Structural basis of γ-secretase inhibition and modulation by small molecule drugs.

Development of γ-secretase inhibitors (GSIs) and modulators (GSMs) represents an attractive therapeutic opportunity for Alzheimer's disease (AD) and cancers. However, how these GSIs and GSMs target γ-secretase has remained largely unknown. Here, we report the cryoelectron microscopy (cryo-EM) structures of human γ-secretase bound individually to two GSI clinical candidates, Semagacestat and Avagacestat, a transition state analog GSI L685,458, and a classic GSM E2012, at overall resolutions of 2.6-3.1 Å. Remarkably, each of the GSIs occupies the same general location on presenilin 1 (PS1) that accommodates the ß strand from amyloid precursor protein or Notch, interfering with substrate recruitment. L685,458 directly coordinates the two catalytic aspartate residues of PS1. E2012 binds to an allosteric site of γ-secretase on the extracellular side, potentially explaining its modulating activity. Structural analysis reveals a set of shared themes and variations for inhibitor and modulator recognition that will guide development of the next-generation substrate-selective inhibitors.

Pines' demon observed as a 3D acoustic plasmon in Sr2RuO4.

The characteristic excitation of a metal is its plasmon, which is a quantized collective oscillation of its electron density. In 1956, David Pines predicted that a distinct type of plasmon, dubbed a 'demon', could exist in three-dimensional (3D) metals containing more than one species of charge carrier1. Consisting of out-of-phase movement of electrons in different bands, demons are acoustic, electrically neutral and do not couple to light, so have never been detected in an equilibrium, 3D metal. Nevertheless, demons are believed to be critical for diverse phenomena including phase transitions in mixed-valence semimetals2, optical properties of metal nanoparticles3, soundarons in Weyl semimetals4 and high-temperature superconductivity in, for example, metal hydrides3,5-7. Here, we present evidence for a demon in Sr2RuO4 from momentum-resolved electron energy-loss spectroscopy. Formed of electrons in the ß and γ bands, the demon is gapless with critical momentum qc = 0.08 reciprocal lattice units and room-temperature velocity v = (1.065 ± 0.12) × 105 m s-1 that undergoes a 31% renormalization upon cooling to 30 K because of coupling to the particle-hole continuum. The momentum dependence of the intensity of the demon confirms its neutral character. Our study confirms a 67-year old prediction and indicates that demons may be a pervasive feature of multiband metals.

Structural basis of Notch recognition by human γ-secretase.

Aberrant cleavage of Notch by γ-secretase leads to several types of cancer, but how γ-secretase recognizes its substrate remains unknown. Here we report the cryo-electron microscopy structure of human γ-secretase in complex with a Notch fragment at a resolution of 2.7 Å. The transmembrane helix of Notch is surrounded by three transmembrane domains of PS1, and the carboxyl-terminal ß-strand of the Notch fragment forms a ß-sheet with two substrate-induced ß-strands of PS1 on the intracellular side. Formation of the hybrid ß-sheet is essential for substrate cleavage, which occurs at the carboxyl-terminal end of the Notch transmembrane helix. PS1 undergoes pronounced conformational rearrangement upon substrate binding. These features reveal the structural basis of Notch recognition and have implications for the recruitment of the amyloid precursor protein by γ-secretase.

Modulation of amyloid precursor protein cleavage by γ-secretase activating protein through phase separation.

Aberrant cleavage of amyloid precursor protein (APP) by γ-secretase is closely associated with Alzheimer's disease (AD). γ-secretase activating protein (GSAP) specifically promotes γ-secretase­mediated cleavage of APP. However, the underlying mechanism remains enigmatic. Here, we demonstrate that the 16-kDa C-terminal fragment of GSAP (GSAP-16K) undergoes phase separation in vitro and forms puncta-like condensates in cells. GSAP-16K exerts dual modulation on γ-secretase cleavage; GSAP-16K in dilute phase increases APP­C-terminal 99-residue fragment (C99) cleavage toward preferred production of ß-amyloid peptide 42 (Aß42), but GSAP-16K condensates reduce APP-C99 cleavage through substrate sequestration. Notably, the Aß42/Aß40 ratio is markedly elevated with increasing concentrations of GSAP-16K. GSAP-16K stably associates with APP-C99 through specific sequence elements. These findings mechanistically explain GSAP-mediated modulation of γ-secretase activity that may have ramifications on the development of potential therapeutics.

Generic character of charge and spin density waves in superconducting cuprates.

