Search Results (148)

The human mitochondrial Lon protease (LonP1) is a central regulator of mitochondrial DNA copy number and metabolic reprogramming. However, the structural basis for how LonP1 recognizes native physiological substrates remains elusive. Here, we present the high-resolution cryo-EM structure of the human LonP1 hexamer actively engaging its native substrate, TFAM. The reconstruction reveals a distinct bipartite search-and-shred mechanism. Unlike its bacterial homologs, the human N-terminal domain (NTD) adopts a compact architecture acting as a selective vestibule to recruit and initially unfold the substrate tertiary structure. Subsequently, the polypeptide is threaded through the central channel via a hand-over-hand mechanism driven by a spiral array of aromatic pore-loops. This structural framework provides a mechanistic rationale for the spatial segregation of LonP1 and offers a template for targeting mitochondrial proteostasis in human diseases. Full article

Arthritis in K/BxN mice is provoked by pathogenic autoantibodies to glucose-6-phosphate isomerase (G6PI), which is a ubiquitously expressed enzyme that is present in cells, in the circulation and on articular cartilage. When G6PI autoantibodies (auto-Abs) deposit on the articular cartilage of K/BxN mice, arthritis ensues due to the activation of various components of the innate immune system. Recent studies have investigated the in vivo efficacy of recombinant fragment-crystallizable (Fc) protein-based therapeutics. Many recombinant Fc proteins evaluated provide protection against inflammation in mouse models of arthritis, such as the K/BxN serum-transfer model. More recently, rFc-µTP-L309C, a recombinant human IgG1-Fc with an additional point mutation at position L309C fused to the human IgM tailpiece to form a hexamer, has been shown to ameliorate the arthritis in K/BxN mice. Additional studies have shown that rFc-µTP-L309C has multiple effects that work together to ameliorate the arthritis, including inhibition of neutrophil migration into the joint, inhibition of IL-1β production, downregulation of Th1 and Th17 cells, and increases in T regulatory cells and synovial fluid IL-10. In this work, rFc-µTP-L309C was shown to effectively prevent arthritis in the K/BxN serum-transfer model, significantly downregulate inflammatory cytokines/chemokines, and ameliorate the arthritis in the endogenous K/BxN model. This amelioration of the arthritis was associated with a significant decrease in autoantibody levels, which was independent of the neonatal Fc receptor (FcRn). rFc-µTP-L309C was shown to specifically inhibit G6PI autoantibody secretion from B-cells with a concomitant increase in TGFβ and decrease in B-cell activating factor (BAFF). These new findings suggest that rFc-µTP-L309C may provide a therapeutic benefit for other antibody-mediated autoimmune diseases through its effects on B-cells. Full article

The rapid evolution of coronaviruses (CoVs) requires researchers to develop specific yet broad-spectrum detection methods to monitor their constant genomic changes. The goal of the present study was to establish a current pan-coronavirus RT-PCR system capable of detecting a wide variety of CoVs and useful for the investigation of virus diversity and host spectrum. For optimization, one-step and two-step nested RT-PCRs with three RT enzymes were examined, amplifying a ~600 bp long product of the RNA-dependent RNA polymerase. As templates, the in vitro transcribed RNA of ten pathogenic CoVs (SARS-CoV, SARS-CoV-2, NL-63, OC43, feline CoV, porcine epidemic diarrhea virus or PEDV, transmissible gastroenteritis virus or TGEV, canine CoV, bat CoV, and infectious bronchitis virus) were applied instead of the often-used DNA standards. A limit of detection of 5–50 copies/reaction was achieved with a random hexamer-primed two-step RT-PCR and a touchdown cycling profile, representing a lower detection limit and higher specificity compared to previously published primer sets. Swine origin pooled samples (n = 121), collected from apparently healthy herds in Hungary, were tested with the novel RT-PCR system. Sequences of porcine respiratory CoV/TGEV and porcine hemagglutinating encephalomyelitis virus were identified in 24 oral fluid and nasal swab pools, demonstrating the circulation of these viruses in this country, as well as the suitability of the new PCR for their detection. The results highlighted the importance of adequate RT enzyme selection and the use of RNase inhibitors in sample preparation and conservation. Full article

