Biology subjects

Yin, Y.

Publications and source records attributed to Yin, Y..

At least 127 records · Page 7Linked to original sources

bioRxiv2023

Imaging Mass Cytometry-Driven Spatial Analysis and Biomarker Discovery in Hodgkin Lymphoma

The biology of tumors is suffused with spatial interactions, such as tumor-immune signaling through localized cytokine/ligand secretion, cell-cell contacts, and checkpoint ligand/receptor signaling. Hodgkin Lymphoma (HL) can serve as a study paradigm for tumor microenvironment (TME) architecture as the defining pathological feature is the scarcity of the malignant Hodgkin and Reed Sternberg (HRS) cells, leaving a diverse and predominantly immune cell rich tumor microenvironment (TME) with complex tumor-immune interactions. Previous studies have identified TME features that are prognostic and predictive, however these studies did not consider the entirety of TME cellular ecosystems, including precisely defined immune cell subsets with opposing inflammatory and immune-suppressive effects, as a determinant for differential clinical course of HL patients. Here we use Imaging Mass Cytometry (IMC) with 42 antibody markers to profile tumors from 93 patients with HL. Our cohort consists of relapsed/refractory HL with matched diagnostic and relapsed biopsies, and we present a bioinformatic pipeline to profile 10 major cell lineages and their subtypes including spatial interaction mapping. Our pipeline identifies putative biomarker candidates with a focus on "rosettes" - local aggregates of immune cells around single tumor cells. In addition to validating existing biomarkers centered on CD68+ macrophages, GranzymeB+CD8+ T cells, and others in HL, we propose new biomarkers based on localized interactions between HRS cells and aggregating CD4+ and CD8+ T cells and macrophages involving the immune checkpoints PD1/PDL1, LAG3, and Galectin9. This study serves as a broad tissue imaging resource for multi-timepoint biopsies in HL, and a computational resource and pipeline for users of IMC and other multiplexed imaging studies to perform tissue analysis and biomarker candidate testing with any tissue type.

cancer biology↗

bioRxiv2023

The spatially resolved tumor microenvironment predicts treatment outcome in relapsed/refractory Hodgkin lymphoma

PURPOSEAbout a third of relapsed or refractory classic Hodgkin lymphoma (r/r CHL) patients succumb to their disease after high-dose chemotherapy followed by autologous stem cell transplantation (HDC/ASCT). Here, we aimed to describe spatially resolved tumor microenvironment (TME) ecosystems to establish novel biomarkers associated with treatment failure in r/r CHL. METHODSWe performed imaging mass cytometry (IMC) on 169 paired primary diagnostic and relapse biopsies using a marker panel specific for CHL biology. For each cell type in the TME, we calculated a spatial score measuring the distance of nearest neighbor cells to the malignant Hodgkin Reed Sternberg cells within close interaction range. Spatial scores were used as features in prognostic model development for post-ASCT outcomes. RESULTSHighly multiplexed IMC data revealed shared TME patterns in paired diagnostic and early relapse/refractory CHL samples, whereas TME patterns were more divergent in pairs of diagnostic and late relapse samples. Integrated analysis of IMC and single cell RNA sequencing data identified unique architecture defined by CXCR5+ HRS cells and their strong spatial relationship with CXCL13+ macrophages in the TME. We developed a prognostic assay ( RHL4S) using four spatially resolved parameters, CXCR5+ HRS cells, PD1+CD4+ T cells, tumor-associated macrophages, and CXCR5+ B cells, which effectively separated patients into high-risk vs low-risk groups with significantly different post-ASCT outcomes. The RHL4S assay was validated in an independent r/r CHL cohort using a multicolor immunofluorescence assay. CONCLUSIONSWe identified the interaction of CXCR5+ HRS cells with ligand-expressing CXCL13+ macrophages as a prominent crosstalk axis in relapsed CHL. Harnessing this TME biology, we developed a novel prognostic model applicable to r/r CHL biopsies, RHL4S, opening new avenues for spatial biomarker development.

cancer biology↗

bioRxiv2023

Deciphering molecular heterogeneity and dynamics of neural stem cells in human hippocampal development, aging, and injury