Charge density waves (CDWs) have been observed in nearly all families of copper-oxide superconductors. But the behavior of these phases across different families has been perplexing. In La-based cuprates, the CDW wavevector is an increasing function of doping, exhibiting the so-called Yamada behavior, while in Y- and Bi-based materials the behavior is the opposite. Here, we report a combined resonant soft X-ray scattering (RSXS) and neutron scattering study of charge and spin density waves in isotopically enriched La1.8−xEu0.2SrxCuO4 over a range of doping 0.07≤x≤0.20. We find that the CDW amplitude is temperature independent and develops well above experimentally accessible temperatures. Further, the CDW wavevector shows a nonmonotonic temperature dependence, exhibiting Yamada behavior at low temperature with a sudden change occurring near the spin ordering temperature. We describe these observations using a Landau­Ginzburg theory for an incommensurate CDW in a metallic system with a finite charge compressibility and spin-CDW coupling. Extrapolating to high temperature, where the CDW amplitude is small and spin order is absent, our analysis predicts a decreasing wavevector with doping, similar to Y and Bi cuprates. Our study suggests that CDW order in all families of cuprates forms by a common mechanism.

Molecular Characterization and Expression Changes of the bcl2l13 Gene in Response to Hypoxia in Megalobrama amblycephala.

Hypoxia is a unique environmental stress, which not only reflects the insufficient oxygen supply of cells and tissues, but also occurs in various physiological and pathological environments. Mitophagy as a selective autophagy can recover and utilize damaged organelles and misfolded proteins to ensure normal cell functions and promote cell survival. Bcl2l13 (B-cell lymphoma-2 like 13) is reported to induce mitophagy as a functional mammalian homolog of Atg32. However, the function of the bcl2l13 gene is still unclear in fish. Here the sequence and structure of the bcl2l13 gene in Megalobrama amblycephala were identified and showed that bcl2l13 contained an open reading frame (ORF) of 1458 bp for encoding 485 aa. Amino acid sequence analysis indicated that Bcl2l13, as a typical anti-apoptotic protein of the Bcl2 family, contained four BH domains, one BHNo domain, and one TM domain. Further study showed that Bcl2l13 was mainly located in the mitochondria, while its localization was changed within the whole cell after the TM domain was deleted. Real-time PCR analysis revealed that bcl2l13 showed higher expression levels in early embryos. After hypoxia treatment, the mRNA levels of the bcl2l13 and autophagy-related genes were significantly up-regulated in most detected tissues, and the bcl2l13 transcription was regulated by Hif-1α mediated pathway. Additionally, the transcription activity of the bcl2l13 promoter was further analyzed using luciferase reporter assays and showed the highest activity in the promoter region from -475 to +111. These results indicated that bcl2l13 may play important roles in embryogenesis and hypoxia mediated autophagy in fish.

SAFA facilitates chromatin opening of immune genes through interacting with anti-viral host RNAs.

Regulation of chromatin structure and accessibility determines the transcription activities of genes, which endows the host with function-specific patterns of gene expression. Upon viral infection, the innate immune responses provide the first line of defense, allowing rapid production of variegated antiviral cytokines. Knowledge on how chromatin accessibility is regulated during host defense against viral infection remains limited. Our previous work found that the nuclear matrix protein SAFA surveilled viral RNA and regulated antiviral immune genes expression. However, how SAFA regulates the specific induction of antiviral immune genes remains unknown. Here, through integration of RNA-seq, ATAC-seq and ChIP-seq assays, we found that the depletion of SAFA specifically decreased the chromatin accessibility, activation and expression of virus induced genes. And mutation assays suggested that the RNA-binding ability of SAFA was essential for its function in regulating antiviral chromatin accessibility. RIP-seq results showed that SAFA exclusively bound with antiviral related RNAs following viral infection. Further, we combined the CRISPR-Cas13d mediated RNA knockdown system with ATAC-qPCR, and demonstrated that the binding between SAFA and according antiviral RNAs specifically mediated the openness of the corresponding chromatin and following robust transcription of antiviral genes. Moreover, knockdown of these associated RNAs dampened the accessibility of related genes in an extranuclear signaling pathway dependent manner. Interestingly, VSV infection cleaved SAFA protein at the C-terminus which deprived its RNA binding ability for immune evasion. Thus, our results demonstrated that SAFA and the interacting RNA products collaborated and remodeled chromatin accessibility to facilitate antiviral innate immune responses.

Multiple Charge Density Waves and Superconductivity Nucleation at Antiphase Domain Walls in the Nematic Pnictide Ba_{1-x}Sr_{x}Ni_{2}As_{2}.

