Biology subjects

Xu, J.

Publications and source records attributed to Xu, J..

At least 379 records · Page 21Linked to original sources

bioRxiv2024

Architecture of intact mammalian pyruvate dehydrogenase complex revealed by in-situ structural analysis

The multi-enzyme pyruvate dehydrogenase complex (PDC) links glycolysis to the citric acid cycle by converting pyruvate into acetyl-coenzyme A and is essential for cellular energy metabolism. Although individual components have been characterized, the structure of intact PDC remains unclear, hampering our understanding of its composition and multi-step catalytic reactions. Here, we report the in-situ architecture of intact PDC within mammalian mitochondria by cryo-electron tomography. The organization of peripheral E1 and E3 multimers varies substantially among PDCs. We discovered 1-46 E1 heterotetramers surrounding each PDC core, and up to 12 E3 dimers locating primarily along the pentagonal openings. Furthermore, we observed dynamic interactions of the substrate translocating lipoyl domains (LDs) with both E1 and E2, implicating the mechanism of substrate channeling. Our findings reveal the intrinsic dynamics of PDC peripheral compositions, suggesting an additional model in which the number of assembled E1 heterotetramer, E3 dimer, and functional LDs of PDC may be regulated to further control its catalytic activity.

cell biology↗

bioRxiv2024

In vivo affinity maturation of the HIV-1 Env-binding domain of CD4

Many human proteins have been repurposed as biologics for clinical use. These proteins have been engineered with in vitro techniques that improve affinity for their ligands. However, these approaches do not select against properties that impair efficacy such as protease sensitivity or self-reactivity. Here we engineer the B-cell receptor of primary murine B cells to express a human protein biologic without disrupting their ability to affinity mature. Specifically, CD4 domains 1 and 2 (D1D2) of a half-life enhanced-HIV-1 entry inhibitor CD4-Ig (CD4-Ig-v0) were introduced into the heavy-chain loci of murine B cells, which were then adoptively transferred to wild-type mice. After immunization, transferred B cells proliferated, class switched, affinity matured, and efficiently produced D1D2-presenting antibodies. Somatic hypermutations found in the D1D2-encoding region of engrafted B cells improved binding affinity of CD4-Ig-v0 for the HIV-1 envelope glycoprotein (Env) and the neutralization potency of CD4-Ig-v0 by more than ten-fold across a global panel of HIV-1 isolates, without impairing its pharmacokinetic properties. Thus, affinity maturation of non-antibody protein biologics in vivo can guide development of more effective therapeutics.

bioengineering↗

bioRxiv2024

Charting the Spatial Transcriptome of the Human Cerebral Cortex at Single-Cell Resolution

In our pursuit of creating a comprehensive human cortical atlas to understand human intelligence, we examined the single-nuclei transcriptomes of 307,738 cells alongside spatial transcriptomics data from 46,948 VISIUM spots and 1,355,582 Stereo cells. Atlases reveal distinct expression patterns and spatial arrangements of cortical neural cell types. Glutamatergic neurons exhibit precise laminar patterns, often mirroring expression patterns in adjacent cortical regions. Overlaying our atlas with functional networks delineated substantial correlations between neural cell types and cortical region function. Notably, regions involved in processing sensory information (pain) display a pronounced accumulation of extratelencephalic neurons. Additionally, our atlas enabled precise localization of the thicker layer 4 of the visual cortex and an in-depth study of the stabilize the subplate structure, known as layer 6b, revealed specific marker genes and cellular compositions. Collectively, our research sheds light on the cellular foundations of the intricate and intelligent regions within the human cortex.

neuroscience↗

bioRxiv2024

Mechanism of bacterial predation via ixotrophy

Predation allows bacteria to access alternative substrates in low-nutrient conditions. Ixotrophy has been proposed as a predatory lifestyle of multicellular filamentous bacteria in aquatic environments; however, the molecular mechanism remains unknown. Here we uncover by a multidisciplinary approach that ixotrophy requires the interplay of multiple cellular machineries and a regulatory mechanism. Attacker-prey contacts are established by gliding motility and extracellular grappling hook-like structures that bind prey flagella. Cryo-electron microscopy identifies the grappling hooks as a heptameric assembly of a Type 9 Secretion System substrate. Cryo-electron tomography and functional assays show that killing is mediated by puncturing of the prey cell using a Type 6 Secretion System, possibly triggered by extracellular antennae. Single-cell analyses with stable isotope-labeled prey demonstrate that prey components are taken up by the attacker. Depending on nutrient availability, ixotrophy is switched off by endogenous Insertion Sequence Elements and re-activated through their excision. A marine metagenomic time series provides evidence for coupled dynamics of ixotrophic bacteria and their prey. Our study reveals the complex mechanism of a conserved microbial predatory lifestyle and indicates the need for its regulation in conditions where the expression of costly pathways is dispensable.

