Biology subjects

Lu, X.

Publications and source records attributed to Lu, X..

At least 181 records · Page 10Linked to original sources

bioRxiv2024

Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome

Pseudouridine ({Psi}) is one of the most abundant modifications in cellular RNA. However, its function remains elusive, mainly due to the lack of highly sensitive and accurate detection methods. To address this challenge, we introduced 2-bromoacrylamide-assisted cyclization sequencing (BACS) for quantitative profiling of {Psi} at single-base resolution. Based on novel bromoacrylamide cyclization chemistry, BACS enables a {Psi}-to-C transition. Compared to previous methods, BACS allowed the precise identification of {Psi} positions, especially in densely modified {Psi} regions and consecutive uridine sequences. BACS successfully detected all known {Psi} sites in human rRNA and spliceosomal snRNAs and generated the first quantitative {Psi} map of human snoRNA and tRNA. Furthermore, BACS simultaneously detected adenosine-to-inosine (A-to-I) editing sites and N1-methyladenosine (m1A). Depletion of three key pseudouridine synthases (PUS) enabled us to elucidate the targets and sequence motifs of TRUB1, PUS7, and PUS1 in HeLa cells. We further applied BACS to Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) and identified a highly abundant {Psi}114 site in EBER2. Surprisingly, applying BACS to a panel of RNA viruses demonstrated the absence of {Psi} in their viral transcripts or genomes, shedding light on differences in pseudouridylation between virus families. We anticipate BACS to serve as a powerful tool to uncover the biological importance of {Psi} in future studies.

molecular biology↗

bioRxiv2024

Characterization of Recombinant Human Lactoferrin Expressed in Komagataella Phaffii

We performed a thorough analysis and characterization of multiple batches of Helaina recombinant human lactoferrin (rhLF, Effera) expressed at an industrial scale in a yeast system. Bottom-up LC-MS/MS-based proteomics analysis detected the full sequence of Helaina rhLF protein and confirmed that its amino acid sequence is identical to that of native human LF (Uniprot i.d. P02788). Helaina rhLF had a protein purity of 98% or higher as determined by three orthogonal methods; reversed-phase HPLC, SDS-PAGE, and LC-MS proteomics analysis. N-linked glycans were detected at three known glycosylation sites, namely, Asparagines-156, -497, and -642. The identified N-glycans of Helaina rhLF were predominantly oligomannose structures with five to nine mannoses (M5-M9), which we also report to be present in both the native human and bovine LF. human milk LF (hmLF) possessed lower levels of oligomannose structures and were mainly M5 and M6. Helaina rhLF protein secondary structure was nearly identical to that of hmLF, as revealed by microfluidic modulation spectroscopy. Results of small-angle X-ray scattering (SAXS) and analytical ultracentrifugation analyses confirmed that, like hmLF, Helaina rhLF displayed well-folded globular structures in solution. Reconstructed solvent envelopes of Helaina rhLF, obtained through the SAXS analysis, demonstrated a remarkable fit with the reported crystalline structure of iron-bound native hmLF. Differential scanning calorimetry investigations into the thermal stability of Helaina rhLF revealed two distinct denaturation temperatures at 68.7{+/-}0.9 {degrees}C and 91.9{+/-}0.5 {degrees}C, consistently mirroring denaturation temperatures observed for apo-and holo-hmLF. Overall, the characterization analysis results affirmed that Helaina rhLF was of high purity and exhibited globular structures closely akin to that of hmLF.

biophysics↗

bioRxiv2024

Target-aware Molecule Generation for Drug Design Using a Chemical Language Model

Generative drug design facilitates the creation of compounds effective against pathogenic target proteins. This opens up the potential to discover novel compounds within the vast chemical space and fosters the development of innovative therapeutic strategies. However, the practicality of generated molecules is often limited, as many designs focus on a narrow set of drug-related properties, failing to improve the success rate of subsequent drug discovery process. To overcome these challenges, we develop TamGen, a method that employs a GPT-like chemical language model and enables target-aware molecule generation and compound refinement. We demonstrate that the compounds generated by TamGen have improved molecular quality and viability. Additionally, we have integrated TamGen into a drug discovery pipeline and identified 7 compounds showing compelling inhibitory activity against the Tuberculosis ClpP protease, with the most effective compound exhibiting a half maximal inhibitory concentration (IC50) of 1.9 M. Our findings underscore the practical potential and real-world applicability of generative drug design approaches, paving the way for future advancements in the field.

