Main Subunit Research Articles

INO80 regulates chromatin accessibility to facilitate suppression of sex-linked gene expression during mouse spermatogenesis.

The INO80 protein is the main catalytic subunit of the INO80-chromatin remodeling complex, which is critical for DNA repair and transcription regulation in murine spermatocytes. In this study, we explored the role of INO80 in silencing genes on meiotic sex chromosomes in male mice. INO80 immunolocalization at the XY body in pachytene spermatocytes suggested a role for INO80 in the meiotic sex body. Subsequent deletion of Ino80 resulted in high expression of sex-linked genes. Furthermore, the active form of RNA polymerase II at the sex chromosomes of Ino80-null pachytene spermatocytes indicates incomplete inactivation of sex-linked genes. A reduction in the recruitment of initiators of meiotic sex chromosome inhibition (MSCI) argues for INO80-facilitated recruitment of DNA repair factors required for silencing sex-linked genes. This role of INO80 is independent of a common INO80 target, H2A.Z. Instead, in the absence of INO80, a reduction in chromatin accessibility at DNA repair sites occurs on the sex chromosomes. These data suggest a role for INO80 in DNA repair factor localization, thereby facilitating the silencing of sex-linked genes during the onset of pachynema.

Foxg1 regulates translation of neocortical neuronal genes, including the main NMDA receptor subunit gene, Grin1

BackgroundMainly known as a transcription factor patterning the rostral brain and governing its histogenesis, FOXG1 has been also detected outside the nucleus; however, biological meaning of that has been only partially clarified.ResultsPrompted by FOXG1 expression in cytoplasm of pallial neurons, we investigated its implication in translational control. We documented the impact of FOXG1 on ribosomal recruitment of Grin1-mRNA, encoding for the main subunit of NMDA receptor. Next, we showed that FOXG1 increases GRIN1 protein level by enhancing the translation of its mRNA, while not increasing its stability. Molecular mechanisms underlying this activity included FOXG1 interaction with EIF4E and, possibly, Grin1-mRNA. Besides, we found that, within murine neocortical cultures, de novo synthesis of GRIN1 undergoes a prominent and reversible, homeostatic regulation and FOXG1 is instrumental to that. Finally, by integrated analysis of multiple omic data, we inferred that FOXG1 is implicated in translational control of hundreds of neuronal genes, modulating ribosome engagement and progression. In a few selected cases, we experimentally verified such inference.ConclusionsThese findings point to FOXG1 as a key effector, potentially crucial to multi-scale temporal tuning of neocortical pyramid activity, an issue with profound physiological and neuropathological implications.

Role of Inflammation and the NF-κB Signaling Pathway in Hirschsprung's Disease.

Hirschsprung's disease (HSCR, incidence 1/5000 live births) is caused by the failure of neural crest-derived precursors to migrate, survive, proliferate, or differentiate during the embryonic development of the Enteric Nervous System (ENS), which could be disrupted by many factors, including inflammatory processes. The NF-κB family controls several biological processes, including inflammation, neurogenesis, and cell migration. With the aim of studying the potential role of NF-κB in HSCR, we have analyzed the expression of the NF-κB main subunits and other NF-κB-related genes by RT-qPCR in HSCR tissue samples (sub-divided into ganglionic and aganglionic segments). We found decreased gene expression of the NF-κB main subunit RELA but also of NFKBIA, TNFA, TFGBR2, and ERBB3 in the pathologic distal aganglionic segments compared to the proximal ganglionic segments. Moreover, we could also confirm the lower protein expression of RelA/p65 in the aganglionic distal segments by immunofluorescence staining. Further, we show that the expression of RelA/p65 protein in the proximal segments concurs with lymphocyte infiltration in the bowel tissue, indicating a pro-inflammatory activation of p65 in the proximal ganglionic HSCR tissue in the patients analyzed. All in all, our findings suggest that the modulation of NF-κB signaling in the neuro-enteric system does obviously contribute to the pathological effects of HSCR.

Gallbladder dysfunction caused by MYPT1 ablation triggers cholestasis-induced hepatic fibrosis in mice.