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are re-activated following stroke injury. Together, our findings provide valuable insights into the development, aging, and re-activation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

neuroscience↗

bioRxiv2023

Codon affinity in mitochondrial DNA shapes evolutionary and somatic fitness

Summary ParagraphSomatic variation contributes to biological heterogeneity by modulating cellular proclivity to differentiate, expand, adapt, or die. While large-scale sequencing efforts have revealed the foundational role of somatic variants to drive human tumor evolution, our understanding of the contribution of mutations to modulate cellular fitness in non-malignant contexts remains understudied. Here, we identify a mosaic synonymous variant (m.7076A>G) in the mitochondrial DNA (mtDNA) encoded cytochrome c-oxidase subunit 1 gene (MT-CO1, p.Gly391=), which was present at homoplasmy in 47% of immune cells from a healthy donor. Using single-cell multi-omics, we discover highly specific selection against the m.7076G mutant allele in the CD8+ effector memory T cell compartment in vivo, reminiscent of selection observed for pathogenic mtDNA alleles1, 2 and indicative of lineage-specific metabolic requirements. While the wildtype m.7076A allele is translated via Watson-Crick-Franklin base-pairing, the anticodon diversity of the mitochondrial transfer RNA pool is limited, requiring wobble-dependent translation of the m.7076G mutant allele. Notably, mitochondrial ribosome profiling revealed altered codon-anticodon affinity at the wobble position as evidenced by stalled translation of the synonymous m.7076G mutant allele encoding for glycine. Generalizing this observation, we provide a new ontogeny of the 8,482 synonymous variants in the human mitochondrial genome that enables interpretation of functional mtDNA variation. Specifically, via inter- and intra-species evolutionary analyses, population-level complex trait associations, and the occurrence of germline and somatic mtDNA mutations from large-scale sequencing studies, we demonstrate that synonymous variation impacting codon:anticodon affinity is actively evolving across the entire mitochondrial genome and has broad functional and phenotypic effects. In summary, our results introduce a new ontogeny for mitochondrial genetic variation and support a model where organismal principles can be discerned from somatic evolution via single-cell genomics.

genetics↗

bioRxiv2023

Colocalization of Protein and microRNA Markers Reveals Unique Extracellular Vesicle Sub-Populations for Early Cancer Detection

Extracellular vesicles (EVs) play important roles in cell-cell communication but they are highly heterogeneous, and each vesicle has dimensions smaller than 200 nm thus encapsulates very limited amounts of cargos. We report the technique of NanOstirBar (NOB)-EnabLed Single Particle Analysis (NOBEL-SPA) that utilizes NOBs, which are superparamagnetic nanorods easily handled by a magnet or a rotating magnetic field, to act as isolated "islands" for EV immobilization and cargo confinement. NOBEL-SPA permits rapid inspection of single EV with high confidence by confocal fluorescence microscopy, and can assess the colocalization of selected protein/microRNA (miRNA) pairs in the EVs produced by various cell lines or present in clinical sera samples. Specific EV sub-populations marked by the colocalization of unique protein and miRNA combinations have been revealed by the present work, which can differentiate the EVs by their cells or origin, as well as to detect early-stage breast cancer (BC). We believe NOBEL-SPA can be expanded to analyze the co-localization of other types of cargo molecules, and will be a powerful tool to study EV cargo loading and functions under different physiological conditions, and help discover distinct EV subgroups valuable in clinical examination and therapeutics development.

cancer biology↗

bioRxiv2023

Discovery and Validation of RUNX1 DNA Methylation in Differentiating Papillary Thyroid Cancer from Benign Nodules

Although most thyroid nodules can be diagnosed preoperatively by thyroid ultrasonography and fine-needle aspiration biopsy, it remains a challenge to accurately identify malignancy of thyroid nodules when the biopsy is indeterminate. This study aims to explore a novel biomarker to distinguish benign and malignant thyroid nodules. Tissue samples from patients with Stage I&II papillary thyroid carcinoma (PTC) and benign thyroid nodules (BTN) were collected for genome profiling by methylation EPIC 850K array and RNA-Sequencing. Genes with significantly differential DNA methylation and inverse mRNA expression were filtered out. The altered methylation of RUNX1 gene was validated in two independent case-control studies with a total of 699 formalin fixed paraffin-embedded (FFPE) samples using mass spectrometry and calculated by binary logistic regression analysis. Hypomethylation of RUNX1 gene in PTC patients compared to BTN subjects was verified in Validation (140 PTC vs. 189 BTN, ORs [≥] 1.50 per-10% methylation, P [≤] 4.40E-05, for all measurable CpG sites) and Validation (184 PTC vs. 186 BTN, ORs [≥] 1.72 per-10% methylation, P [≤] 2.38E-11, for all measurable CpG sites). Besides, RUNX1 methylation achieved good accuracy in differentiating early-stage PTC from BTN in Validation (AUC: 0.74) and Validation (AUC: 0.79). Gender- and age-stratified analysis revealed RUNX1 hypomethylation as an important risk factor for thyroid disease in younger women. We disclosed a significant association between RUNX1 hypomethylation and PTC, suggesting RUNX1 methylation based on FFPE tissue samples as a potential biomarker for predicting malignancy of thyroid nodules.