microbiology↗

bioRxiv2024

Control of Munc13-1 Activity by Autoinhibitory Interactions Involving the Variable N-terminal Region

Regulation of neurotransmitter release during presynaptic plasticity underlies varied forms of information processing in the brain. Munc13s play essential roles in release via their conserved C-terminal region, which contains a MUN domain involved SNARE complex assembly, and control multiple presynaptic plasticity processes. Munc13s also have a variable N-terminal region, which in Munc13-1 includes a calmodulin binding (CaMb) domain involved in short-term plasticity and a C2A domain that forms an inhibitory homodimer. The C2A domain is activated by forming a heterodimer with the zinc-finger domain of RIMs, providing a link to RIM-dependent short- and long-term plasticity. However, it is unknown how the functions of the N- and C-terminal regions are integrated, in part because of the difficulty of purifying Munc13-1 fragments containing both regions. We describe for the first time the purification of a Munc13-1 fragment spanning its entire sequence except for a flexible region between the C2A and CaMb domains. We show that this fragment is much less active than the Munc13-1 C-terminal region in liposome fusion assays and that its activity is strongly enhanced by the RIM2 zinc-finger domain together with calmodulin. NMR experiments show that the C2A and CaMb domains bind to the MUN domain and that these interactions are relieved by the RIM2 ZF domain and calmodulin, respectively. These results suggest a model whereby Munc13-1 activity in promoting SNARE complex assembly and neurotransmitter release are inhibited by interactions of the C2A and CaMb domains with the MUN domain that are relieved by RIMs and calmodulin.

biophysics↗

bioRxiv2024

Alzheimer's disease-linked risk alleles elevate microglial cGAS-associated senescence and neurodegeneration in a tauopathy model

The strongest risk factors for Alzheimers disease (AD) include the {chi}4 allele of apolipoprotein E (APOE), the R47H variant of triggering receptor expressed on myeloid cells 2 (TREM2), and female sex. Here, we combine APOE4 and TREM2R47H (R47H) in female P301S tauopathy mice to identify the pathways activated when AD risk is the strongest, thereby highlighting disease-causing mechanisms. We find that the R47H variant induces neurodegeneration in female APOE4 mice without impacting hippocampal tau load. The combination of APOE4 and R47H amplified tauopathy-induced cell-autonomous microglial cGAS-STING signaling and type-I interferon response, and interferon signaling converged across glial cell types in the hippocampus. APOE4-R47H microglia displayed cGAS- and BAX-dependent upregulation of senescence, showing association between neurotoxic signatures and implicating mitochondrial permeabilization in pathogenesis. By uncovering pathways enhanced by the strongest AD risk factors, our study points to cGAS-STING signaling and associated microglial senescence as potential drivers of AD risk.

neuroscience↗

bioRxiv2024

Development and optimization of a diluted whole blood ELISpot assay to test immune function

BackgroundSepsis remains a leading cause of death worldwide with no proven immunomodulatory therapies. Stratifying Patient Immune Endotypes in Sepsis ( SPIES) is a prospective, multicenter observational study testing the utility of ELISpot as a functional bioassay specifically measuring cytokine-producing cells after stimulation to identify the immunosuppressed endotype, predict clinical outcomes in septic patients, and test potential immune stimulants for clinical development. Most ELISpot protocols call for the isolation of PBMC prior to their inclusion in the assay. In contrast, we developed a diluted whole blood (DWB) ELISpot protocol that has been validated across multiple laboratories. MethodsHeparinized whole blood was collected from healthy donors and septic patients and tested under different stimulation conditions to evaluate the impact of blood dilution, stimulant concentration, blood storage, and length of stimulation on ex vivo IFN{gamma} and TNF production as measured by ELISpot. ResultsWe demonstrate a dynamic range of whole blood dilutions that give a robust ex vivo cytokine response to stimuli. Additionally, a wide range of stimulant concentrations can be utilized to induce cytokine production. Further modifications demonstrate anticoagulated whole blood can be stored up to 24 hours at room temperature without losing significant functionality. Finally, we show ex vivo stimulation can be as brief as 4 hours allowing for a substantial decrease in processing time. ConclusionsThe data demonstrate the feasibility of using ELISpot to measure the functional capacity of cells within DWB under a variety of stimulation conditions to inform clinicians on the extent of immune dysregulation in septic patients.

