Site-specific Interactions Research Articles

Hsa-miR-214-3p inhibits breast cancer cell growth and improves the tumor immune microenvironment by downregulating B7H3.

Immune checkpoint inhibitors play an important role in the treatment of solid tumors, but the currently used immune checkpoint inhibitors targeting programmed cell death-1 (PD-1), programmed cell death ligand-1 (PD-L1), and cytotoxic T-lymphocyte antigen-4 (CTLA-4) show limited clinical efficacy in many breast cancers. B7H3 has been widely reported as an immunosuppressive molecule, but its immunological function in breast cancer patients remains unclear. We analyzed the expression of B7H3 in breast cancer samples using data from the Cancer Genome Atlas Program (TCGA) and the Gene Expression Omnibus (GEO) databases. MicroRNAs were selected using the TarBase, miRTarBase, and miRBase databases. The regulatory role of the microRNA hsa-miR-214-3p on B7H3 was investigated through dual-luciferase reporter assays, which identified the specific action sites of interaction. The expression levels of B7H3 and hsa-miR-214-3p in human breast cancer tissues and adjacent normal tissues were quantified using Western blotting and quantitative PCR (qPCR). In vitro experiments were performed to observe the effects of modulating the expression of B7H3 or hsa-miR-214-3p on breast cancer cell proliferation and apoptosis. Additionally, the regulatory impact of hsa-miR-214-3p on B7H3 was examined. Enzyme-linked immunosorbent assays (ELISA) and flow cytometry were employed to assess the effects of co-cultured breast cancer cells and normal human peripheral blood mononuclear cells (PBMCs) on immune cells and associated cytokines. In breast cancer tissues, the expression level of B7H3 is inversely correlated with that of hsa-miR-214-3p, as well as with the regulatory effects on breast cancercell behavior. Hsa-miR-214-3p was found to inhibit breast cancer cell growth by downregulating B7H3. Importantly, our research identified, for the first time, two binding sites for hsa-miR-214-3p on the 3' UTR of B7H3, both of which exert similar effects independently. Co-culture experiments revealed that hsa-miR-214-3p obstructs the suppressive function of B7H3 on CD8+ T cells and natural killer cells. This study confirms the existence of two hsa-miR-214-3p binding sites on the 3' UTR of B7H3, reinforcing the role of hsa-miR-214-3p as a regulatory factor for B7H3. In breast cancer, hsa-miR-214-3p reduces tumor cell proliferation and enhances the tumor immune microenvironment by downregulating B7H3. These findings suggest new potential targets for the clinical treatment of breast cancer.

Transcriptomic, mutational and structural bioinformatics approaches to explore the therapeutic role of FAP in predominant cancer types

Fibroblast activating protein (FAP) is a cell surface marker of cancer-associated fibroblasts with a distinct pro-tumorigenic role. The present study analyzed the pan-cancer expression; and clinical and mutational profiles of the FAP coding gene. Molecular dynamics simulation (MDS) deciphered the backbone dynamics and energetics of FAP. Virtual screening and subsequent pharmacokinetic-profiling (PK) filtered lead molecules, which were subjected to molecular docking. MDS projected a stable trajectory for the protein, as dynamics evidenced by low residue-level fluctuations, stable backbone dynamics, and energetics. Around five stabilization and deleterious mutations in the catalytic domain were identified. The low binding energy (BE) profiles from molecular docking studies screened the top five lead molecules for site-specific intermolecular interaction studies. Lead-16 (ZINC000245289699) exhibited a significant BE and inhibition constant of −6.87 kcal/mol and 12.27 μM, respectively, across FAP and its mutants. Interestingly, the docked complexes of Lead-16 interacted with the catalytic triad residues (S624, D702, and H734). The docked complexes of Lead-16 with FAP showed lower average root-mean-square fluctuations compared to the unbound protein, suggesting a stable ligand–protein complex. The tumor-specific expression and its critical overall survival suggest the inhibitors of FAP for potential cancer therapeutic intervention and hindering tumor microenvironment-driven cancer progression.