biochemistry↗

bioRxiv2023

Broadly neutralizing antibody induction by non-stabilized SARS-CoV-2 Spike mRNA vaccination in nonhuman primates

Immunization with mRNA or viral vectors encoding spike with diproline substitutions (S-2P) has provided protective immunity against severe COVID-19 disease. How immunization with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike elicits neutralizing antibodies (nAbs) against difficult-to-neutralize variants of concern (VOCs) remains an area of great interest. Here, we compare immunization of macaques with mRNA vaccines expressing ancestral spike either including or lacking diproline substitutions, and show the diproline substitutions were not required for protection against SARS-CoV-2 challenge or induction of broadly neutralizing B cell lineages. One group of nAbs elicited by the ancestral spike lacking diproline substitutions targeted the outer face of the receptor binding domain (RBD), neutralized all tested SARS-CoV-2 VOCs including Omicron XBB.1.5, but lacked cross-Sarbecovirus neutralization. Structural analysis showed that the macaque broad SARS-CoV-2 VOC nAbs bound to the same epitope as a human broad SARS-CoV-2 VOC nAb, DH1193. Vaccine-induced antibodies that targeted the RBD inner face neutralized multiple Sarbecoviruses, protected mice from bat CoV RsSHC014 challenge, but lacked Omicron variant neutralization. Thus, ancestral SARS-CoV-2 spike lacking proline substitutions encoded by nucleoside-modified mRNA can induce B cell lineages binding to distinct RBD sites that either broadly neutralize animal and human Sarbecoviruses or recent Omicron VOCs. One Sentence SummaryNon-stabilized SARS-CoV-2 Spike mRNA vaccination activated B cells that target either conserved epitopes on SARS-CoV-2 Omicron variants of concern, or cross-neutralizing epitopes on pre-emergent Sarbecoviruses.

immunology↗

bioRxiv2023

Engineering immunogens that select for specific mutations in HIV broadly neutralizing antibodies

Vaccine development targeting rapidly evolving pathogens such as HIV-1 requires induction of broadly neutralizing antibodies (bnAbs) with conserved paratopes and mutations, and, in some cases, the same Ig-heavy chains. The current trial-and-error search for immunogen modifications that improve selection for specific bnAb mutations is imprecise. To precisely engineer bnAb boosting immunogens, we used molecular dynamics simulations to examine encounter states that form when antibodies collide with the HIV-1 Envelope (Env). By mapping how bnAbs use encounter states to find their bound states, we identified Env mutations that were predicted to select for specific antibody mutations in two HIV-1 bnAb B cell lineages. The Env mutations encoded antibody affinity gains and selected for desired antibody mutations in vivo. These results demonstrate proof-of-concept that Env immunogens can be designed to directly select for specific antibody mutations at residue-level precision by vaccination, thus demonstrating the feasibility of sequential bnAb-inducing HIV-1 vaccine design.

immunology↗

bioRxiv2023

ProTokens: A Machine-Learned Language for Compact and Informative Encoding of Protein 3D Structures

Understanding functions of proteins and designing proteins committed to specific functions in silico are highly valuable for science, industry and therapeutics. However, there is a long-standing divergence in how to present function-related protein structures to the machine learning models: Although the 1-dimensional (1D) representation of proteins via Anfinsens tokens (i.e., amino acids) is sufficient in principle and more machine-friendly, it is less successful in structure-oriented protein design compared to symmetry-constrained 3D representation (i.e., atom coordinates). Aiming to bridge the gap between 1D and 3D protein representations and harvest the advantages of the two, we develop probabilistic tokenization theory for metastable protein structures. We present an unsupervised learning strategy, which conjugates inverse folding with structure prediction, to encode protein structures into artificial amino-acid tokens (ProTokens) and decode them back to atom coordinates. We show that tokenizing protein structures variationally can lead to compact and informative representations. Compared to amino acids -- the Anfinsens tokens -- ProTokens are easier to detokenize and more descriptive of finer conformational ensembles. Therefore, protein structures can be efficiently compressed, stored, aligned and compared in the form of ProTokens. By unifying the discrete and continuous representations of protein structures, ProTokens also enable all-atom protein structure design via various generative models without the concern of symmetry or modality mismatch, and allows scalable foundation models to perceive, process and explore the microscopic structures of biomolecules effectively.

bioinformatics↗

bioRxiv2023

CRISPR-Cas13d screens identify KILR a breast cancer risk-associated lncRNA that regulates DNA replication and repair.