The incidence of gallbladder diseases is as high as 20%, but whether gallbladder diseases contribute to hepatic disorders remains unknown. Here, we established an animal model of gallbladder dysfunction and assessed the role of a diseased gallbladder in cholestasis-induced hepatic fibrosis (CIHF). Mice with smooth muscle-specific deletion of Mypt1, the gene encoding the main regulatory subunit of myosin light chain phosphatase (myosin phosphatase target subunit 1 [MYPT1]), had apparent dysfunction of gallbladder motility. This dysfunction was evidenced by abnormal contractile responses, namely, inhibited cholecystokinin 8-mediated contraction and nitric oxide-resistant relaxation. As a consequence, the gallbladder displayed impaired bile filling and biliary tract dilation comparable to the alterations in CIHF. Interestingly, the mutant animals also displayed CIHF features, including necrotic loci by the age of 1 month and subsequently exhibited progressive fibrosis and hyperplastic/dilated bile ducts. This pathological progression was similar to the phenotypes of the animal model with bile duct ligation and patients with CIHF. The characteristic biomarker of CIHF, serum alkaline phosphatase activity, was also elevated in the mice. Moreover, we observed that the myosin phosphatase target subunit 1 protein level was able to be regulated by several reagents, including lipopolysaccharide, exemplifying the risk factors for gallbladder dysfunction and hence CIHF. We propose that gallbladder dysfunction caused by myosin phosphatase target subunit 1 ablation is sufficient to induce CIHF in mice, resulting in impairment of the bile transport system.

Nucleotide metabolism-related host proteins RNA polymerase II subunit and uridine phosphorylase 1 interacting with porcine epidemic diarrhea virus N proteins affect viral replication.

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that targets pig intestines to cause disease. It is globally widespread and causes huge economic losses to the pig industry. PEDV N protein is the protein that constitutes the core of PEDV virus particles, and most of it is expressed in the cytoplasm, and a small part can also be expressed in the nucleus. However, the role of related proteins in host nucleotide metabolic pathways in regulating PEDV replication have not been fully elucidated. In this study, PEDV-N-labeled antibodies were co-immunoprecipitated and combined with LC-MS to screen for host proteins that interact with N proteins. Bioinformatics analyses showed that the selected host proteins were mainly enriched in metabolic pathways. Moreover, co-immunoprecipitation and confocal microscopy confirmed that the second-largest subunit of RNA polymerase II (RPB2) and uridine phosphorylase 1 (UPP1) interacted with the N protein. RPB2 is the main subunit of RNA polymerase II and plays an important role in eukaryotic transcription. UPP1 is an enzyme that catalyzes reversible phosphorylation of uridine to uracil and ribo-1-phosphate to promote catabolism and bio anabolism. RPB2 overexpression significantly promoted viral replication, whereas UPP1 overexpression significantly inhibited viral replication. Studies on interactions between the PEDV N and host proteins are helpful in elucidating the pathogenesis and immune escape mechanism of PEDV.

Rapidly repeated visual stimulation induces long-term potentiation of VEPs and increased content of membrane AMPA and NMDA receptors in the V1 cortex of cats.

Studies report that rapidly repeated sensory stimulation can evoke LTP-like improvement of neural response in the sensory cortex. Whether this neural response potentiation is similar to the classic LTP induced by presynaptic electrical stimulation remains unclear. This study examined the effects of repeated high-frequency (9 Hz) versus low-frequency (1 Hz) visual stimulation on visually-evoked field potentials (VEPs) and the membrane protein content of AMPA / NMDA receptors in the primary visual cortex (V1) of cats. The results showed that repeated high-frequency visual stimulation (HFS) caused a long-term improvement in peak-to-peak amplitude of V1-cortical VEPs in response to visual stimuli at HFS-stimulated orientation (SO: 90°) and non-stimulated orientation (NSO: 180°), but the effect exhibited variations depending on stimulus orientation: the amplitude increase of VEPs in response to visual stimuli at SO was larger, reached a maximum earlier and lasted longer than at NSO. By contrast, repeated low-frequency visual stimulation (LFS) had not significantly affected the amplitude of V1-cortical VEPs in response to visual stimuli at both SO and NSO. Furthermore, the membrane protein content of the key subunit GluA1 of AMPA receptors and main subunit NR1 of AMPA receptors in V1 cortex was significantly increased after HFS but not LFS when compared with that of control cats. Taken together, these results indicate that HFS can induce LTP-like improvement of VEPs and an increase in membrane protein of AMPA and NMDA receptors in the V1 cortex of cats, which is similar to but less specific to stimulus orientation than the classic LTP.