Persistent luminescent ZnAl-LDH nanosheets for all-weather degradation of nitenpyram and tetracycline: Superoxide radical induction, life cycle analysis and engineering applications

Photocatalysts designed for all-weather applications, capable of efficient charge carrier separation, are set to transform the adoption of photocatalysis-driven advanced oxidation processes in wastewater management. In this investigation, an energy-storing ZnAl-LDH@SrAl2O4:Eu2+, Dy3+ (SALDH) was synthesized successfully, optimized for the photocatalytic selective degradation of NTP and tetracycline hydrochloride (TC) under varying weather conditions, with 100 % degradation of both NTP and TC within 75 min. Remarkably, SALDH demonstrates energy storage capabilities that markedly reduce energy demands relative to traditional photocatalytic approaches. The primary reactive species identified were O2−, and the Fukui index, computed via density-functional theory (DFT), identified the specific interaction sites of O2− on NTP molecules. Utilizing the energy retention properties of SALDH, a novel, environmentally friendly device was engineered for processing organic pollutants in agricultural wastewater. This system harness the energy from solar radiation to induce the oxidation of pollutants. The experiments that were performed in the both artificially prepared lab-scale and actual agricultural WWT plants indicated the degradation efficiencies of 36 % and 37 %, respectively. As seen during the day, both SALDH and persulfate were capable of effectively decomposing organic contaminants; at night, the SA’s luminescence maintained the oxidative ability of ZnAl-LDH. Meanwhile, SALDH combines the corrosion resistance of ZnAl-LDH with good metal compatibility, enabling SALDH to effectively improve the photostability of long afterglow. This work contributes to the existing knowledge on the degradation of organic pollutants by efficient photocatalysts and points to possible implementations in water treatment processes.

Heterotypic Site-Specific Interactions between WNK4, TSC22D2, and NRBP1 Promote Kinase Activity and Co-localization in WNK Bodies

Heterotypic Site-Specific Interactions between WNK4, TSC22D2, and NRBP1 Promote Kinase Activity and Co-localization in WNK Bodies

Small-molecule properties define partitioning into biomolecular condensates

Biomolecular condensates regulate cellular function by compartmentalizing molecules without a surrounding membrane. Condensate function arises from the specific exclusion or enrichment of molecules. Thus, understanding condensate composition is critical to characterizing condensate function. Whereas principles defining macromolecular composition have been described, understanding of small-molecule composition remains limited. Here we quantified the partitioning of ~1,700 biologically relevant small molecules into condensates composed of different macromolecules. Partitioning varied nearly a million-fold across compounds but was correlated among condensates, indicating that disparate condensates are physically similar. For one system, the enriched compounds did not generally bind macromolecules with high affinity under conditions where condensates do not form, suggesting that partitioning is not governed by site-specific interactions. Correspondingly, a machine learning model accurately predicts partitioning using only computed physicochemical features of the compounds, chiefly those related to solubility and hydrophobicity. These results suggest that a hydrophobic environment emerges upon condensate formation, driving the enrichment and exclusion of small molecules.

Competitive Effects of Anions on Protein Solvation by Aqueous Ionic Liquids.

The present study utilizes molecular dynamics simulations to examine how different anions compete for protein solvation in aqueous solutions of ionic liquids (ILs). Ubiquitin is used as model protein and studied in IL mixtures sharing the same cation, 1-ethyl-3-methylimidazolium (EMIM), and two different anions in the same solution, from combinations of dicyanamide (DCA), chloride (Cl), nitrate (NO3), and tetrafluoroborate (BF4). Our findings reveal that specific interactions between anions and the protein are paramount in IL solvation, but that combinations of anions are not additive. For example, DCA exhibits a remarkable ability to form hydrogen bonds with the protein, resulting in a significantly stronger preferential binding to the protein than other anions. However, the combination of DCA with NO3, which also forms hydrogen bonds with the protein, results in a smaller preferential solvation of the protein than the combination of DCA with chloride ions, which are weaker binders. Thus, combining anions with varying affinities for the protein surface modulates the overall ion accumulation through nonadditive mechanisms, highlighting the importance of the understanding of competition for specific interaction sites, cooperative binding, bulk-solution affinity, and overall charge compensations, on the overall solvation capacity of the solution. Such knowledge may allow for the design of novel IL-based processes in biotechnology and material science, where fine-tuning protein solvation is crucial for optimizing performance and functionality.