Serum lectins in vertebrates play crucial roles in innate immunity as recognition molecules for pathogen-associated molecular patterns (PAMPs). In mammals, two major lectins, mannose-binding lectin (MBL) and ficolin, both containing N-terminal collagen-like domains, activate the lectin pathway of complement. While MBL and ficolin recognize distinct PAMPs, their counterparts in teleosts are less understood. To date, MBL and galactose-binding lectin (GalBL) have been identified in teleosts, but the presence of ficolin remains unclear. In this study, we purified a 31-kDa serum lectin from common carp that displayed carbohydrate-binding specificity similar to that of mammalian ficolin. Unexpectedly, this lectin lacked an N-terminal collagenous domain and showed the highest similarity to mammalian microfibril-associated glycoprotein 4 (MFAP4), suggesting that the lectin is distinct from fibulin. Biochemical analyses revealed that carp MFAP4-like lectin (MFAP4Lec) protein forms a hexamer in serum, specifically binds GlcNAc and GalNAc, and recognizes the fish pathogen Vibrio anguillarum. The binding was competitively inhibited by GlcNAc but not by EDTA, indicating Ca²⁺-independent recognition. These findings suggest that MFAP4Lec functions as a novel serum lectin in teleost fish, serving as a recognition molecule for bacterial pathogens in innate immunity. Full article

The past decade has seen significant advancement in our understanding of DNA replication-coupled chromatin assembly, especially parental histone recycling that is essential for epigenetic inheritance. Leading strand-specific and lagging strand-specific pathways have been found to promote the transfer of parental histones H3-H4 to nascent DNA. It is now clear that the replisome initially characterized as the machinery that carries out the duplication of genomic DNA is also responsible for parental histone recycling. A series of replisome components including CMG (Cdc45-MCM-GINS) replicative helicase, DNA polymerases Polε, Polδ, Polα-primase, and FPC (Fork Protection Complex) that promote parental histone recycling exhibit histone-binding activities. Structural analyses of native and reconstituted replisomes, together with AlphaFold modeling of histone (H3-H4)₂ tetramer binding by replisome components, provided a framework for understanding the molecular mechanisms of parental histone recycling. A working model has emerged in which the mobile histone chaperone FACT (Facilitates Chromatin Transcription) binds parental histone (H3-H4)₂ tetramer or (H3-H4)₂-(H2A-H2B) hexamer on the front of the replication fork, and escorts it across the replisome to the daughter strands in the wake of the replication fork. In this model, parental histones transiently associate with the histone-binding modules in the replisome as steppingstones during their movement. Future studies are needed to elucidate the spatiotemporal coordination of the functions of replisome factors in parental histone transfer. Full article

This review article focuses first on the historical development of present understanding of gap junction channel architecture, one of its goals being to enlighten younger generations of scientists about the early steps of this field that begun over half a century ago. Early findings on gap junction architecture are reviewed as follows. The channels cross the membrane and project from the membrane surfaces; they are made of six subunits (hexamers) and show dimples on both ends, which represent inner and outer openings of the channel. Images of the central dimples on both channel ends (channel pores) seen in freeze-fracture replicas correspond to the electron-opaque spots visible in negatively stained sections and in isolated junctions. The channels are linked to each other extracellularly. Calmodulin (CaM) is a major accessory protein of gap junctions that is involved in channel gating and gap junction formation and is also likely to play a key role in determining different patterns of channel aggregation. Full article