Reorganization of Actin Cytoskeleton and Reduction of Cortical Elasticity in Podocytes Lacking Acid Ceramidase: Role of Sphingomyelinase-Like Phosphodiesterase 3b

Podocytopathy and associated nephrotic syndrome have been reported in a mouse strain (Asah1fl/fl/Podocre) with a podocyte-specific deletion of α subunit (the main catalytic subunit) of acid ceramidase (AC). Nevertheless, the pathogenic mechanism of podocytopathy in these mice remains poorly understood. Given that sphingomyelinase-like phosphodiesterase 3b (SMPDL3b) has been recently reported to play a vital role in maintaining the actin cytoskeleton and normal function of podocytes, the present study tested whether Asah1 gene deletion-induced podocytopathy is attributed to altered SMPDL3b activity and reorganized actin cytoskeleton in podocytes. By Western blot analysis and SMPDL3b enzyme activity assay, we demonstrated that Asah1 gene knockout remarkably decreased both expression and function of SMPDL3b in podocytes isolated from Asah1fl/fl/Podocre mice compared to podocytes of wild type (WT/WT) mice. As a monoclonal antibody which can bind to and stabilize SMPDL3b on podocytes, rituximab was found to significantly recover SMPDL3b expression and activity in podocytes lacking Asah1 gene. Correspondingly, Asah1 gene deletion reduced podocin expression in podocytes, which was attenuated by rituximab treatment. Phalloidin staining of F-actin showed elevation of cortical actin and reduction of stress fiber in podocytes lacking Asah1 gene. Such actin cytoskeleton redistribution was significantly attenuated by rituximab. Moreover, atom force microscopy was used to study the impact of actin cytoskeleton remodeling on mechanical properties of podocytes. Asah1 gene knockout was found to increase podocyte height but decrease cortical elasticity in these cells. These pathological changes were significantly inhibited by rituximab, suggesting the restoration of actin cytoskeleton in podocytes lacking Asah1 gene by rituximab. In vivo, intraperitoneal injection of rituximab markedly attenuated proteinuria and albuminuria in Asah1fl/fl/Podocre mice compared to control groups. In summary, our findings suggest that alteration of SMPDL3b activity and reorganization of actin cytoskeleton in podocytes may contribute to the pathogenesis of podocytopathy in mice with podocyte-specific Asah1 gene deletion. This study was supported by NIH grants DK054927 and DK120491. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Extraction, Purification, and Characterization of Olive (Olea europaea L., cv. Chemlal) Polyphenol Oxidase.

Among fruits susceptible to enzymatic browning, olive polyphenol oxidase (OePPO) stood out as being unisolated from a natural source until this study, wherein we successfully purified and characterized the enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of heated and nonheated OePPO revealed distinct molecular weights of 35 and 54 kDa, respectively, indicative of its oligomeric nature comprising active and C-terminal subunits. OePPO displayed latency, fully activating with 5 mM SDS under optimal conditions of pH 7.5 and 15 °C. The enzyme demonstrated monophenolase activity and showcased the highest efficiency toward hydroxytyrosol. Despite its low optimal temperature, OePPO exhibited high thermal resistance, maintaining stability up to 90 °C. However, beyond this threshold, the oligomeric enzyme disassociated, yielding a denatured main subunit and C-terminal fragments. Six OePPO genes were found in the fruits. Tryptic digestion identified the enzyme as mature OePPO1 (INSDC OY733096), while mass spectrometry detected the active form mass alongside several C-terminal fragments, revealing potential cleavage sites (Gly407, Tyr408).

Cadmium phosphates Cd2(PO4)OH and Cd5(PO4)2(OH)4 crystallizing in mineral structures

