules has led to not only new β-lactamase inhibitors but potentially a new class of antibiotics by directly targeting PBPs. With the cautiously optimistic successes of a number of new β-lactamase inhibitor chemotypes and many questions remaining to be answered about the structure and function of cell wall transpeptidases, non-β-lactam transpeptidase inhibitors may usher in the next exciting phase of drug discovery in this field. [ABSTRACT FROM AUTHOR]

Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaMmediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis. [ABSTRACT FROM AUTHOR]

Atherosclerotic cardiovascular disease is the leading cause of mortality worldwide, and hypercholesterolemia is a central risk factor for atherosclerosis. This study evaluated the effects of Totum-070, a plant-based polyphenol-rich supplement, in hamsters with high-fat diet (HFD)-induced dyslipidemia. The molecular mechanisms of action were explored using human Caco2 enterocytes. Totum-070 supplementation reduced the total cholesterol (−41%), non-HDL cholesterol (−47%), and triglycerides (−46%) in a dose-dependent manner, compared with HFD. HFD-induced hepatic steatosis was also significantly decreased by Totum-070, an effect associated with the reduction in various lipid and inflammatory gene expression. Upon challenging with olive oil gavage, the post-prandial triglyceride levels were strongly reduced. The sterol excretion in the feces was increased in the HFD-Totum-070 groups compared with the HFD group and associated with reduction of intestinal cholesterol absorption. These effects were confirmed in the Caco2 cells, where incubation with Totum-070 inhibited cholesterol uptake and apolipoprotein B secretion. Furthermore, a microbiota composition analysis revealed a strong effect of Totum-070 on the alpha and beta diversity of bacterial species and a significant decrease in the Firmicutes to Bacteroidetes ratio. Altogether, our findings indicate that Totum-070 lowers hypercholesterolemia by reducing intestinal cholesterol absorption, suggesting that its use as dietary supplement may be explored as a new preventive strategy for cardiovascular diseases. [ABSTRACT FROM AUTHOR]

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A–D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors. [ABSTRACT FROM AUTHOR]

Cytolethal distending toxins (CDTs) are intracellular-acting bacterial genotoxins generated by a diverse group of mucocutaneous human pathogens. CDTs must successfully bind to the plasma membrane of host cells in order to exert their modulatory effects. Maximal toxin activity requires all three toxin subunits, CdtA, CdtB, and CdtC, which, based primarily on high-resolution structural data, are believed to preassemble into a tripartite complex necessary for toxin activity. However, biologically active toxin has not been experimentally demonstrated to require assembly of the three subunits into a heterotrimer. Here, we experimentally compared concentration-dependent subunit interactions and toxin cellular activity of the Campylobacter jejuni CDT (Cj-CDT). Coimmunoprecipitation and dialysis retention experiments provided evidence for the presence of heterotrimeric toxin complexes, but only at concentrations of Cj-CdtA, Cj-CdtB, and Cj-CdtC several logs higher than required for Cj-CDTmediated arrest of the host cell cycle at the G2/M interface, which is triggered by the endonuclease activity associated with the catalytic Cj-CdtB subunit. Microscale thermophoresis confirmed that Cj-CDT subunit interactions occur with low affinity. Collectively, our data suggest that at the lowest concentrations of toxin sufficient for arrest of cell cycle progression, mixtures of Cj-CdtA, Cj-CdtB, and Cj-CdtC consist primarily of non-interacting, subunit monomers. The lack of congruence between toxin tripartite structure and cellular activity suggests that the widely accepted model that CDTs principally intoxicate host cells as preassembled heterotrimeric structures should be revisited. [ABSTRACT FROM AUTHOR]

One strategy to combat antimicrobial resistance is the discovery of new classes of antibiotics. Most antibiotics will at some point interact with the bacterial membrane to either interfere with its integrity or to cross it. Reliable and efficient tools for determining the dissociation constant for membrane binding (KD) and the partitioning coefficient between the aqueous- and membrane phases (KP) are therefore important tools for discovering and optimizing antimicrobial hits. Here we demonstrate that microscale thermophoresis (MST) can be used for label-free measurement of KD by utilising the intrinsic fluorescence of tryptophan and thereby removing the need for chromophore labelling. As proof of principle, we have used the method to measure the binding of a set of small cyclic AMPs to large unilamellar vesicles (LUVs) and two types of lipid nanodiscs assembled by styrene maleic acid (SMA) and quaternary ammonium SMA (SMA-QA). The measured KD values correlate well with the corresponding measurements using surface plasmon resonance (SPR), also broadly reflecting the tested AMPs' minimal inhibition concentration (MIC) towards S. aureus and E. coli. We conclude that MST is a promising method for fast and cost-efficient detection of peptide-lipid interactions or mapping of sample conditions in preparation for more advanced studies that rely on expensive sample preparation, labelling and/or instrument time. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated that bis-(3',5')-cyclic diguanosine monophosphate (bis-3',5'-c-di-GMP) is a ubiquitous second messenger employed by bacteria. Here, we report that 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) controls the important biological functions, quorum sensing (QS) signaling systems and virulence in Ralstonia solanacearum through the transcriptional regulator RSp0980. This signal specifically binds to RSp0980 with high affinity and thus abolishes the interaction between RSp0980 and the promoters of target genes. In-frame deletion of RSp0334, which contains an evolved GGDEF domain with a LLARLGGDQF motif required to catalyze 2',3'-cGMP to (2',5')(3',5')-cyclic diguanosine monophosphate (2',3'-c-di-GMP), altered the abovementioned important phenotypes through increasing the intracellular 2',3'-cGMP levels. Furthermore, we found that 2',3'-cGMP, its receptor and the evolved GGDEF domain with a LLARLGGDEF motif also exist in the human pathogen Salmonella typhimurium. Together, our work provides insights into the unusual function of the GGDEF domain of RSp0334 and the special regulatory mechanism of 2',3'-cGMP signal in bacteria. Nucleotide second messengers are employed by many bacterial species to regulate various cellular processes. Here, the authors demonstrate that 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) controls the important biological functions and virulence in Ralstonia solanacearum by abolishing the interaction between a transcriptional regulator and the promoters of target genes. [ABSTRACT FROM AUTHOR]