immunology↗

bioRxiv2024

Noncaloric monosaccharides induce excessive sprouting angiogenesis in zebrafish via foxo1a-marcksl1a signal

Artificially sweetened beverages containing noncaloric monosaccharides were suggested as healthier alternatives to sugar-sweetened beverages. Nevertheless, the potential detrimental effects of these noncaloric monosaccharides on blood vessel function remain inadequately understood. We have established a zebrafish model that exhibits significant excessive angiogenesis induced by high glucose, resembling the hyperangiogenic characteristics observed in proliferative diabetic retinopathy (PDR). Utilizing this model, we observed that glucose and noncaloric monosaccharides could induce excessive formation of blood vessels, especially intersegmental vessels (ISVs). The excessively branched vessels were observed to be formed by ectopic activation of quiescent endothelial cells (ECs) into tip cells. Single-cell transcriptomic sequencing analysis of the endothelial cells in the embryos exposed to high glucose revealed an augmented ratio of capillary ECs, proliferating ECs, and a series of upregulated proangiogenic genes. Further analysis and experiments validated that reduced foxo1a mediated the excessive angiogenesis induced by monosaccharides via up-regulating the expression of marcksl1a. This study has provided new evidence showing the negative effects of noncaloric monosaccharides on the vascular system and the underlying mechanisms.

pharmacology and toxicology↗

bioRxiv2024

Binding profiles for 954 Drosophila and C. elegans transcription factors revealtissue specific regulatory relationships

A catalog of transcription factor (TF) binding sites in the genome is critical for deciphering regulatory relationships. Here we present the culmination of the modERN (model organism Encyclopedia of Regulatory Networks) consortium that systematically assayed TF binding events in vivo in two major model organisms, Drosophila melanogaster (fly) and Caenorhabditis elegans (worm). We describe key features of these datasets, comprising 604 TFs identifying 3.6M sites in the fly and 350 TFs identifying 0.9 M sites in the worm. Applying a machine learning model to these data identifies sets of TFs with a prominent role in promoting target gene expression in specific cell types. TF binding data are available through the ENCODE Data Coordinating Center and at https://epic.gs.washington.edu/modERNresource, which provides access to processed and summary data, as well as widgets to probe cell type-specific TF-target relationships. These data are a rich resource that should fuel investigations into TF function during development.

genomics↗

bioRxiv2024

A basally active cGAS-STING pathway limits SARS-CoV-2 replication in a subset of ACE2 positive airway cell models

Host factors that define the cellular tropism of SARS-CoV-2 beyond the cognate ACE2 receptor are poorly defined. Here we report that SARS-CoV-2 replication is restricted at a post-entry step in a number of ACE2-positive airway-derived cell lines due to tonic activation of the cGAS-STING pathway mediated by mitochondrial DNA leakage and naturally occurring cGAS and STING variants. Genetic and pharmacological inhibition of the cGAS-STING and type I/III IFN pathways as well as ACE2 overexpression overcome these blocks. SARS-CoV-2 replication in STING knockout cell lines and primary airway cultures induces ISG expression but only in uninfected bystander cells, demonstrating efficient antagonism of the type I/III IFN-pathway in productively infected cells. Pharmacological inhibition of STING in primary airway cells enhances SARS-CoV-2 replication and reduces virus-induced innate immune activation. Together, our study highlights that tonic activation of the cGAS-STING and IFN pathways can impact SARS-CoV-2 cellular tropism in a manner dependent on ACE2 expression levels.

microbiology↗

bioRxiv2024

The J-Like Protein ARC6 Regulates Chloroplast FtsZ-Ring Assembly through Fine-tuning ARC3 Activity in Arabidopsis

Chloroplast division is initiated by the establishment of the stromal FtsZ ring (Z ring). Assembly and positioning of the Z ring are governed by the chloroplast Min system, which inhibits Z-ring formation everywhere but the middle of the chloroplast. ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), the core component of this system, is a direct inhibitor of Z-ring assembly. Regulation of ARC3 activity is vital thus for chloroplast division. Here, we report that ARC6, which localizes on the chloroplast inner envelope membrane, interacts with ARC3 and acts upstream of ARC3 during chloroplast division. We show that the C-terminal MORN domain of ARC3, demonstrated previously to prevent ARC3-FtsZ interaction, binds to the J-like domain (JLD) of ARC6, enabling full-length ARC3 to interact with FtsZ proteins and activating the inhibitory activity of ARC3 on the assembly of FtsZ filaments. Overexpression of a JLD-deleted version of ARC6 causes disruption of Z-ring formation in an ARC3-dependent manner. Finally, we reveal that ARC6 recruits ARC3 to the middle of the chloroplast. Our findings suggest a model whereby ARC6 regulates the assembly and positioning of the Z ring through fine-tuning the inhibitory activity of ARC3 at the chloroplast division site. One Sentence SummaryThe chloroplast membrane protein ARC6 recruits ARC3 to the chloroplast division site and regulates the assembly of the FtsZ ring by fine-tuning ARC3 activity through its J-like domain. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plcell/pages/General-Instructions) is: Cheng Chen (cgchen@sjtu.edu.cn).