Single crystals of two basic cadmium phosphates, dicadmium orthophosphate hydroxide, Cd2(PO4)OH, and pentacadmium bis(orthophosphate) tetrakis(hydroxide), Cd5(PO4)2(OH)4, were obtained under hydrothermal conditions. Cd2(PO4)OH adopts the triplite [(Mn,Fe)2(PO4)F] structure type. Its asymmetric unit comprises two Cd, one P and five O sites, all situated at the general Wyckoff position 8 f of space group I2/a; two of the O atoms are positionally disordered over two sites, and the H atom could not be localized. Disregarding the disorder, distorted [CdO6] polyhedra form a tri-periodic network by edge-sharing with neighbouring [CdO6] units and by vertex-sharing with [PO4] units. The site associated with the OH group is coordinated by four Cd atoms in a distorted tetrahedral manner forming 1 ∞[(OH)Cd4/2] chains parallel to [001]. The oxygen environment around the OH site suggests multiple acceptor atoms for possible O—H...O hydrogen-bonding interactions and is the putative reason for the disorder. Cd5(PO4)2(OH)4 adopts the arsenoclasite [Mn5(AsO4)2(OH)4] structure type. Its asymmetric unit comprises five Cd, two P, and twelve O sites all located at the general Wyckoff position 4 a of space group P212121; the H atoms could not be localized. The crystal structure of Cd5(PO4)2(OH)4 can be subdivided into two main sub-units. One consists of three edge-sharing [CdO6] octahedra, and the other of two edge- and vertex-sharing [CdO6] octahedra. Each sub-unit forms corrugated ribbons extending parallel to [100]. The two types of ribbons are linked into the tri-periodic arrangement through vertex-sharing and through common [PO4] tetrahedra. Quantitative structure comparisons are made with isotypic M 5(XO4)2(OH)4 crystal structures (M = Cd, Mn, Co; X = P, As, V).

The IL-1β-p65 axis stimulates quiescent odontogenic epithelial cell rests via TGF-β signalling to promote cell proliferation of the lining epithelia in radicular cysts: A laboratory investigation.

Cyst formation of the jaws is frequently accompanied by the proliferation of odontogenic epithelial cells located in the periodontal ligament (PDL), which consists of heterozygous cells and includes the most fibroblasts. The lining epithelium of radicular cyst, an odontogenic cyst of inflammatory origin, is derived from the proliferation of the remnants of the Hertwig epithelial root sheath (odontogenic epithelial cell rests of Malassez; ERMs) in the PDL. ERMs are maintained at a lower proliferative state under physiological conditions, but the regulatory mechanisms underlying the inflammation-dependent enhanced-proliferative capabilities of ERMs are not fully understood. The aim of this study was to evaluate the effects of cytokine pathway association between TGF-β signalling and IL-1β signalling on the regulation of odontogenic epithelial cell proliferation using radicular cyst pathological specimens and odontogenic epithelial cell lines. Immunofluorescence analyses were performed to clarify the expression levels of Smad2/3 and Ki-67 in ERMs of 8-week-old mouse molar specimens. In radicular cyst (n = 52) and dentigerous cysts (n = 6) specimens from human patients, the expression of p65 (a main subunit of NF-κB), Smad2/3 and Ki-67 were investigated using immunohistochemical analyses. Odontogenic epithelial cells and PDL fibroblastic cells were co-cultured with or without an inhibitor or siRNAs. Odontogenic epithelial cells were cultured with or without TGF-β1 and IL-1β. The proliferative capabilities and Smad2 phosphorylation levels of odontogenic epithelial cells were examined. Immunohistochemically, Smad2/3-positivity was increased, and p65-positivity and Ki-67-positivity were decreased both in ERMs and in the epithelial cells in dentigerous cysts, a non-inflammatory developmental cyst. In contrast, p65-positive cells, along with the expression of Ki-67, were increased and Smad2/3-positive cells were decreased in the lining epithelia of radicular cysts. Co-culture experiments with odontogenic epithelial cells and PDL fibroblastic cells revealed that PDL cells-derived TGF-β1/2 and their downstream signalling suppressed odontogenic epithelial cell proliferation. Moreover, TGF-β1 stimulation induced Smad2 phosphorylation and suppressed odontogenic epithelial cell proliferation, while IL-1β stimulation reversed these phenotypes through p65 transactivation. These results suggest that IL-1β-p65 signalling promotes odontogenic epithelial cell proliferation through suppressing TGF-β-Smad2 signalling, which would be involved in the pathogenesis of radicular cysts.