Long noncoding RNAs (lncRNAs) have surpassed the number of protein-coding genes, yet the majority have no known function. We previously discovered >800 lncRNAs at regions identified by breast cancer genome-wide association studies (GWAS). Here, we performed a pooled CRISPR-Cas13d RNA knockdown screen to identify which of these lncRNAs altered cell proliferation. We found that KILR, a lncRNA that functions as a tumor suppressor, safeguards breast cells against uncontrolled proliferation. The half-life of KILR is significantly reduced by the risk haplotype, revealing an alternative mechanism by which variants alter cancer risk. We showed that KILR sequesters RPA1, a subunit of the RPA complex, required for DNA replication and repair. Reduced KILR expression promotes cell proliferation by increasing the available pool of RPA1 and the speed of DNA replication. Our findings confirm lncRNAs as mediators of breast cancer risk, emphasize the need to annotate noncoding transcripts in relevant cell types when investigating GWAS variants and provide a scalable platform for mapping phenotypes associated with lncRNAs.

cancer biology↗

bioRxiv2023

Implantable nanophotonic neural probes for integrated patterned photostimulation and electrophysiology recording

Optogenetics has transformed neuroscience by allowing precise manipulation of neural circuits with light [1-5]. However, a central difficulty has been to deliver spatially shaped light and record deep within the brain without causing damage or significant heating. Current approaches form the light beam in free space and record the neural activity using fluorescence imaging or separately inserted electrodes [6-9], but attenuation limits optical penetration to around 1 mm of the brain surface [10]. Here, we overcome this challenge with foundry-fabricated implantable silicon neural probes that combine microelectrodes for electrophysiology recordings with nanophotonic circuits that emit light with engineered beam profiles and minimal thermal impact. Our experiments reveal that planar light sheets, emitted by our neural probes, excited more neurons and induced greater firing rate fatigue in layers V and VI of the motor and somatosensory cortex of Thy1-ChR2 mice at lower output intensities than low divergence beams. In the hippocampus of an epilepsy mouse model, we induced seizures, a network-wide response, with light sheets without exceeding the[~] 1{whitebullet}C limit for thermally induced electrophysiological responses [11-13]. These findings show that optical spatial profiles can be tailored for optogenetic stimulation paradigms and that the probes can photostimulate and record neural activity at single or population levels while minimizing thermal damage to brain tissue. The neural probes, made in a commercial silicon photonics foundry on 200-mm silicon wafers, demonstrate the manufacturability of the technology. The prospect of monolithically integrating additional well-established silicon photonics devices, such as wavelength and polarization multiplexers, temperature sensors, and optical power monitors, into the probes holds the potential of realizing more versatile, implantable tools for multimodal brain activity mapping.

bioengineering↗

bioRxiv2023

NLRP3 Cys126 palmitoylation by ZDHHC7 Promotes Inflammasome Activation

NACHT-, leucine-rich-repeat- (LRR), and pyrin domain-containing protein 3 (NLRP3) mediates inflammasome activation in response to multiple pathogen and damage-associated molecular patterns in macrophages. Hyperactivation of NLRP3 inflammasome contributes to many human chronic inflammatory diseases. Understanding how NLRP3 inflammasome is regulated can potentially provide new strategies to treat inflammatory diseases. Here, we demonstrated that NLRP3 is palmitoylated on Cys126 by palmitoyl-acyltransferase ZDHHC7 in macrophages, which is critical for NLRP3-mediated inflammasome activation. Perturbating NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation, diminishes NLRP3 activation and the consequential Caspase-1 and Gasdermin D (GSDMD) cleavage, and IL-1{beta} and IL-18 secretion in mouse primary macrophages and human macrophages. Furthermore, NLRP3 Cys126 palmitoylation is vital for inflammasome activation in vivo, as Zdhhc7 knockout, pharmacological inhibition, or NLRP3 C126A mutation protects mice from LPS-induced endotoxic shock and monosodium urate (MSU)-induced peritonitis. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN (dTGN), which is indispensable for the recruitment and oligomerization of adaptor protein ASC after inflammasome activation. The activation of NLRP3 by ZDHHC7-mediated Cys126 palmitoylation is different from the previously reported inhibitory effect by ZDHHC12-mediated Cys841 palmitoylation, highlighting the versatile regulatory roles of S-palmitoylation. Therefore, our study identifies a new regulatory mechanism of NLRP3 activation and suggests targeting ZDHHC7 or NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders. HighlightsO_LINLRP3 Cys126 is palmitoylated by ZDHHC7. C_LIO_LIZDHHC7 promotes NLRP3 activation in macrophages, which can be inhibited by ZDHHCs inhibitors, 2-bromopalmitate and MY-D4. C_LIO_LICys126 palmitoylation of NLRP3 is critical for NLRP3 activation. C_LIO_LINLRP3 TGN/dTGN localization depends on ZDHHC7-mediated Cys126 palmitoylation, which is crucial for ASC recruitment and inflammasome assembly. C_LIO_LINLRP3 inflammasome activation by ZDHHC7 differs from ZDHHC12-mediated NLRP3 inhibition. C_LI