How superconductivity interacts with charge or nematic order is one of the great unresolved issues at the center of research in quantum materials. Ba_{1-x}Sr_{x}Ni_{2}As_{2} (BSNA) is a charge ordered pnictide superconductor recently shown to exhibit a sixfold enhancement of superconductivity due to nematic fluctuations near a quantum phase transition (at x_{c}=0.7) [1]. The superconductivity is, however, anomalous, with the resistive transition for 0.4

CBRPP: a new RNA-centric method to study RNA-protein interactions.

RNA and protein are interconnected biomolecules that can influence each other's life cycles and functions through physical interactions. Abnormal RNA-protein interactions lead to cell dysfunctions and human diseases. Therefore, mapping networks of RNA-protein interactions is crucial for understanding cellular processes and pathogenesis of related diseases. Different practical protein-centric methods for studying RNA-protein interactions have been reported, but few robust RNA-centric methods exist. Here, we developed CRISPR-based RNA proximity proteomics (CBRPP), a new RNA-centric method to identify proteins associated with an endogenous RNA of interest in native cellular context without pre-editing of the target RNA, cross-linking or RNA-protein complexes manipulation in vitro. CBRPP is based on a fusion of dCas13 and proximity-based labelling (PBL) enzyme. dCas13 can deliver PBL enzyme to the target RNA with high specificity, while PBL enzyme labels the surrounding proteins of the target RNA, which are then identified by mass spectrometry.

Carbon nanotube-loaded Nafion film electrochemical sensor for metal ions: europium.

A Nafion film loaded with novel catalyst-free multiwalled carbon nanotubes (MWCNTs) was used to modify a glassy carbon (GC) electrode to detect trace concentrations of metal ions, with europium ion (Eu(3+)) as a model. The interaction between the sidewalls of MWCNTs and the hydrophobic backbone of Nafion allows the MWCNTs to be dispersed in Nafion, which was then coated as a thin film on the GC electrode surface. The electrochemical response to Eu(3+) was found to be ∼10 times improved by MWCNT concentrations between 0.5 and 2 mg/mL, which effectively expanded the electrode surface into the Nafion film and thereby reduced the diffusion distance of Eu(3+) to the electrode surface. At low MWCNT concentrations of 0.25 and 0.5 mg/mL, no significant improvement in signal was obtained compared with Nafion alone. Scanning electron microscopy and electrochemical impedance spectroscopy were used to characterize the structure of the MWCNT-Nafion film, followed by electrochemical characterization with Eu(3+) via cyclic voltammetry and preconcentration voltammetry. Under the optimized conditions, a linear range of 1-100 nM with a calculated detection limit of 0.37 nM (signal/noise = 3) was obtained for determination of Eu(3+) by Osteryoung square-wave voltammetry after a preconcentration time of 480 s.

Identification and characterization of endogenous retroviruses upon SARS-CoV-2 infection.

Endogenous retroviruses (ERVs) derived from the long terminal repeat (LTR) family of transposons constitute a significant portion of the mammalian genome, with origins tracing back to ancient viral infections. Despite comprising approximately 8% of the human genome, the specific role of ERVs in the pathogenesis of COVID-19 remains unclear. In this study, we conducted a genome-wide identification of ERVs in human peripheral blood mononuclear cells (hPBMCs) and primary lung epithelial cells from monkeys and mice, both infected and uninfected with SARS-CoV-2. We identified 405, 283, and 206 significantly up-regulated transposable elements (TEs) in hPBMCs, monkeys, and mice, respectively. This included 254, 119, 68, and 28 ERVs found in hPBMCs from severe and mild COVID-19 patients, monkeys, and transgenic mice expressing the human ACE2 receptor (hACE2) and infected with SARS-CoV-2. Furthermore, analysis using the Genomic Regions Enrichment of Annotations Tool (GREAT) revealed certain parental genomic sequences of these up-regulated ERVs in COVID-19 patients may be involved in various biological processes, including histone modification and viral replication. Of particular interest, we identified 210 ERVs specifically up-regulated in the severe COVID-19 group. The genes associated with these differentially expressed ERVs were enriched in processes such as immune response activation and histone modification. HERV1_I-int: ERV1:LTR and LTR7Y: ERV1:LTR were highlighted as potential biomarkers for evaluating the severity of COVID-19. Additionally, validation of our findings using RT-qPCR in Bone Marrow-Derived Macrophages (BMDMs) from mice infected by HSV-1 and VSV provided further support to our results. This study offers insights into the expression patterns and potential roles of ERVs following viral infection, providing a valuable resource for future studies on ERVs and their interaction with SARS-CoV-2.