genetics↗

bioRxiv2023

NULISA: a novel proteomic liquid biopsy platform with attomolar sensitivity and high multiplexing

The blood proteome holds great promise for precision medicine but poses substantial challenges due to the low abundance of most plasma proteins and the vast dynamic range across the proteome. We report a novel proteomic technology - NUcleic acid Linked Immuno-Sandwich Assay (NULISA) - that incorporates a dual capture and release mechanism to suppress the assay background and improves the sensitivity of the proximity ligation assay by over 10,000-fold to the attomolar level. It utilizes pairs of antibodies conjugated to DNA oligonucleotides that enable immunocomplex purification and generate reporter DNA containing target- and sample-specific barcodes for a next-generation sequencing-based, highly multiplexed readout. A 200-plex NULISA targeting 124 cytokines and chemokines and 80 other immune response-related proteins demonstrated superior sensitivity for detecting low-abundance proteins and high concordance with other immunoassays. The ultrahigh sensitivity allowed the detection of previously difficult-to-detect, but biologically important, low-abundance biomarkers in patients with autoimmune diseases and COVID-19. Fully automated NULISA addresses longstanding challenges in proteomic analysis of liquid biopsies and makes broad and in-depth proteomic analysis accessible to the general research community and future diagnostic applications.

genomics↗

bioRxiv2023

Homeobox Transcription Factor HbxA Influences Expression of over One ThousandGenes in the Model Fungus Aspergillus nidulans

In fungi, conserved homeobox-domain (HD) proteins are transcriptional regulators governing development. In Aspergillus species, several HD transcription factor genes have been identified, among them, hbxA/hbx1. For instance, in the opportunistic human pathogen Aspergillus fumigatus, hbxA is involved in conidial production and germination, as well as virulence and secondary metabolism (SM), including production of fumigaclavines, fumiquinazolines, and chaetominine. In the agriculturally important fungus Aspergillus flavus, disruption of hbx1 results in fluffy aconidial colonies unable to produce sclerotia. hbx1 also regulates production of aflatoxins, cyclopiazonic acid and aflatrem. Furthermore, transcriptome studies revealed that hbx1 has a broad effect on the A. flavus genome, including numerous genes involved in SM. These studies underline the importance of the HbxA/Hbx1 regulator, not only in developmental processes but also in the biosynthesis of a broad number of fungal natural products, including potential medical drugs and mycotoxins. To gain further insight into the regulatory scope of HbxA in Aspergilli, we studied its role in the model fungus Aspergillus nidulans. Our present study of the A. nidulans hbxA-dependent transcriptome revealed that more than one thousand genes are differentially expressed when this regulator was not transcribed at wild-type levels, among them numerous transcription factors, including those involved in development as well as in SM regulation. Furthermore, our metabolomics analyses revealed that production of several secondary metabolites, some of them associated with A. nidulans hbxA-dependent gene clusters, was also altered in deletion and overexpression hbxA strains compared to the wild type, including synthesis of nidulanins A, B and D, versicolorin A, sterigmatocystin, austinol, dehydroaustinol, and three unknown novel compounds.

genetics↗

bioRxiv2023

Broadly effective ACE2 decoy proteins protect mice from lethal SARS-CoV-2 infection