immunology↗

bioRxiv2023

Architecture and activation of human muscle phosphorylase kinase

The study of phosphorylase kinase (PhK)-regulated glycogen metabolism has contributed to the fundamental understanding of protein phosphorylation. Here we present the high-resolution cryo-electron microscopy structures of human muscle PhK. The 1.3-megadalton PhK 4{beta}4{gamma}4{delta}4 hexadecamer consists of a tetramer of tetramer, wherein four {beta}{gamma}{delta} modules are connected by the central {beta}4 scaffold. The - and {beta}-subunits possess glucoamylase-like domains, but exhibit no detectable enzyme activities. The -subunit serves as a bridge between the {beta}-subunit and the {gamma}{delta} subcomplex, and facilitates the {gamma}-subunit to adopt an autoinhibited state. Ca2+-free calmodulin ({delta}-subunit) binds to the {gamma}-subunit in a compact conformation. Upon binding of Ca2+, a conformational change occurs, allowing for the de-inhibition of the {gamma}-subunit through a spring-loaded mechanism. We also reveal an ADP-binding pocket in the {beta}-subunit, which plays a role in allosterically enhancing PhK activity. These results provide unprecedented molecular insights of this important kinase complex.

biochemistry↗

bioRxiv2023

Impaired Mitochondrial Calcium Homeostasis Induces Failed Remodeling of the Spiral Artery in Preeclampsia

Withdrawal StatementThe authors have withdrawn their manuscript owing to further revisions and additional experiments are needed to ensure the accuracy and completeness of the research findings. As a result, we believe it is in the best interest of the scientific community and the integrity of the manuscript to withdraw it at this time. Therefore, the authors do not wish this work to be cited as a reference for the project. If you have any questions, please contact the corresponding author.

pathology↗

bioRxiv2023

Identification and characterization of a beta-1,4 Galactosidase from Elizabethkingia meningoseptica and its application on living cell surface

The biological function of terminal galactose on glycoprotein is an open field of research. Although progress had being made on enzymes that can remove the terminal galactose on glycoproteins, there is a lack of report on galactosidases that can work directly on living cells. In this study, a unique beta 1,4 galactosidase was isolated from Elizabethkingia meningoseptica (Em). It exhibited favorable stability at various temperatures (4-37) and pH (5-8) levels and can remove {beta}-1, 4 linked galactoses directly from glycoproteins. Using Alanine scanning, we found that two acidic residues (Glu-468, and Glu-531) in the predicted active pocket are critical for galactosidase activity. In addition, we also demonstrated that it could cleave galactose residues present on living cell surface. As the enzyme has a potential application for living cell glycan editing, we named it glycan editing galactosidase I or geGalaseI. In summary, our findings lay the groundwork for prospective investigations by presenting a prompt and gentle approach for the removal of galactose moieties from cell surface.

biochemistry↗

bioRxiv2023

Epigenetic insights into GABAergic development in Dravet Syndrome iPSC and therapeutic implications