plant biology↗

bioRxiv2024

Downstream-of-gene (DoG) transcripts contribute to an imbalance in the cancer cell transcriptome

Downstream-of-gene (DoG) transcripts are an emerging class of noncoding RNAs. However, it remains largely unknown how DoG RNA production is regulated and whether alterations in DoG RNA signatures exist in major cancers. Here, through transcriptomic analyses of matched tumors and non-neoplastic tissues and cancer cell lines, we reveal a comprehensive catalogue of DoG RNA signatures. Through separate lines of evidence, we support the biological importance of DoG RNAs in carcinogenesis. First, we reveal DoG RNAs are tissue-specific and differentially expressed in tumors versus paired normal tissues with their respective host genes involved in tumor promoting versus tumor suppressor pathways. Second, increased DoG RNA number and length is associated with poor patient prognosis. Third, depletion of essential enzyme Topoisomerase I in colon cancer alters RNA polymerase II chromatin engagement leading to termination defects and induction of DoG RNAs. Our results underlie the significance of DoG RNAs in diversifying the cancer transcriptome.

genomics↗

bioRxiv2023

Non-equilibrium distribution of kinesin-streptavidin complexes revealed by Mass Photometry

Kinesin-streptavidin complexes are widely used in microtubule-based active-matter studies. The stoichiometry of the complexes is empirically tuned but experimentally challenging to determine. Here, mass photometry measurements reveal heterogenous distributions of kinesin-streptavidin complexes. Our binding model indicates that heterogeneity arises from both the kinesin-streptavidin mixing ratio and the kinesin-biotinylation efficiency.

biophysics↗

bioRxiv2023

Modulation of the spatiotemporal dynamics of striatal direct pathway neurons and motor output by mGluR5

Striatal spiny-projection neurons (SPNs) integrate glutamatergic inputs from the motor cortex and thalamus with neuromodulatory signals to regulate motor output. In vivo Ca2+ imaging has demonstrated that spatially overlapping ensembles of direct and indirect pathway SPNs (dSPNs, iSPNs) are co-active during spontaneous movement. This co-activity is statistically greater among nearby neurons, correlates with behavioral state, and changes in an SPN-type-specific manner under pathological conditions. How this co-activity arises and whether is important for movement are not well understood. Co-activity likely arises from shared excitatory inputs, the strength of which are scaled by well-characterized mechanisms of synaptic plasticity. Here, we show that the Group I metabotropic glutamate receptor 5 (mGluR5), which regulates the strength of corticostriatal synapses, is a key mediator of behaviorally relevant SPN co-activity. Pharmacological modulation of mGluR5 signaling bidirectionally altered movement and co-activity, but not the absolute level of activity in dSPNs. Targeted deletion of mGluR5 in dSPNs recapitulated the effects on spatiotemporal neural dynamics and movement, consistent with a striatum-specific effect of mGluR5 modulation. Targeted deletion of mGluR5 also produced changes in the synaptic properties of dSPNs. Separate from any effects on overall activity, our results show that excitatory synaptic modulation influences motor function by coordinating the spatial co-activation of dSPNs in vivo.

neuroscience↗

bioRxiv2023

Evolution of resistance to KRASG12C inhibitor in a non-small cell lung cancer responder

Despite initial therapeutic successes, most patients with non-small cell lung cancer (NSCLC) who carry the KRASG12C mutation ultimately exhibit resistance to targeted treatments. To improve our comprehension of how acquired resistance develops, we present an unprecedented longitudinal case study profiling the transcriptome of peripheral blood mononuclear cells (PBMCs) over 5 months from an NSCLC patient with the KRASG12C mutation and initial response to sotorasib followed by resistance and death. Single-cell RNA sequencing analysis uncovered notable fluctuations in immune cell populations throughout treatment with sotorasib. Specifically, we observed a decline in circulating CD8+CD161hi T cells correlating with periods of therapeutic response, followed by a resurgence during phases of nonresponse. This study established a high-resolution atlas detailing the evolutionary trajectory of resistance to sotorasib and characterizes a CD8+CD161hi T cells population in KRASG12C mutation patient.