As SARS-CoV-2 variants have been causing increasingly serious drug resistance problem, development of broadly effective and hard-to-escape anti-SARS-CoV-2 agents is in urgent need. Here we describe further development and characterization of two SARS-CoV-2 receptor decoy proteins, ACE2-Ig-95 and ACE2-Ig-105/106. We found that both proteins had potent and robust in vitro neutralization activities against diverse SARS-CoV-2 variants including Omicron, with an average IC50 of up to 37 pM. In a stringent lethal SARS-CoV-2 infection mouse model, both proteins lowered lung viral load by up to [~]1000 fold, prevented the emergence of clinical signs in >75% animals, and increased animal survival rate from 0% (untreated) to >87.5% (treated). These results demonstrate that both proteins are good drug candidates for protecting animals from severe COVID-19. In a head-to-head comparison of these two proteins with five previously-described ACE2-Ig constructs, we found that two of these constructs, each carrying five surface mutations in the ACE2 region, had partial loss of neutralization potency against three SARS-CoV-2 variants. These data suggest that extensively mutating ACE2 residues near the RBD-binding interface should be avoided or performed with extra caution. Further, we found that both ACE2-Ig-95 and ACE2-Ig-105/106 could be produced to gram/liter level, demonstrating the developability of them as biologic drug candidates. Stress-condition stability test of them further suggests that more studies are required in the future to improve the stability of these proteins. These studies provide useful insight into critical factors for engineering and preclinical development of ACE2 decoys as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. Abstract ImportanceEngineering soluble ACE2 proteins that function as a receptor decoy to block SARS-CoV-2 infection is a very attractive approach to broadly effective and hard-to-escape anti-SARS-CoV-2 agents. This study here describes development of two antibody-like soluble ACE2 proteins that broadly block diverse SARS-CoV-2 variants including Omicron. In a stringent COVID-19 mouse model, both proteins successfully protected >87.5% animals from lethal SARS-CoV-2 infection. In addition, a head-to-head comparison of the two constructs developed in this study with five previously-described ACE2 decoy constructs were performed here. Two previously-described constructs with relatively more ACE2-surface mutations were found with less robust neutralization activities against diverse SARS-CoV-2 variants. Further, the developability of the two proteins as biologic drug candidates was also assessed here. This study provides two broadly anti-SARS-CoV-2 drug candidates and useful insight into critical factors for engineering and preclinical development of ACE2 decoy as broadly effective therapeutics against diverse ACE2-utilizing coronaviruses. TweetTwo antibody-like ACE2 decoy proteins could block diverse SARS-CoV-2 variants and prevent animals from severe COVID-19.

microbiology↗

bioRxiv2023

Decontamination of ambient and margin noise in droplet-based single cell protein expression data with DecontPro

Assays such as CITE-seq can measure the abundance of cell surface proteins on individual cells using antibody derived tags (ADTs). However, many ADTs have high levels of background noise that can obfuscate down-stream analyses. Using an exploratory analysis of PBMC datasets, we find that some droplets that were originally called "empty" due to low levels of RNA contained high levels of ADTs and likely corresponded to neutrophils. We identified a novel type of artifact in the empty droplets called a "spongelet" which has medium levels of ADT expression and is distinct from ambient noise. ADT expression levels in the spongelets correlate to ADT expression levels in the background peak of true cells in several datasets suggesting that they can contribute to background noise along with ambient ADTs. We then developed DecontPro, a novel Bayesian hierarchical model that can decontaminate ADT data by estimating and removing contamination from these sources. DecontPro outperforms other decontamination tools in removing aberrantly expressed ADTs while retaining native ADTs and in improving clustering specificity. Overall, these results suggest that identification of empty drops should be performed separately for RNA and ADT data and that DecontPro can be incorporated into CITE-seq workflows to improve the quality of downstream analyses.

bioinformatics↗

bioRxiv2022

Single-cell multi-omics reveals dynamics of purifying selection of pathogenic mitochondrial DNA across human immune cells

Cells experience intrinsic and extrinsic pressures that affect their proclivity to expand and persist in vivo. In congenital disorders caused by loss-of-function mutations in mitochondrial DNA (mtDNA), metabolic vulnerabilities may result in cell-type specific phenotypes and depletion of pathogenic alleles, contributing to purifying selection. However, the impact of pathogenic mtDNA mutations on the cellular hematopoietic landscape is not well understood. Here, we establish a multi-omics approach to quantify deletions in mtDNA alongside cell state features in single cells derived from Pearson syndrome patients. We resolve the interdependence between pathogenic mtDNA and lineage, including purifying selection against deletions in effector/memory CD8 T-cell populations and recent thymic emigrants and dynamics in other hematopoietic populations. Our mapping of lineage-specific purifying selection dynamics in primary cells from patients carrying pathogenic heteroplasmy provides a new perspective on recurrent clinical phenotypes in mitochondrial disorders, including cancer and infection, with potential broader relevance to age-related immune dysfunction.