Performance and characterization of snail adhesive mucus as a bioflocculant against toxic Microcystis

Toxic Microcystis blooms are widespread in aquatic bodies, posing major threats to aquatic and human life. Recently, bioflocculants have attracted considerable attention as a promising biomaterial for Microcystis management. In search of a novel organism that can produce an efficient bioflocculant for controlling harmful algae sustainably, the native gastropod Cipangopaludina chinensis was co-cultured continuously with toxic Microcystis under different initial algal cell densities. The bioflocculation effect of snail mucus on toxic Microcystis, microcystin removal, and toxin accumulation in snails was investigated. In addition, the properties of the adhesive mucus were characterized using microscopic, X-ray diffraction, infrared spectroscopy, and polysaccharide and proteome analyses. Microcystis cells were captured and flocculated by the snail mucus; removal efficiencies of up to 89.9% and 84.8% were achieved for microalgae and microcystin-leucine arginine (MC-LR), respectively, when co-cultured with C. chinensis for only one day. After nine-day exposure, less than 5.49 µg/kg DW microcystins accumulated in the snails, indicating safety for human consumption. The snail mucus contained 104.3 µg/mg protein and 72.7 µg/mg carbohydrate, which provide several functional groups beneficial for Microcystis bioflocculation. The main monosaccharide subunits of polysaccharides are galactose, galactosamine, glucosamine, fucose, glucose, and mannose. Most of them are key components of polysaccharides in many bioflocculants. Gene Ontology analysis indicated the protein enrichment in binding processes and catalytic activity, which may account for Microcystis bioflocculation via protein binding or enzymatic reactions. The findings indicate that native C. chinensis secretes adhesive mucus that can act as bioflocculant for toxic Microcystis from ambient water and can be an effective and eco-friendly tool for Microcystis suppression.

Epigenetic Modifiers MMSET and EZH2 Physically Interact and Cooperate to Support Multiple Myeloma Pathophysiology

Multiple myeloma (MM) is a malignant B cell dyscrasia characterized by the accumulation of clonal plasma cells (PC) within the bone marrow (1). Genome sequencing studies have revealed great heterogeneity and genomic instability, a complex mutational landscape and a branching pattern of clonal evolution (2-4). Epigenetic factors are also involved in MM initiation, progression, and occurrence of chemoresistance (5). Among them, EZH2 and MMSET are associated with poor prognosis values and contribute to MM disease. EZH2 is the main catalytic subunit of the Polycomb Repressive Complex 2 (PRC2) and deposits H3K27me3 on chromatin (6), whereas MMSET, coded by the gene NSD2, which is the target oncogene of t(4;14) primary translocation, is a histone methyltransferase that methylates H3K36me2 (7). Using proteomics analysis of EZH2-immune precipitates, we identified a constitutive interaction between EZH2 and MMSET independently of t(4;14) translocation and mutational status of EZH2 in MM cells. Furthermore, MAK-683, an allosteric inhibitor of EZH2, disrupted this interaction, but not EPZ-6438, a catalytic inhibitor of EZH2, suggesting that MMSET-EZH2 interaction may be mediated by other PRC2 factors, but is independent of the PRC2 activity. Analysis of EZH2 expression levels and t(4;14) translocation status in 3 independent cohorts of MM patients (CoMMPass (N=674), UAMS-TT2 (N=345), Mtp-Daratumumab (N=51)) identified a worse overall survival of patients with high EZH2 expression combined with t(4;14) chromosomal translocation than patients with only one of these genetic events. We identified 355 differentially expressed genes in this “ EZH2 High t(4;14) +” subgroup compared to other patients. GSEA analysis of up-regulated genes in this subgroup identified a significant enrichment for genes involved in cell cycle, KRAS signaling, Cell-Cell signaling, Hypoxia and Epithelial-Mesenchymal transition. These results indicate that EZH2-MMSET interaction is of clinical interest in t(4;14) MM patients. To better understand the molecular relationship between EZH2 and MMSET, we generated MMSET-depleted clones by CRISPR-Cas9. MMSET depletion did not significantly alter cell proliferation and survival, but reduced the clonogenic capacity of MM cells compared to non-depleted cells. Moreover, after IL-6 or serum starvation, IL-6 and IGF-1 sensitivity was significantly affected in MMSET-depleted clones compared to the parental cell line, indicating that MMSET overexpression confers a cell growth advantage to MM cells under stress conditions and an independence of growth factors produced by bone marrow microenvironment Additionally, MMSET depletion induced changes in PRC2 nuclear localization in MM cells, a decrease of H3K36me3 and a concomitant increase in H3K27me3 levels, which altered the transcriptional program of cells showing, among others, a specific enrichment in genes up-regulated in t(4;14) MM subgroup of patients. Finally, we demonstrated that disrupting EZH2-MMSET interaction with MAK-683 may be of therapeutic interest, since MAK-683 combination with conventional drugs used in MM treatment, including melphalan and Panobinostat or BRD4 inhibitors, synergized to kill MM cells. Taken together, our data identified an “ EZH2 High t(4;14) +” signature in MM patients with a poor outcome who may benefit from combination therapy of MAK-683 with melphalan or epidrugs.