Dravet syndrome (DS) is a devastating early onset refractory epilepsy syndrome caused by variants in the SCN1A gene. A disturbed GABAergic interneuron function is implicated in the progression to DS but the underlying developmental and pathophysiological mechanisms remain elusive, in particularly at the chromatin level. In this study, we utilized induced pluripotent stem cells (iPSCs) derived from DS cases and healthy donors to model disease- associated epigenetic abnormalities of GABAergic development. Employing the ATAC-Seq technique, we assessed chromatin accessibility at multiple time points (Day 0, Day 19, Day 35, and Day 65) of GABAergic differentiation. Additionally, we elucidated the effects of the commonly used anti-seizure drug valproic acid (VPA) on chromatin accessibility in GABAergic cells. The distinct dynamics in chromatin profile of DS iPSC predicted accelerated early GABAergic development, evident at D19, and diverged further from the pattern in control iPSC with continued differentiation, indicating a disrupted GABAergic maturation. Exposure to VPA at D65 reshaped the chromatin landscape at a variable extent in different iPSC-lines and rescued the observed dysfunctional development in some DS iPSC-GABA. This study provides the first comprehensive investigation on the chromatin landscape of GABAergic differentiation in DS-patient iPSC, offering valuable insights into the epigenetic dysregulations associated with interneuronal dysfunction in DS. Moreover, our detailed analysis of the chromatin changes induced by VPA in iPSC-GABA holds the potential to improve development of personalized and targeted anti-epileptic therapies.

cell biology↗

bioRxiv2023

A Scalable Staining Strategy for Whole-Brain Connectomics

Mapping the complete synaptic connectivity of a mammalian brain would be transformative, revealing the pathways underlying perception, behavior, and memory. Serial section electron microscopy, via membrane staining using osmium tetroxide, is ideal for visualizing cells and synaptic connections but, in whole brain samples, faces significant challenges related to chemical treatment and volume changes. These issues can adversely affect both the ultrastructural quality and macroscopic tissue integrity. By leveraging time-lapse X-ray imaging and brain proxies, we have developed a 12-step protocol, ODeCO, that effectively infiltrates osmium throughout an entire mouse brain while preserving ultrastructure without any cracks or fragmentation, a necessary prerequisite for constructing the first comprehensive mouse brain connectome.

neuroscience↗

bioRxiv2023

Cytoskeleton remodeling induced by SMYD2 methyltransferase drives breast cancer metastasis

Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cells dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cells ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulates lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation loose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

cancer biology↗

bioRxiv2023

Probing Molecular Diversity and Ultrastructure of Brain Cells with Fluorescent Aptamers

Detergent-free immunolabeling has been proven feasible for correlated light and electron microscopy, but its application is restricted by the availability of suitable affinity reagents. Here we introduce CAptVE, a method using slow off-rate modified aptamers for cell fluorescence labeling on ultrastructurally reconstructable electron micrographs. CAptVE provides labeling for a wide range of biomarkers, offering a pathway to integrate molecular analysis into recent approaches to delineate neural circuits via connectomics.

neuroscience↗

bioRxiv2023

Acod1 Expression in Cancer Cells Promotes Immune Evasion through the Generation of Inhibitory Peptides

Targeting PD-1 is an important component of many immune checkpoint blockade (ICB) therapeutic approaches. However, ICB is not an efficacious strategy in a variety of cancer types, in part due to immunosuppressive metabolites in the tumor microenvironment (TME). Here, we find that PD-1-resistant cancer cells produce abundant itaconate (ITA) due to enhanced levels of aconitate decarboxylase (Acod1). Acod1 has an important role in the resistance to PD-1, as decreasing Acod1 levels in PD-1 resistant cancer cells can sensitize tumors to PD-1 therapy. Mechanistically, cancer cells with high Acod1 inhibit the proliferation of naive CD8+ T cells through the secretion of inhibitory factors. Surprisingly, inhibition of CD8+ T cell proliferation is not dependent on secretion of ITA, but is instead a consequence of the release of small inhibitory peptides. Our study suggests that strategies to counter the activity of Acod1 in cancer cells may sensitize tumors to ICB therapy.

cancer biology↗

bioRxiv2023

An optogenetic method for the controlled release of single molecules

We developed a system for optogenetic release of single molecules in live cells. We confined soluble and transmembrane proteins to the Golgi apparatus via a photocleavable protein and released them by short pulses of light. Our method allows for the controlled delivery of functional proteins to cytosol and plasma membrane in amounts compatible with single molecule imaging, greatly simplifying access to single molecule microscopy of any protein in live cells. Furthermore, we could reconstitute cellular functions such as ion conductance by delivering BK and VRAC ion channels to the plasma membrane. Finally, we could induce NF-kB signaling in T-Lymphoblasts stimulated by IL-1 by controlled release of a signaling protein that had been knocked-out in the same cells. We observed light induced formation of functional inflammatory signaling complexes that could trigger IKK phosphorylation in single cells. We thus developed an optogenetic method for the reconstitution and investigation of cellular function at the single molecule level.

biophysics↗