genetics↗

bioRxiv2022

Heavy-chain CDR3-engineered B cells facilitate in vivo evaluation of HIV-1 vaccine candidates

V2-glycan/apex broadly neutralizing antibodies (bnAbs) recognize a closed quaternary epitope of the HIV-1 envelope glycoprotein (Env). This closed structure is necessary to elicit apex antibodies and useful to guide maturation of other bnAb classes. To compare antigens designed to maintain this conformation, apex-specific responses were monitored in mice engrafted with a diverse repertoire of B cells expressing the HCDR3 of the apex bnAb VRC26.25. Engineered B cells affinity matured, guiding improvement of VRC26.25 itself. We found that soluble Env (SOSIP) variants differed significantly in their ability to raise anti-apex responses. A transmembrane SOSIP (SOSIP-TM) delivered as an mRNA-lipid nanoparticle elicited more potent neutralizing responses than multimerized SOSIP proteins. Importantly, SOSIP-TM elicited neutralizing sera from B cells engineered with the predicted VRC26.25-HCDR3 progenitor, which also affinity matured. Our data show that HCDR3-edited B cells facilitate efficient in vivo comparisons of Env antigens and highlight the potential of an HCDR3-focused vaccine approach.

immunology↗

bioRxiv2022

Brassinosteroid gene regulatory networks at cellular resolution

Brassinosteroids (BRs) are plant steroid hormones that regulate diverse processes such as cell division and cell elongation. BRs control thousands of genes through gene regulatory networks that vary in space and time. By using time-series single-cell RNA-sequencing to identify BR-responsive gene expression specific to different cell types and developmental stages of the Arabidopsis root, we uncovered the elongating cortex as a site where BRs trigger a shift from proliferation to elongation associated with increased expression of cell wall-related genes. Our analysis revealed HAT7 and GTL1 as BR-responsive transcription factors that regulate cortex cell elongation. These results establish the cortex as an important site for BR-mediated growth and unveil a BR signaling network regulating the transition from proliferation to elongation, illuminating new aspects of spatiotemporal hormone response.

plant biology↗

bioRxiv2022

AcaFinder: genome mining for anti-CRISPR associated genes

Anti-CRISPR (Acr) proteins are encoded by (pro)viruses to inhibit their hosts CRISPR-Cas systems. Genes encoding Acr and Aca (Acr associated) proteins often co-localize to form acr-aca operons. Here, we present AcaFinder as the first Aca genome mining tool. AcaFinder can: (i) predict Acas and their associated acr-aca operons using guilt-by-association (GBA); (ii) identify homologs of known Acas using an HMM (Hidden Markov model) database; (iii) take input genomes for potential prophages, CRISPR-Cas systems, and self-targeting spacers (STSs); and (iv) provide a standalone program (https://github.com/boweny920/AcaFinder) and a web server (http://aca.unl.edu/Aca). AcaFinder was applied to mining over 16,000 prokaryotic and 142,000 gut phage genomes. After a multi-step filtering, 36 high-confident new Aca families were identified, which is three times of the 12 known Aca families. Seven new Aca families were from major human gut bacteria (Bacteroidota, Actinobacteria, Fusobacteria) and their phages, while most known Aca families were from Proteobacteria and Firmicutes. A complex association network between Acrs and Acas was revealed by analyzing their operonic co-localizations. It appears very common in evolution that the same aca genes can recombine with different acr genes and vice versa to form diverse acr-aca operon combinations. ImportanceAt least four bioinformatics programs have been published for genome mining of Acrs since 2020. In contrast, no bioinformatics tools are available for automated Aca discovery. As the self-transcriptional repressor of acr-aca operons, Aca can be viewed as anti-anti-CRISPRs, with a great potential in the improvement of CRISPR-Cas technology. Although all the 12 known Aca proteins contain a conserved Helix-Turn-Helix (HTH) domain, not all HTH-containing proteins are Acas. However, HTH-containing proteins with an adjacent Acr homologs encoded in the same genetic operon are likely Aca proteins. AcaFinder implements this guilt-by-association (GBA) idea and the idea of using HMMs of known Acas for homologs into one software package. Applying AcaFinder in screening prokaryotic and gut phage genomes reveals a complex acr-aca operonic co-localization network between different families of Acrs and Acas.