Introduction/Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) poses a threat to global public health with a mortality rate of up to 30%. However, there is currently no commercialized SFTSV vaccine. This study focused on the construction of DNA vaccines with different structures based on the surface glycoproteins Gn and Gc to identify the immunodominant conformations. Methods: The DNA vaccines encoding secretory proteins including Gn or Gc monomer, heterodimer of Gn and Gc (dimer), two forms of hexamer composed of the Gn and Gc heterodimer (hexamer-1 and hexamer-2) or ferritin nanoparticles of Gn, and non-secretory proteins including Gn (Gn-TM) and Gc (Gc-TM) were constructed. Western blot confirmed the expression level and the specificity of those DNA vaccines. After vaccinating mice with those DNA vaccines, its induced humoral and cellular immunity were comprehensively evaluated. Results: The DNA vaccines were constructed successfully. The DNA vaccines of Gn and polymers including dimer, hexamer-2, and ferritin nanoparticles inducing stronger binding antibody, neutralizing antibody, and antibody-dependent cellular cytotoxicity (ADCC) activity. The neutralizing antibody induced by these constructs was also cross-recognized by other five SFTSV pseudovirus strains. However, the T cell response induced by Gc, dimer or hexamer-2 DNA vaccines were significantly higher than those in most other groups, including Gn. Conclusion: The DNA vaccines encoding dimer or hexamer-2 demonstrated superior immunogenicity over other conformations, after taking the results of humoral and cellular responses into account. This study revealed the advantages of using polymer conformations in SFTSV vaccine design and provided new targets in SFTSV vaccine development. Full article

Viral oncolysis is considered a promising cancer treatment method because of its good tolerability and durable anti-tumor effects. Compared with other oncolytic viruses, Newcastle disease virus (NDV) has some distinct advantages. As an RNA virus, NDV does not recombine with the host genome, making it safer compared with DNA viruses and retroviruses; NDV can induce syncytium formation, allowing the virus to spread among cells without exposure to host neutralizing antibodies; and its genome adheres to the hexamer genetic code rule (genome length as a multiple of six nucleotides), ensuring accurate replication, low recombination rates, and high genetic stability. Although wild-type NDV has a killing effect on various tumor cells, its oncolytic effect and working mechanism are diverse, increasing the complexity of generating engineered oncolytic viruses with NDV. This study aims to employ whole-genome CRISPR-Cas9 knockout screening and RNA sequencing to identify putative key regulatory factors involved in the interaction between NDV and human colon cancer HCT116 cells and map their global interaction networks. The results suggests that NDV infection disrupts cellular homeostasis, thereby exerting oncolytic effects by inhibiting cell metabolism and proliferation. Meanwhile, the antiviral immune response triggered by NDV infection, along with the activation of anti-apoptotic signaling pathways, may be responsible for the limited oncolytic efficacy of NDV against HCT116 cells. These findings not only enhance our understanding of the oncolytic mechanism of NDV against colonic carcinoma but also provide potential strategies and targets for the development of NDV-based engineered oncolytic viruses. Full article

Human Interleukin-6 (hIL-6) is a pro inflammatory cytokine that binds to its receptor, IL-6Rα followed by binding to gp130 and subsequent dimerization to form a hexamer signaling complex. As a critical inflammation mediator, hIL-6 is associated with a diverse range of diseases and monoclonal antibodies in clinical use that either target IL-6Rα or hIL-6 to inhibit signaling. Here, we perform high-throughput structure-based computational screening using ensemble docking for small-molecule antagonists for which the target conformations were taken from 600 ns long molecular dynamics simulations of the apo protein. Prior knowledge of the contact sites from binary complex studies and experimental work was incorporated into the docking studies. The top 20 scoring ligands from the in silico studies after post analysis were subjected to in vitro functional assays. Among these compounds, the ligand with the second-highest calculated binding affinity experimentally showed an ~84% inhibitory effect on IL6-induced STAT3 reporter activity at 10 μM concentration. This finding may pave the way for designing small-molecule inhibitors of hIL-6 of therapeutic significance. Full article