Trimethylamine N-oxide (TMAO) doubly locks the hydrophobic core and surfaces of protein against desiccation stress.

Interactions between proteins and osmolytes are ubiquitous within cells, assisting in response to environmental stresses. However, our understanding of protein-osmolyte interactions underlying desiccation tolerance is limited. Here, we employ solid-state NMR (ssNMR) to derive information about protein conformation and site-specific interactions between the model protein, SH3, and the osmolyte trimethylamine N-oxide (TMAO). The data show that SH3-TMAO interactions maintain key structured regions during desiccation and facilitate reversion to the protein's native state once desiccation stress is even slightly relieved. We identify 10 types of residues at 28 sites involved in the SH3-TMAO interactions. These sites comprise hydrophobic, positively charged, and aromatic amino acids located in SH3's hydrophobic core and surface clusters. TMAO locks both the hydrophobic core and surface clusters through its zwitterionic and trimethyl ends. This double locking is responsible for desiccation tolerance and differs from ideas based on exclusion, vitrification, and water replacement. ssNMR is a powerful tool for deepening our understanding of extremely weak protein-osmolyte interactions and providing insight into the evolutionary mechanism of environmental tolerance.

PGRMC1 promotes NSCLC stemness phenotypes by disrupting TRIM56-mediated ubiquitination of AHR

Cancer stem cells (CSCs) are responsible for tumor chemoresistance, and the aryl hydrocarbon receptor (AHR) is indispensable for maintaining CSC characteristics. Here, we aimed to investigate how the interaction between progesterone receptor membrane component 1 (PGRMC1) and AHR contributes to the maintenance of CSC phenotypes in non-small cell lung cancer (NSCLC). Clinical data and tissue microarray analyses indicated that patients with elevated PGRMC1 expression had poorer prognoses. Moreover, PGRMC1 overexpression enhanced CSC phenotypes and chemotherapy resistance in vitro and in vivo by modulating AHR ubiquitination. We then determined the specific interaction sites between PGRMC1 and AHR. Mass spectrometry screening identified tripartite motif containing 56 (TRIM56) as the E3 ligase targeting AHR. Notably, PGRMC1 overexpression inhibited the interaction between TRIM56 and AHR. Overall, our study revealed a regulatory mechanism that involves PGRMC1, AHR, and TRIM56, providing insights for developing CSC-targeting strategies in NSCLC treatment.

Structure and Dynamics of Drk-SH2 Domain and Its Site-Specific Interaction with Sev Receptor Tyrosine Kinase.

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three β strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.

Dual Antimicrobial-Anticancer Potential, Hydrolysis, and DNA/BSA Binding Affinity of a Novel Water-Soluble Ruthenium-Arene Ethylenediamine Schiff base (RAES) Organometallic

The need for a systematic approach in developing new metal-based drugs with dual anticancer-antimicrobial properties is emphasized by the vulnerability of cancer patients to bacterial infections. In this context, a novel organometallic assembly was designed, featuring ruthenium(II) coordination with p-cymene, one chlorido ligand, and a bidentate neutral Schiff base derived from 4-methoxybenzaldehyde and N,N-dimethylethylenediamine. The compound was extensively characterized in both solid-state and solution, employing single crystal X-ray diffraction, nuclear magnetic resonance, infrared, ultraviolet–visible spectroscopy, and density functional theory, alongside Hirshfeld surface analysis. The hydrolysis kinetic was thoroughly investigated, revealing the important role of the chloro-aqua equilibrium in the dynamics of binding with deoxyribonucleic acid and bovine serum albumin. Notably, the aqua species exhibited a pronounced affinity for deoxyribonucleic acid, engaging through electrostatic and hydrogen bonding interactions, while the chloro species demonstrated groove-binding properties. Interaction with albumin revealed distinct binding mechanisms. The aqua species displayed covalent binding, contrasting with the ligand-like van der Waals interactions and hydrogen bonding observed with the chloro specie. Molecular docking studies highlighted site-specific interactions with biomolecular targets. Remarkably, the compound exhibited wide spectrum moderate antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans, coupled with low micromolar cytotoxic activity against human colorectal adenocarcinoma cells and significant activity against human leukemic monocyte lymphoma cells. The presented findings encourage further development of this compound, promising avenues for its evolution into a versatile therapeutic agent targeting both infectious diseases and cancer.