Stochastic chain termination in bacterial pilus assembly

Adhesive type 1 pili from uropathogenic Escherichia coli strains are filamentous, supramolecular protein complexes consisting of a short tip fibrillum and a long, helical rod formed by up to several thousand copies of the major pilus subunit FimA. Here, we reconstituted the entire type 1 pilus rod assembly reaction in vitro, using all constituent protein subunits in the presence of the assembly platform FimD, and identified the so-far uncharacterized subunit FimI as an irreversible assembly terminator. We provide a complete, quantitative model of pilus rod assembly kinetics based on the measured rate constants of FimD-catalyzed subunit incorporation. The model reliably predicts the length distribution of assembled pilus rods as a function of the ratio between FimI and the main pilus subunit FimA and is fully consistent with the length distribution of membrane-anchored pili assembled in vivo. The results show that the natural length distribution of adhesive pili formed via the chaperone-usher pathway results from a stochastic chain termination reaction. In addition, we demonstrate that FimI contributes to anchoring the pilus to the outer membrane and report the crystal structures of (i) FimI in complex with the assembly chaperone FimC, (ii) the FimI-FimC complex bound to the N-terminal domain of FimD, and (iii) a ternary complex between FimI, FimA and FimC that provides structural insights on pilus assembly termination and pilus anchoring by FimI.

Naturally occurring PAE206K point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century. In this study, we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients. Sequence comparison of the three main protein subunits (PB2, PB1, and PA) of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution (E206K) was responsible for the observed impaired replication phenotype. Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo. Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature (39.5 ​°C). Hence, this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Conformationally constrained potent inhibitors for enhancer of zeste homolog 2 (EZH2)

The enhancer of zeste homolog 2 (EZH2) plays the role of the main catalytic subunit of polycomb repressive complex 2 (PRC2) that catalyzes the methylation of histone H3 lysine 27 (H3K27). Overexpression or mutation of EZH2 has been observed in many types of hematologic malignancies and solid tumors, such as myeloma, lymphoma, prostate, breast, kidney, and lung cancers. EZH2 has been demonstrated as a promising therapeutic target for the treatment of tumors. Based on the structure of 1 (EPZ-6438), a series of novel conformationally constrained derivatives were designed and synthesized aiming to improve the EZH2 inhibition activity, especially for mutated EZH2. Structure and activity relationship (SAR) exploration and optimization at both enzymatic and cellular levels led to the discovery of 28. In vitro, 28 displayed potent EZH2 inhibition activity with an IC50 value of 0.95 nM, which is comparable to EPZ-6438 (1). 28 exhibited high anti-proliferation activity against different lymphoma cell lines including WSU-DLCL2, Pfeiffer and Karpas-422 (IC50 = 2.36, 1.73, and 1.82 nM, respectively). In vivo, 28 showed acceptable pharmacokinetic characteristics (oral bioavailability F = 36.9%) and better efficacy than 1 in both Pfeiffer and Karpas-422 xenograft mouse models, suggesting that it can be further developed as a potential therapeutic candidate for EZH2-associated cancers.