bioinformatics↗

bioRxiv2022

The gut microbiota promotes liver regeneration through hepatic membrane phospholipid synthesis

Background & AimsHepatocyte growth and proliferation is dependent on the synthesis of membrane phospholipids. Lipid synthesis, in turn, requires short chain fatty acids (SCFA) generated by bacterial fermentation, delivered through the gut- liver axis. We therefore hypothesized that dysbiotic insults like antibiotics treatment not only negatively affect gut microbiota, but also impair hepatic lipid synthesis and liver regeneration. MethodsStable isotope labelling and 70% partial hepatectomy (PHx) was carried out in C57Bl/6J wildtype mice, in mice treated with broad-spectrum antibiotics, in germfree mice and gnotobiotic mice colonized with minimal microbiota. Microbiome was analysed by 16S rRNA gene sequencing and microbial culture. Gut content, liver and blood were tested by lipidomics mass spectrometry, qRT-PCR, immunoblot and immunohistochemistry for expression of proliferative and lipogenic markers. Matched biopsies from hyperplastic and hypoplastic liver tissue of human patients subjected to portal vein embolization were analysed by qRT-PCR for lipogenic enzymes and results were correlated with liver volumetry. ResultsThree days of antibiotics treatment induced persistent dysbiosis with significantly decreased beta-diversity and richness, but massive increase of Proteobacteria, accompanied by decreased colonic SCFA. After PHx, antibiotics- treated mice showed delayed liver regeneration, increased mortality, impaired hepatocyte proliferation and decreased hepatic phospholipid synthesis. Expression of the key lipogenic enzyme SCD1 was upregulated after PHx, but delayed by antibiotics-treatment. Germfree mice essentially recapitulated the phenotype of antibiotics-treatment. Importantly, phospholipid synthesis, hepatocyte proliferation, liver regeneration and survival were rescued in gnotobiotic mice colonized with a minimal SCFA-producing microbial community. SCD1 was required for human hepatoma cell proliferation, and its hepatic expression was associated with liver regeneration and hyperproliferation in human patients. ConclusionGut microbiota are pivotal for hepatic membrane phospholipid synthesis and liver regeneration. Lay SummaryGut microbiota affects the liver lipid metabolism through the gut-liver axis, and microbial metabolites promote liver regeneration. Perturbations of the microbiome, e.g., by antibiotics treatment, impair the production of bacterial metabolites, which serve as building blocks for new membrane lipids in liver cells. As a consequence, hepatocyte growth and proliferation, and ultimately, liver regeneration and survival after liver surgery is impaired. HighlightsO_LIPartial hepatectomy in mice pretreated with antibiotics is associated with impaired hepatocyte proliferation and phospholipid synthesis, delayed liver regeneration and increased mortality C_LIO_LIThe delay in liver regeneration and impaired lipogenesis upon antibiotics treatment is preceded by dysbiosis of gut microbiota, increase of Proteobacteria and decreased short-chain fatty acids in the gut C_LIO_LIPartial hepatectomy in germfree mice essentially phenocopies the detrimental effects of antibiotic treatment C_LIO_LILiver regeneration and mortality, as well as phospholipid synthesis and hepatocyte proliferation in germfree mice are fully rescued upon colonisation with a minimal gut bacterial consortium capable of short-chain fatty acid production C_LIO_LIIn human patients, the intrahepatic expression of lipid synthesis enzymes positively correlates with proliferation and liver regeneration in the clinic C_LIO_LIThus, liver regeneration is affected by composition of gut microbiota C_LIO_LIClinically, pre-operative analysis of the gut microbiome may serve as biomarker to determine the extent of liver resections C_LI

cell biology↗

bioRxiv2022

Pathway and mechanism of tubulin folding mediated by TRiC/CCT conjugated with its ATPase cycle revealed by cryo-EM

The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. Our structural and XL-MS analyses suggested a gradual upward translocation and stabilization of tubulin within the TRiC chamber accompanying TRiC ring closure. Remarkably, in the closed TRiC-tubulin-S3 map resolved to 3.1-[A]-resolution, we captured a near-natively folded tubulin. We found the near-natively folded tubulin engaging through its N and C domains mainly with the A and I domains of the CCT3/6/8 subunits through electrostatic and hydrophilic interactions, while the tubulin I domain was found to remain dynamic. Moreover, we also showed the potential role of TRiC C-terminal tails in substrate stabilization and folding. Our study delineates the pathway and molecular mechanism of TRiC-mediated tubulin folding conjugated with TRiC ATPase cycle, and may also inform the design of therapeutic agents targeting TRiC-tubulin interactions.