Ion and Molecular Sieving With Ultrathin Polydopamine Nanomembranes.

In contrast to biological cell membranes, it is still a major challenge for synthetic membranes to efficiently separate ions and small molecules due to their similar sizes in the sub-nanometer range. Inspired by biological ion channels with their unique channel wall chemistry that facilitates ion sieving by ion-channel interactions, the first free-standing, ultrathin (10-17nm) nanomembranes composed entirely of polydopamine (PDA) are reported here as ion and molecular sieves. These nanomembranes are obtained via an easily scalable electropolymerization strategy and provide nanochannels with various amine and phenolic hydroxyl groups that offer a favorable chemical environment for ion-channel electrostatic and hydrogen bond interactions. They exhibit remarkable selectivity for monovalent ions over multivalent ions and larger species with K+/Mg2+ of ≈4.2, K+/[Fe(CN)6]3- of ≈10.3, and K+/Rhodamine B of ≈273.0 in a pressure-driven process, as well as cyclic reversible pH-responsive gating properties. Infrared spectra reveal hydrogen bond formation between hydrated multivalent ions and PDA, which prevents the transport of multivalent ions and facilitates high selectivity. Chemically rich, free-standing, and pH-responsive PDA nanomembranes with specific interaction sites are proposed as customizable high-performance sieves for a wide range of challenging separation requirements.

Heat-activated growth of metastable and length-defined DNA fibers expands traditional polymer assembly

Biopolymers such as nucleic acids and proteins exhibit dynamic backbone folding, wherein site-specific intramolecular interactions determine overall structure. Proteins then hierarchically assemble into supramolecular polymers such as microtubules, that are robust yet dynamic, constantly growing or shortening to adjust to cellular needs. The combination of dynamic, energy-driven folding and growth with structural stiffness and length control is difficult to achieve in synthetic polymer self-assembly. Here we show that highly charged, monodisperse DNA-oligomers assemble via seeded growth into length-controlled supramolecular fibers during heating; when the temperature is lowered, these metastable fibers slowly disassemble. Furthermore, the specific molecular structures of oligomers that promote fiber formation contradict the typical theory of block copolymer self-assembly. Efficient curling and packing of the oligomers – or ‘curlamers’ – determine morphology, rather than hydrophobic to hydrophilic ratio. Addition of a small molecule stabilises the DNA fibers, enabling temporal control of polymer lifetime and underscoring their potential use in nucleic-acid delivery, stimuli-responsive biomaterials, and soft robotics.

Self-assembly of nanocrystal checkerboard patterns via non-specific interactions

Checkerboard lattices—where the resulting structure is open, porous, and highly symmetric—are difficult to create by self-assembly. Synthetic systems that adopt such structures typically rely on shape complementarity and site-specific chemical interactions that are only available to biomolecular systems (e.g., protein, DNA). Here we show the assembly of checkerboard lattices from colloidal nanocrystals that harness the effects of multiple, coupled physical forces at disparate length scales (interfacial, interparticle, and intermolecular) and that do not rely on chemical binding. Colloidal Ag nanocubes were bi-functionalized with mixtures of hydrophilic and hydrophobic surface ligands and subsequently assembled at an air–water interface. Using feedback between molecular dynamics simulations and interfacial assembly experiments, we achieve a periodic checkerboard mesostructure that represents a tiny fraction of the phase space associated with the polymer-grafted nanocrystals used in these experiments. In a broader context, this work expands our knowledge of non-specific nanocrystal interactions and presents a computation-guided strategy for designing self-assembling materials.