MOI is a comprehensive database collecting processed multi-omics data associated with viral infection.

Viral infections pose significant public health challenges, exemplified by the global impact of COVID-19 caused by SARS-CoV-2. Understanding the intricate molecular mechanisms governing virus-host interactions is pivotal for effective intervention strategies. Despite the burgeoning multi-omics data on viral infections, a centralized database elucidating host responses to viruses remains lacking. In response, we have developed a comprehensive database named 'MOI' (available at http://www.fynn-guo.cn/ ), specifically designed to aggregate processed Multi-Omics data related to viral Infections. This meticulously curated database serves as a valuable resource for conducting detailed investigations into virus-host interactions. Leveraging high-throughput sequencing data and metadata from PubMed and Gene Expression Omnibus (GEO), MOI comprises over 3200 viral-infected samples, encompassing human and murine infections. Standardized processing pipelines ensure data integrity, including bulk RNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). MOI offers user-friendly interfaces presenting comprehensive cell marker tables, gene expression data, and epigenetic landscape charts. Analytical tools for DNA sequence conversion, FPKM calculation, differential gene expression, and Gene Ontology (GO)/ Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment enhance data interpretation. Additionally, MOI provides 16 visualization plots for intuitive data exploration. In summary, MOI serves as a valuable repository for researchers investigating virus-host interactions. By centralizing and facilitating access to multi-omics data, MOI aims to advance our understanding of viral pathogenesis and expedite the development of therapeutic interventions.

SENP1 exacerbates acute myeloid leukemia by enhancing BECN1-dependent autophagy through PTBP1 deSUMOylation.

Genetic association between SUMO-specific protease 1 (SENP1) and acute myeloid leukemia (AML) has been validated. However, the mechanism by which SENP1 affects AML proliferation, apoptosis, and autophagy remains unknown. The levels of SENP1 and polypyrimidine tract-binding protein 1 (PTBP1) were measured in AML patients, AML cell lines, and xenograft tissues. The effects of SENP1 on AML proliferation, apoptosis, and BECN1-dependent autophagy were assessed through in vitro and in vivo loss- or gain-of-function experiments. SUMOylation analysis using immunoprecipitation (IP), RNA pull-down, RIP, and RNA stability assays were used to explore the molecular mechanism of SENP1 in AML development. The SENP1 level was elevated in AML samples. Silencing SENP1 impeded the development of AML, as evidenced by the inhibition of proliferation and promotion of G1 phase arrest and apoptosis resulting from SENP1 depletion in AML cells. Moreover, silencing of SENP1 restrained BECN1-depentent autophagy in AML cells. In addition, the overexpression of BECN1 or PTBP1 partially neutralized the effect of SENP1 knockdown on AML cell behavior. Mechanistically, SENP1 mediated PTBP1 deSUMOylation, which then directly interacted with BECN1 mRNA and enhanced its stability. In vivo experiments further confirmed the repressive effects of SENP1 suppression on AML development. Collectively, the SENP1/PTBP1/BECN1 signaling axis has been identified as a significant therapeutic target for enhancing AML treatment.

Molecular mechanism of substrate recognition and cleavage by human γ-secretase.

Successive cleavages of amyloid precursor protein C-terminal fragment with 99 residues (APP-C99) by γ-secretase result in amyloid-ß (Aß) peptides of varying lengths. Most cleavages have a step size of three residues. To elucidate the underlying mechanism, we determined the atomic structures of human γ-secretase bound individually to APP-C99, Aß49, Aß46, and Aß43. In all cases, the substrate displays the same structural features: a transmembrane α-helix, a three-residue linker, and a ß-strand that forms a hybrid ß-sheet with presenilin 1 (PS1). Proteolytic cleavage occurs just ahead of the substrate ß-strand. Each cleavage is followed by unwinding and translocation of the substrate α-helix by one turn and the formation of a new ß-strand. This mechanism is consistent with existing biochemical data and may explain the cleavages of other substrates by γ-secretase.

XAF1 promotes anti-RNA virus immune responses by regulating chromatin accessibility.