Background/Objectives: Recombinant Fc proteins have been produced that have a protective effect in mouse models of arthritis, such as the K/BxN rheumatoid arthritis model. We have previously shown that a recombinant human IgG1 Fc with a point mutation at position 309, replacing a leucine with a cysteine, fused to the human IgM tailpiece to form a human IgG1 Fc hexamer, rFc-µTP-L309C, effectively prevents neutrophil infiltration into the joints and ameliorates arthritis in the K/BxN serum transfer model and in the endogenous chronic arthritis K/BxN model. We have now investigated the effect of rFc-µTP-L309C on T-cells in the K/BxN chronic arthritis mouse model. Methods: PBMCs were isolated from the spleen, lymph nodes and joint synovial fluid from K/BxN mice having severe chronic arthritis that had been treated with 200 mg/kg rFc-µTP-L309C or human serum albumin (HSA). Flow cytometry was used to isolate the activated CD4⁺CD44⁺ T-cells and T-regulatory cells (Tregs). Intracellular staining was used to identify Th1 and Th17 T-cell subsets, and CD4⁺CD25⁺FoxP3⁺ Tregs. ELISA was used to measure levels of IL-10 and TGF-β in synovial fluid. Results: We find that amelioration of the arthritis occurs after treatment with rFc-µTP-L309C and results in a decrease in Th1 cells’ production of IFNγ and Th17 cells’ production of IL-17. Amelioration also results in decreased production of GM-CSF. Moreover, amelioration results in increased Tregs and IL-10 production in the synovial fluid. Conclusions: rFc-µTP-L309C reduces the inflammatory T-cells and increases the regulatory anti-inflammatory T-cells in the chronic arthritis K/BxN mouse model. This effect explains, in part, the ability of rFc-µTP-L309C to ameliorate the arthritis and reduce damage on the articular cartilage of K/BxN mice. Full article

Backgroud: Glutamate dehydrogenase (GDH) is involved in the metabolism of glutamate and ammonia. It is regulated by multiple ligand variants, and hyper-active GDH mutants have been reported for hyperinsulinism hyperammonemia syndrome (HHS). Methods: Here, we constructed the wild-type human GDH and three human GDH454 mutants and investigated their degradation activity and performance under different GDH inhibitors. Results: Protein activity test and SDS-PAGE analysis of the purified proteins showed that the GDH454 mutant from HHS has weaker GDH enzymatic activity but greater resistance to trypsin hydrolysis than the wild type. Interestingly, using the biomolecular interactions technique, it showed that the GDH454 mutant has 10⁹ times weaker affinity for trypsin and 10-fold weaker for epigallocatechin gallate (EGCG) than the wild-type GDH. Subsequently, native-PAGE gel analysis demonstrated that EGCG could break down the GDH hexamer into monomers and form a complex with trypsin to enhance the degradation of both types of GDH. Conclusions: EGCG showed good affinity to both the wild-type and the mutant GDH proteins, promoting protein degradation; this provides a new strategy for the treatment of HHS and other hyper-active GDH-related diseases. Full article

The HIV-1 capsid is one of virology’s most iconic structures, yet how it assembles has long remained elusive. Remarkably, the capsid is made from just a single protein, CA, which forms a lattice of ~250 hexamers and exactly 12 pentamers. Conical capsids form inside budded virions during maturation, but early efforts to reproduce this in vitro resulted instead in open-ended tubes with a purely hexameric lattice. The missing component in capsid assembly was finally identified as the metabolite inositol hexakisphosphate (IP6). Simply mixing soluble CA protein with IP6 is sufficient to drive the spontaneous assembly of conical capsids with a similar size and shape to those inside of infectious virions. Equally important, IP6 stabilises capsids once formed, increasing their stability from minutes to hours. Indeed, such is the dependence of HIV-1 on IP6 that the virus actively packages it into virions during production. These discoveries have stimulated work from multiple labs into the role and importance of IP6 in HIV-1 replication, and is the subject of this review. Full article