Probing Protein Structural Changes in Alzheimer's Disease via Quantitative Cross-linking Mass Spectrometry.

Alzheimer's disease (AD) is a progressive neurological disorder featuring abnormal protein aggregation in the brain, including the pathological hallmarks of amyloid plaques and hyperphosphorylated tau. Despite extensive research efforts, understanding the molecular intricacies driving AD development remains a formidable challenge. This study focuses on identifying key protein conformational changes associated with the progression of AD. To achieve this, we employed quantitative cross-linking mass spectrometry (XL-MS) to elucidate conformational changes in the protein networks in cerebrospinal fluid (CSF). By using isotopically labeled cross-linkers BS3d0 and BS3d4, we reveal a dynamic shift in protein interaction networks during AD progression. Our comprehensive analysis highlights distinct alterations in protein-protein interactions within mild cognitive impairment (MCI) states. This study accentuates the potential of cross-linked peptides as indicators of AD-related conformational changes, including previously unreported site-specific binding between α-1-antitrypsin (A1AT) and complement component 3 (CO3). Furthermore, this work enables detailed structural characterization of apolipoprotein E (ApoE) and reveals modifications within its helical domains, suggesting their involvement in MCI pathogenesis. The quantitative approach provides insights into site-specific interactions and changes in the abundance of cross-linked peptides, offering an improved understanding of the intricate protein-protein interactions underlying AD progression. These findings lay a foundation for the development of potential diagnostic or therapeutic strategies aimed at mitigating the negative impact of AD.

NMR insights into β-Lactamase activity of UVI31+ Protein from Chlamydomonas reinhardtii

β-Lactamases (EC 3.5.2.6) confer resistance against β-lactam group-containing antibiotics in bacteria and higher eukaryotes, including humans. Pathogenic bacterial resistance against β-lactam antibiotics is a primary concern for potential therapeutic developments and drug targets. Here, we report putative β-lactamase activity, sulbactam binding (a β-lactam analogue) in the low μM affinity range, and site-specific interaction studies of a 14 kDa UV- and dark-inducible protein (abbreviated as UVI31+, a BolA homologue) from Chlamydomonas reinhartii. Intriguingly, the solution NMR structure of UVI31 + bears no resemblance to other known β-lactamases; however, the sulbactam binding is found at two sites rich in positively charged residues, mainly at the L2 loop regions and the N-terminus. Using NMR spectroscopy, ITC and MD simulations, we map the ligand binding sites in UVI31 + providing atomic-level insights into its β-lactamase activity. Current study is the first report on β-lactamase activity of UVI31+, a BolA analogue, from C. reinhartii. Furthermore, our mutation studies reveal that the active site serine-55 is crucial for β-lactamase activity.

Biochanin obstructs human serum albumin from non-enzymatic glycation: an in vitro approach

Various serum proteins, like Human Serum Albumin (HSA) and others, are susceptible to glycation and the formation of Advanced Glycation End Products (AGEs). Diabetes and other diseases are associated with AGE development. Recently, isoflavones have been studied for their therapeutic benefits. In the present study, we glycated HSA with Methylglyoxal (MGO) with and without the test compound, i.e., Biochanin A (BCA), to test its antiglycating capacity. We studied the biochemical and biophysical effects of glycation on HSA with and without BCA and also took the help of the in silico technique. Analytical methods included intrinsic and extrinsic fluorescence, polyacrylamide gel electrophoresis (PAGE), UV spectroscopy, far UV circular dichroism, and others. For structural comprehension, TEM and SEM were used. Molecular docking and simulation were employed to observe BCA-HSA's site-specific interaction. Since HSA is a therapeutically relevant protein involved in many disorders, this study’s findings are important. Communicated by Ramaswamy H. Sarma