cancer biology↗

bioRxiv2023

Structural elucidation of how ARF small GTPases induce membrane tubulation for vesicle fission

The ADP-Ribosylation Factor (ARF) small GTPases have been found to act in vesicle fission through a direct ability to tubulate membrane. Here, we have used cryo-electron microscopy (EM) to solve the structure of an ARF6 protein lattice assembled on tubulated membrane to 3.9 [A] resolution. ARF6 forms tetramers that polymerize into helical arrays to form this lattice. We identify, and confirm functionally, protein contacts critical for this lattice formation. The solved structure also suggests how the ARF amphipathic helix is positioned in the lattice for membrane insertion, and how a GTPase-activating protein (GAP) docks onto the lattice to catalyze ARF-GTP hydrolysis in completing membrane fission. As ARF1 and ARF6 are structurally conserved, we have also modeled ARF1 onto the ARF6 lattice, which has allowed us to pursue the reconstitution of Coat Protein I (COPI) vesicles to confirm more definitively that the ARF lattice acts in vesicle fission. Our findings are notable for having achieved the first detailed glimpse of how a small GTPase bends membrane and having provided a molecular understanding of how an ARF protein acts in vesicle fission.

cell biology↗

bioRxiv2023

Ferritinophagy is a Druggable Vulnerability of Quiescent Leukemic Stem Cells

Acute myeloid leukemia (AML) remains a challenging hematological malignancy with poor prognosis and limited treatment options. Leukemic stem cells (LSCs) contributes to therapeutic failure, post-therapy relapse and adverse outcome. Here, we investigated the role of quiescence and its associated molecular mechanisms in AML pathogenesis and LSCs functions, and identified potential vulnerabilities for therapeutic intervention. We found that LSC-enriched quiescent cell population exhibited a distinct gene set of prognostic significance in AML patients. Furthermore, this quiescent cells subset displayed heightened autophagic activity with a reliance on ferritinophagy, a selective form of autophagy mediated by Nuclear Receptor Coactivator 4 (NCOA4) regulating iron bioavailability. Inhibition of NCOA4 genetically or chemically showed potent anti-leukemic effects, particularly targeting the LSC compartment. These findings uncover that ferritinophagy inhibition may represent a promising therapeutic strategy for patients with AML. One Sentence SummaryTargeting quiescent leukemic stem cells via NCOA4-dependent ferritinophagy inhibition may improve therapeutic outcomes in acute myeloid leukemia.

cancer biology↗

bioRxiv2023

The α/β hydrolase domain-containing protein 1 (ABHD1) acts as a lysolipid lipase and is involved in lipid droplet formation

Lipid droplets (LDs) are the major sites of lipid and energy homeostasis. However, few LD biogenesis proteins have been identified. Here, using Chlamydomonas as a model, we show that ABHD1, a member of the /{beta} hydrolase domain-containing protein family, is a novel type of LD-associated protein which stimulates LD formation through two distinct actions on the LD surface, one enzymatic and the other structural. ABHD1 was localized to LD surface in Chlamydomonas cells. The knockout mutants contained similar amounts of triacylglycerols (TAG) but their LDs showed an increased content in lyso- derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS). Over-expression of ABHD1 in Chlamydomonas induced LD formation and boosted TAG content, suggesting a key role in LD biogenesis. The purified recombinant ABHD1 protein hydrolyzed lyso-DGTS, producing a free fatty acid and a glyceryltrimethylhomoserine moiety. In vitro experiments using droplet- embedded vesicles showed that ABHD1 promoted LD emergence. Taken together, these results identify ABHD1 as a new player in LD formation by its lipase activity on lyso-DGTS and by its distinct biophysical property. This study further suggests that lipases targeted to LDs and able to act on their polar lipid coat may be interesting tools to promote LD assembly in eukaryotic cells. Significant statementLipid droplets are subcellular organelles specialized for triacylglycerol storage. Their dynamic turnover is key to managing energy homeostasis in response to cell cycle states and environmental cues. To gain insights into LD biogenesis, we characterized a putative /{beta}- hydrolase (ABHD1) in the model algae Chlamydomonas reinhardtii and show it is located at the LD surface. We found that ABHD1 overexpression promotes LD formation and acts as a lipase mainly on lyso derivatives of the betaine lipid diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), the major lipid constituent of the LD hemi-membrane. We also show that ABHD1 has a remarkable biophysical property favoring LD budding. This work thus identifies a novel type of lipase acting on betaine lipid and provides a first example of a protein with a dual function nvolved in LD formation.

biochemistry↗