Hypoxia-inducible factors: details create a picture. Part I. HIF-1

The review presents a comparative analysis of scientific data on the structural and functional characteristics of subunits (HIF-1α and HIF-1β) of hypoxia-inducible transcription factor-1 (HIF-1). Differences between the main regulatory HIF-1α subunit and the constitutively expressed HIF-1β subunit, sensitivity to endo- and exogenous regulators of their stability, and intracellular content depending on the cell's oxygen supply state (normoxia and hypoxia conditions) are described. In normoxia, the intracellular content of HIF-1α is determined by oxygen-dependent and oxygen-independent mechanisms. Oxygen-dependent enzymatic degradation of HIF-1α occurs by PHD-dependent hydroxylation, VHL-dependent ubiquitination, and FIH-1-dependent hydroxylation. Oxygen-independent pathways of HIF-1α pool regulation include: 1) HIF-1α gene transcription (Notch and/or NF-KB-dependent, STAT3 and Sp1 cytokine-dependent), 2) mRNA translation (cap-dependent or IRES-dependent, as well as cytokine-dependent activation of the PI-3K/AKT pathway activation under the effect of growth factors and vasoactive hormones), 3) protein-protein interactions, 4) various mechanisms of post-translational modification. Changes in Krebs cycle enzyme activity and active oxygen forms confer HIF-1α stability through PHD activity inhibition and reduction of ubiquitin-proteasome degradation. PHD-independent post-translational stabilizers of HIF-1α are: cytosolic reductase NQO1, sirtuin-2, prostaglandin E2, activated protein kinase C1 receptor competing with heat shock protein 90, human Hdm2 (a natural inhibitor of p53), glycogen synthase kinase 3β, and negative modifiers are enzymes - methyltransferase SET7/9, lysine-specific demethylase-1, sex-like kinase 3, β-arrestin-2, casein kinase-1. Under hypoxic conditions, non-hydroxylated HIF-1α subunits migrate to the nucleus where they heterodimerize with HIF-1β, HIF-1α/β heterodimers bind the main 5'-(A/G)CGTG-3' consensus sequence within the hypoxia-reaction element (HRE) of the target genes, and recruit co-activators (p300, histone modifying enzymes, histone readers, chromatin remodeling proteins, and mediator proteins for target genes transcription enhancement with the aid of RNA polymerase II), resulting in the formation of HIF-1, acting as a transcription factor for the target genes providing metabolic reprogramming from oxidative phosphorylation to anaerobic glycolysis (genes encoding glucose transporters (GLUT1 and GLUT3), genes for glycolytic enzymes hexokinase 1 and 2 (HK1 and HK2), phosphoglycerate kinase 1), as well as genes for erythropoietin, vascular endothelial growth factor and its receptors FLT1 and FLK1, endothelin 1 and angiopoietin 1, resulting in adaptation to hypoxia.

Investigating the Spectroscopy of the Gas Phase Guanine-Cytosine Pair: Keto versus Enol Configurations.

We report on a vibrational study of the guanine-cytosine dimer tautomers using state-of-the-art quasiclassical trajectory and semiclassical vibrational spectroscopy. The latter includes possible quantum mechanical effects. Through an accurate comparison to the experimental spectra, we are able to shine a light on the hydrogen bond network of one of the main subunits of DNA and put the experimental assignment on a solid footing. Our calculations corroborate the experimental conclusion that the global minimum Watson-and-Crick structure is not detected in the spectra, and there is no evidence of tunnel-effect-based double proton hopping. Our accurate assignment of the spectral features may also serve as a basis for the development of precise force fields to study the guanine-cytosine dimer.

Functional evaluation allows ACMG/AMP-based re-classification of CNGA3 variants associated with achromatopsia

PurposeCNGA3 encoding the main subunit of the cyclic nucleotide-gated ion channel in cone photoreceptors is one of the major disease-associated genes for achromatopsia. Most CNGA3 variants are missense variants with the majority being functionally uncharacterized and therefore hampering genetic diagnosis. In light of potential gene therapy, objective variant pathogenicity assessment is essential. MethodsWe established a medium-throughput aequorin-based luminescence bioassay allowing mutant CNGA3 channel function assessment via quantification of CNGA3 channel-mediated calcium influx in a cell culture system, thereby enabling American College of Medical Genetics and Genomics/Association for Molecular Pathology–based variant re-classification. ResultsWe provide functional read-out obtained for 150 yet uncharacterized CNGA3 missense substitutions of which 55 were previously categorized as variants of uncertain significance (VUS) identifying 25 as functionally normal and 125 as functionally abnormal. These data enabled the American College of Medical Genetics and Genomics/ Association for Molecular Pathology–based variant re-classification of 52/55 VUS as either benign, likely benign, or likely pathogenic reaching a VUS re-classification rate of 94.5%. ConclusionOur aequorin-based bioassay allows functionally ensured clinical variant interpretation for 150 CNGA3 missense variants enabling and supporting VUS re-classification and assuring molecular diagnosis to patients affected by CNGA3-associated achromatopsia, hereby identifying patients eligible for future gene therapy trials on this disease.