Abstract 4641: Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells

Abstract Among the myriad of molecular alterations associated with cancer, loss of the normal tumor suppressive functions of p53, the “guardian of the genome,” is the most consistent. Thus, the possibility of targeting oncogenic, mutant p53 proteins has long been a therapeutic goal. However, that aspiration has not been fully realized, leaving patients vulnerable to aggressive disease that is resistant to chemotherapy and to targeted agents such as PARP inhibitors and immunotherapy owing to the loss of wild type p53 functionality and the gain of new oncogenic functions when mutant p53 is expressed. An effective therapeutic strategy is urgently needed for p53 mutant cancers, and our team is developing novel analogues of curcumin with the potential to convert mutant p53 to a wild type conformation and restore its tumor suppressive functions. In this study, we investigated the effects of AKT-100, which was developed from the parent molecule and curcumin analogue HO-3867. Effects were compared to another drug believed to reactivate p53 functionality, APR-246, using CyQUANT IC50 cell proliferation assays and bulk and single cell RNA sequencing. Structural analyses are underway investigating the specific sites of interaction of AKT-100 with various mutant forms of p53. Our findings indicate that, first, AKT-100 exhibits impressive cell killing in multiple ovarian and serous endometrial cancer cells at concentrations ranging from 100-300 nM and is synergistic with the PARP inhibitor olaparib. Second, RNA sequencing demonstrates the reactivation of wild type p53 genes associated with normal cell cycle regulation (induction of CDKN1A encoding p21 and GADD45A), apoptosis (induction of PMAIP1 encoding Noxa and DR5 encoding Death Receptor 5), and the inhibition of DNA replication and multiple alternative mechanisms of DNA repair employed by malignant cells. Third, RNA sequencing indicates that AKT-100 acts through both p53-dependent and independent pathways to inhibit cancer cell proliferation. Gene expression is impacted in ways that portend a therapeutic effect as well as highlight potential resistance pathways (e.g. significant induction of PLK5 encoding polo like kinase 5) that can be further targeted to create synthetic lethal drug combinations with AKT-100 chosen specifically to target the various mutant forms of p53. Taken together, these results identify novel curcumin analogues in early development with the potential to improve therapy for p53 mutant cancers. Through this strategy it is also possible to identify and block resistance pathways that are activated by different forms of mutant p53 in response to AKT-100 and to create synthetic lethal precision drug combinations. Citation Format: Kimberly K. Leslie, Jamie L. Padilla, Geneva L. Williams, Hua-Ying Fan, Cristian G. Bologa, Eric R. Prossnitz, Jun-Yong Choe, Lane E. Smith, Alexander Goss, Robert J. Lake, Avinash D. Sahu. Preclinical evaluation of the novel curcumin Analogue AKT-100 demonstrates therapeutic effectiveness in p53-mutated cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4641.

CRISPR du-HITI an attractive approach to targeting Long Noncoding RNA HCP5 as inhibitory factor for proliferation of ovarian cancer cell.