biochemistry↗

bioRxiv2022

Structural basis of plp2-mediated cytoskeletal protein folding by TRiC/CCT

The eukaryotic chaperonin TRiC/CCT assists the folding of ~10% cytosolic proteins. The essential cytoskeletal proteins tubulin and actin are the obligate substrates of TRiC and their folding involves cochaperone and co-factors. Here, through cryo-EM analysis, we present a more complete picture of yeast TRiC-assisted tubulin and actin folding in the ATPase-cycle, under the coordination of cochaperone plp2. Our structures revealed that in the open C1 and C2 states, plp2 and substrates tubulin/actin engage with TRiC inside its chamber, one per ring. Noteworthy, we captured a ternary TRiC-plp2-tubulin complex in the closed C3 state, engaged with a full-length {beta}-tubulin in the native folded state even loaded with a GTP, and with a plp2 occupying the opposite ring, not reported before. Another closed C4 state revealed an actin in the intermediate state of folding and a plp2 occupying the other ring. Intriguingly, along with TRiC ring closure, we captured a large translocation of plp2 within TRiC chamber coordinating substrate translocation on the CCT6 hemisphere, potentially facilitating substrate stabilization and folding. Our findings provide structural insights into the folding mechanism of the major cytoskeletal proteins tubulin/actin under the coordination of the complex biogenesis machinery TRiC and plp2, and could extend our understanding on the links between cytoskeletal proteostasis and related human diseases.

biophysics↗

bioRxiv2022

A Caenorhabditis elegans nck-1 and filamentous actin-regulating protein pathway mediates a key cellular defense against bacterial pore-forming proteins

Pore-forming proteins (PFPs) comprise the largest single class of bacterial protein virulence factors and are expressed by many human and animal bacterial pathogens. Cells that are attacked by these virulence factors activate epithelial intrinsic cellular defenses (or INCEDs) to prevent the attendant cellular damage, cellular dysfunction, osmotic lysis, and organismal death. Several conserved PFP INCEDs have been identified using the nematode Caenorhabditis elegans and the nematicidal PFP Cry5B, including mitogen-activated protein kinase (MAPK) signaling pathways. Here we demonstrate that the gene nck-1, which has homologs from Drosophila to humans and links cell signaling with localized F-actin polymerization, is required for INCED against small-pore PFPs in C. elegans. Reduction/loss of nck-1 function results in C. elegans hypersensitivity to PFP attack, a hallmark of a gene required for INCEDs against PFPs. This requirement for nck-1-mediated INCED functions cell-autonomously in the intestine and is specific to PFPs but not other tested stresses. Genetic interaction experiments indicate that nck-1-mediated INCED against PFP attack is independent of the major MAPK PFP INCED pathways. Proteomics and cell biological and genetic studies further indicate that nck-1 functions with F-actin cytoskeleton modifying genes like arp2/3, erm-1, and dbn-1 and that nck-1/arp2/3 promote pore repair at the membrane surface and protect against PFP attack independent of p38 MAPK. Consistent with these findings, PFP attack causes significant changes in the amount of actin cytoskeletal proteins and in total amounts of F-actin in the target tissue, the intestine. nck-1 mutant animals appear to have lower F-actin levels than wild-type C. elegans. Studies on nck-1 and other F-actin regulating proteins have uncovered a new and important role of this pathway and the actin cytoskeleton in PFP INCED and protecting an intestinal epithelium in vivo against PFP attack. Author SummaryThe mechanism of action for a significant number of bacterial protein toxins is the formation of pores in the membrane of target cells. Host cells contain programmed defenses against such attacks. Here we use the model system of the roundworm Caenorhabditis elegans and crystal proteins produced by Bacillus thuringiensis to demonstrate a new defense pathway mediated by the activity of the NCK-1 protein. Consistent with its known function in mammalian cells, the NCK-1-mediated defense involves various actin-interacting proteins and affects the kinetics of pore repair. This pathway is novel in its independence from MAPK signaling. Furthermore, the NCK-1 activity is found to be generally needed for defense against multiple pore-forming proteins and yet is specific to defense against such proteins but not against other environmental stressors.

microbiology↗