A rapid induction of antiviral genes is critical for eliminating viruses, which requires activated transcription factors and opened chromatins to initiate transcription. However, it remains elusive how the accessibility of specific chromatin is regulated during infection. Here, we found that XAF1 functioned as an epigenetic regulator that liberated repressed chromatin after infection. Upon RNA virus infection, MAVS recruited XAF1 and TBK1. TBK1 phosphorylated XAF1 at serine-252 and promoted its nuclear translocation. XAF1 then interacted with TRIM28 with the guidance of IRF1 to the specific locus of antiviral genes. XAF1 de-SUMOylated TRIM28 through its PHD domain, which led to increased accessibility of the chromatin and robust induction of antiviral genes. XAF1-deficient mice were susceptible to RNA virus due to impaired induction of antiviral genes. Together, XAF1 acts as an epigenetic regulator that promotes the opening of chromatin and activation of antiviral immunity by targeting TRIM28 during infection.

The collateral activity of RfxCas13d can induce lethality in a RfxCas13d knock-in mouse model.

BACKGROUND: The CRISPR-Cas13 system is an RNA-guided RNA-targeting system and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. RESULTS: Here, we find that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which may result from the collateral activity of RfxCas13d rather than the loss of target gene function or off-target effects. Mechanistically, we show that RfxCas13d exhibits collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAKα-JNK/p38-immediate early gene pathway. CONCLUSIONS: These findings provide new mechanistic insights into the collateral activity of RfxCas13d in mammalian cells and warn that the biosafety of the CRISPR-Cas13 system needs further evaluation before application to clinical treatments.

Carbohydrate-based label-free detection of Escherichia coli ORN 178 using electrochemical impedance spectroscopy.

A label-free biosensor for Escherichia coli (E. coli) ORN 178 based on faradaic electrochemical impedance spectroscopy (EIS) was developed. α-Mannoside or ß-galactoside was immobilized on a gold disk electrode using a self-assembled monolayer (SAM) via a spacer terminated in a thiol functionality. Impedance measurements (Nyquist plot) showed shifts due to the binding of E. coli ORN 178, which is specific for α-mannoside. No significant change in impedance was observed for E. coli ORN 208, which does not bind to α-mannoside. With increasing concentrations of E. coli ORN 178, electron-transfer resistance (R(et)) increases before the sensor is saturated. After the Nyquist plot of E. coli/mixed SAM/gold electrode was modeled, a linear relationship between normalized R(et) and the logarithmic value of E. coli concentrations was found in a range of bacterial concentration from 10(2) to 10(3) CFU/mL. The combination of robust carbohydrate ligands with EIS provides a label-free, sensitive, specific, user-friendly, robust, and portable biosensing system that could potentially be used in a point-of-care or continuous environmental monitoring setting.

A simple electrochemiluminesecence aptasenor using a GCE/NCQDs/aptamers for detection of Pb.

An electrochemiluminescence (ECL) aptasensor was prepared to detect Pb2+ with nitrogen-doped carbon quantum dots (NCQDs) as ECL materials. To prepare the working electrode, NCQDs with carboxyl groups were loaded on a glassy carbon electrode (GCE) and then Pb2+ aptamers were covalently bound to the NCQDs to form a stable GCE/NCQDs/aptamers. On addition of Pb2+, the chain aptamers change to a pb2+ G-quadruplex conformation, which lead to a large decrease in the ECL intensity. The variation of intensity and the logarithm of the Pb2+ concentration had a good linear relationship (R2 = 0.998). The detection range was wide (50 pM to 387.9 nM) with a low detection limit (18.9 pM). In interference experiments, the ECL Pb2+ aptasensor did not suffer from interference and it had good stability. The NCQDs ECL aptasensor can detect Pb2+ quickly and accurately, and provides a fast and efficient method for detection of Pb2+. Compared with literatures, the Pb2+ aptasensor has simpler preparation process, lower cost; furthermore, it is more environmentally friendly.

Molecular basis for isoform-selective inhibition of presenilin-1 by MRK-560.

Inhibition of γ-secretase activity represents a potential therapeutic strategy for Alzheimer's disease (AD). MRK-560 is a selective inhibitor with higher potency for Presenilin 1 (PS1) than for PS2, the two isoforms of the catalytic subunit of γ-secretase, although the underlying mechanism remains elusive. Here we report the cryo-electron microscopy (cryo-EM) structures of PS1 and PS2-containing γ-secretase complexes with and without MRK-560 at overall resolutions of 2.9-3.4 Å. MRK-560 occupies the substrate binding site of PS1, but is invisible in PS2. Structural comparison identifies Thr281 and Leu282 in PS1 to be the determinant for isoform-dependent sensitivity to MRK-560, which is confirmed by swapping experiment between PS1 and PS2. By revealing the mechanism for isoform-selective inhibition of presenilin, our work may facilitate future drug discovery targeting γ-secretase.