This research provides a glimmer of hope that the knockout of HCP5 leads to a therapy response to considerably prolong the life of patients with OC. RT-PCR evaluated the expression of lncRNA HCP5 in the ovarian cancer OVCAR-3 cell line. CRISPR knockout cell lines validated by western blot. Small genomic deletions at the targeted locus were induced. CCK-8 colony formation assays were used to analyze the effect of HCP5 knockout on the proliferation capacity of OVCAR-3 cells. Transwell migration and invasion assayed. Furthermore, the Sphere-formation assay isolated the most aggressive population of cancer stem cells. Bioinformatic analysis showed a significant correlation between lncRNA HCP5 up-regulation and OVCAR-3 cell proliferation. The ChIP technique assesses specific sites of interaction between transcription factors and DNA. Real-time PCR assays explored the relationship between HCP5, Hsa-miR-9-5p, CXCR4, CDH1, caspase-3, p53, bcl2 and survivin. PCR carried out amplification of the 448-bp band for sgRNA1 and sgRNA2 after the use of particular primers for HCP5. the number of breast cancer cells that moved to the bottom chamber reduced considerably after transfection with PX461-sgRNA1/2 vectors compared to the Blank control groups (P < 0.05). MTT assay designated growth curves that showed the rate of OVCAR-3 growth was significantly repressed (***P < 0.001) when compared with control OVCAR-3 cells after HCP5 knockdown. Also, the survival results of W.T cells in 24, 48 and 72 h showed 92%, 87% and 85%, respectively. This is while the cells of the CRISPR/Cas9 group in which LncRNA HCP5 was knocked out had 42% (*P < 0.05), 23%(**P < 0.01) and 14% (**P < 0.01) survival, respectively. The expression levels of caspase-3, Hsa-miR-9-5p, P53 genes in the HCP5 deletion of CRISPR/Cas9 group significantly increased than the W.T. control group; the deletion group showed a considerable reduction in HCP5 expression compared to the blank control group (3.6-fold, p < 0.01). Whereas BCL2, SURVIVIN, CXCR4, CDH1 genes expression markedly increased than in HCP5 knockout cells (5.8-fold, p < 0.05). These results indicate that CRISPR/Cas9-mediated HCP5 disruption on OVCAR-3 cell lines promotes anti-tumor biomarkers, suppressing ovarian cancer progression. Consistent with these results, HCP5 is one of the most critical lnc for the efficient proliferation and migration of OVCAR-3 cell lines.

NME3 is a gatekeeper for DRP1-dependent mitophagy in hypoxia

NME3 is a member of the nucleoside diphosphate kinase (NDPK) family localized on the mitochondrial outer membrane (MOM). Here, we report a role of NME3 in hypoxia-induced mitophagy dependent on its active site phosphohistidine but not the NDPK function. Mice carrying a knock-in mutation in the Nme3 gene disrupting NME3 active site histidine phosphorylation are vulnerable to ischemia/reperfusion-induced infarction and develop abnormalities in cerebellar function. Our mechanistic analysis reveals that hypoxia-induced phosphatidic acid (PA) on mitochondria is essential for mitophagy and the interaction of DRP1 with NME3. The PA binding function of MOM-localized NME3 is required for hypoxia-induced mitophagy. Further investigation demonstrates that the interaction with active NME3 prevents DRP1 susceptibility to MUL1-mediated ubiquitination, thereby allowing a sufficient amount of active DRP1 to mediate mitophagy. Furthermore, MUL1 overexpression suppresses hypoxia-induced mitophagy, which is reversed by co-expression of ubiquitin-resistant DRP1 mutant or histidine phosphorylatable NME3. Thus, the site-specific interaction with active NME3 provides DRP1 a microenvironment for stabilization to proceed the segregation process in mitophagy.

Intrinsically disordered Pseudomonas chaperone FapA slows down the fibrillation of major biofilm-forming functional amyloid FapC.

Functional bacterial amyloids play a crucial role in the formation of biofilms, which mediate chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC amyloid fibrillar protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution nuclear magnetic resonance (NMR), we recently showed that both FapC and FapA are intrinsically disordered proteins (IDPs). Here, the secondary structure propensities (SSPs) are compared to alphafold (DeepMind, protein structure prediction tool/algorithm: https://alphafold.ebi.ac.uk/) models. We further demonstrate that the FapA chaperone interacts with FapC and significantly slows down the formation of FapC fibrils. Our NMR titrations reveal ~ 18% of the resonances show FapA-induced chemical shift perturbations (CSPs), which has not been previously observed, the largest being for A82, N201, C237, C240, A241, and G245. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the repeat-1 (R1)/loop-2 (L2) and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, ~ 90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC, the largest being for S63, A69, A80, and I92. A temperature-dependent effect of FapA was observed on FapC by thioflavin T (ThT) and NMR experiments. This study provides a detailed understanding of the interaction between the FapA and FapC, shedding light on the regulation and slowing down of amyloid formation, and has important implications for the development of therapeutic strategies targeting biofilms and associated infections.