A non-linear (non-additive) increase in stability of hexamers follows an increase in the total number of (i) aad (a double proton acceptor) plus add (a double proton donor) waters commonly linked with anticooperativity and (ii) the total number of intermolecularly delocalized electrons (^intermolN^deloc) in the 3D space occupied by a hexamer. Subsequently, we obtained nearly a perfect linear correlation between increase in the cluster stability and ^intermolN^deloc. Individual water molecules that act as either aad or add (i) delocalize the largest number of electrons throughout a cluster; (ii) are involved in the strongest attractive, hence energy-stabilizing intermolecular interaction with the remaining five waters; (iii) have the most significant quantum component of the intermolecular interaction energy and (iv) relative to six non-interacting water molecules, stabilize a hexamer the most, as quantified by a purposely derived mol-FAMSEC energy term. Clearly, the all-body approach used in the unified, molecular-wide and electron density (MOWeD)-based concept of chemical bonding contradicts the commonly accepted view that aad and add water molecules are involved in anticooperativity in 3D water hexamers. Consequently, we propose here a general definition of cooperativity that should be applicable to any n-membered molecular cluster, namely the quantifiable, classical physics- and quantum-based cooperativity phenomenon is synonymous with the intermolecular all-body delocalization of electrons, leading to the increase in stability of individual molecules on an n-membered cluster formation. Full article

Janus nanorods are a special class of nanorods composed of two distinct surface regions, one hydrophilic and one hydrophobic. This amphiphilic characteristic makes them promising candidates for stabilizing water–oil interfaces. Oily wastewater (OWW) contamination, resulting from industrial activities such as petroleum extraction and refining and vegetable oil processing, poses significant risks to ecosystems, water resources, and public health. Traditional surfactants used in enhanced oil recovery (EOR) and wastewater treatment often introduce secondary pollution due to their persistence and toxicity. In this work, we investigate the interfacial behavior of Janus NRs under two different conditions: a thin oil film surrounded by water and a nanoconfined system with purely repulsive walls. Using dissipative particle dynamics (DPD) simulations, we analyze how nanorod length and confinement influence interfacial tension and self-assembly. In bulk systems, shorter NRs (dimers and quadrimers) effectively reduce interfacial tension by adsorbing at the oil–water interface, while longer NRs (hexamers) exhibit bulk aggregation, limiting their surfactant efficiency. In contrast, under nanoconfinement, all NR sizes increase interfacial tension due to steric constraints, with longer NRs preferentially adsorbing onto the solid–liquid interface. These results pave the way for the rational design of nanostructured materials for applications in enhanced oil recovery, wastewater treatment, and membrane filtration. Full article

Perfringolysin O (PFO) is a prototypical member of a large family of pore-forming toxins (PFTs) that are potent virulence factors for many pathogenic bacteria. One of the most enigmatic properties of these PFTs is how structural changes are coordinated between different subunits within a single complex. Moreover, there are conflicting data in the literature, with gel electrophoresis results apparently showing that pores are only complete rings, whereas microscopy images clearly also show incomplete-ring pores. Here, we developed a novel multi-stack gel electrophoretic assay to finely separate PFO pore complexes and found that this assay indeed resolves both complete- and incomplete-ring pores. However, unexpectedly, we found that the stoichiometries of these complexes are predominantly integral multiples of six subunits. High-resolution atomic force microscopy images of PFO pore complexes also reveal a predominant hexameric-based stoichiometry. We also observed this hexameric-based stoichiometry at the prepore stage and identified a mutant that is kinetically trapped at a hexameric state. Thus, overall, these results reveal a previously unknown hexameric-based structural hierarchy in the PFO complexes. We suggest that the structural coordination within the hexamers is different than between the hexamers and is thus a critical feature of the structural coordination of the complex as a whole. Full article