Biology subjects

Hui, E.

Publications and source records attributed to Hui, E..

At least 19 recordsLinked to original sources

bioRxiv2026

Declining ribosome function limits oocyte longevity

Protein synthesis declines with age across many tissues, but whether this decline actively contributes to physiological aging remains unclear. Oocytes may be particularly vulnerable because they rely on ribosomes synthesized during growth and maintained for prolonged periods without continual replacement. Here we show that declining translational capacity is a causal component of oocyte aging. An unbiased genetic screen identified ribosome dosage as a major determinant of oocyte longevity, and experimentally reducing ribosome abundance was sufficient to accelerate the loss of developmental competence and prematurely recapitulate meiotic defects that arise during natural aging, while also producing defects in early embryonic development. Ribosomes isolated from aged Drosophila oocytes exhibit ~50% lower translational activity despite only a modest reduction in ribosome abundance and preserved gross structure and composition, with both ribosomal subunits contributing to this functional decline. Protein synthesis also declines markedly in aged metaphase-I mouse oocytes. Together, these findings identify long-lived translational machinery as an intrinsic vulnerability of the oocyte and establish declining ribosome function as a mechanistic contributor to reproductive aging.

cell biology↗

bioRxiv2026

Chaperoning through time - defining DNAJA2 co-chaperone client selectivity via pulse-SILAC and BioID.

Molecular chaperones are a major driving force ensuring efficient protein folding and regulating proteostasis in the cell. However, it remains unclear how their clients are selected in most cases, especially after the release of nascent protein chains from ribosomes. Here, we present a novel approach that combines pulse metabolic labelling with SILAC and BioID mass spectrometry to temporally resolve protein-protein interactions of a co-chaperone. Using the Hsp70 co-chaperone DNAJA2 as a benchmark, we reveal that two distinct pools of proteins are enriched. In particular, DNAJA2 displays a preferential association with highly structured recently synthesized proteins enriched in {beta}-strands. In contrast, pre-existing proteins captured in our assay exhibit higher intrinsic disorder. In both cases, these proteins tend to be longer and contain a lower net charge compared to the proteome. Notably, these preferential associations are retained upon heat-shock, while interactions with the "older" pool of proteins become more prevalent under these conditions. Through this methodology, we gain novel insights on how co-chaperone-client interactions may occur over the lifespan of a protein to preserve proteostasis.

biochemistry↗

bioRxiv2026

BAG6 and RNF126 promote the degradation of cytosolic misfolded proteins that contain buried degron motifs

Missense mutations account for the majority of catalogued human disease-associated variants, and many are predicted to destabilize proteins and promote their degradation. To characterize the pathways responsible for recognizing and clearing such variants, we employed a two-pronged approach to identify both quality control components mediating turnover of misfolded proteins and the sequence elements within their substrates that drive this process. Using a panel of unstable cytosolic missense variants in proximity-labeling and RNAi-based experiments, we identified the BAG6-RNF126 pathway as contributing to the clearance of a subset of these substrates. Applying a tile-based approach to a model cytosolic protein, we uncovered strong potential degrons, including a C-terminal degron degraded in part in a BAG6- and RNF126-dependent manner. Modeling supports that this degron can be accommodated by BAG6. Together, our findings add to the growing body of evidence implicating the BAG6-RNF126 pathway as a key mediator of cytosolic protein quality control.

molecular biology↗

bioRxiv2025

Recombinant Laccase Production Causes Alterations of the S. cerevisiae Proteome that are Dependent on the Strain Origins

Saccharomyces cerevisiae yeast is a widely used recombinant protein production host. Recombinant protein expression requires adaptation of the host cell proteome to accommodate recombinant expression. However, this adaptation has not been well characterized. A better understanding of the adaptation to recombinant protein expression may inform us of pathways important to the process of expression. The proteome of a laboratory yeast was measured each of the 4 days of recombinant laccase expression to determine the adaptations of the proteome. Whereas a sizeable portion of the proteome had altered levels in response to nutrient depletion in batch growth, a smaller portion of the changes was specific to the laccase expression. By comparing yeast strains of different origins and laccase production capacities, we found that each strain tends to display a distinct response to heterologous expression, regardless of the origin of the laccase. For example, the chaperones Hsp26 and Kar2 were specifically elevated in a whey-derived strain upon laccase expression. Nonetheless, the higher capacity to produce active recombinant laccase in some strains appears to be more strongly associated with small groups of proteins that are constitutively expressed at different levels. These results indicate that strains of different origins each provide a unique cellular milieu that, in some cases, is more favorable for the expression of a given recombinant protein. This study provides potential new targets for strain engineering to improve the yield of select recombinant proteins and provides the first insights into the dynamics of the yeast proteome during recombinant laccase expression. Key PointsO_LIProteomes of S. cerevisiae strains during recombinant laccase expression determined C_LIO_LIChanges to ribosomal & metabolic protein levels occur during recombinant expression C_LIO_LIUnique cellular milieu, rather than proteome shifts, is linked to higher yields C_LI

molecular biology↗

bioRxiv2025

Brain cholesterol metabolites cause significant neurodegeneration in human iPSC-derived neurons

Disrupted cholesterol metabolism is increasingly recognised as a contributing factor in neurodegeneration; however, the specific effects of key brain-derived cholesterol metabolites, 24S-hydroxycholesterol (24S-HC) and 27-hydroxycholesterol (27-HC), remain poorly understood. Using human iPSC-derived i3 cortical neurons, we demonstrate that both 24S-HC and 27-HC significantly impair neuronal calcium signalling by elevating resting calcium levels, reducing spike amplitude, and disrupting network synchrony. These functional deficits are accompanied by widespread organelle dysfunction. Both oxysterols induce mitochondrial fragmentation, decrease spare respiratory capacity, and impair lysosomal degradation. Notably, 27-HC uniquely triggers lysosomal swelling and membrane permeabilisation. Additional signs of cellular stress, including axonal swellings and elevated endoplasmic reticulum calcium levels, were also observed. Furthermore, both 24S-HC and 27-HC were found to directly interact with alpha-synuclein (aSyn), promoting its accumulation in cellular models. In contrast, cholesterol itself had minimal impact, highlighting the distinct toxicity of its hydroxylated metabolites. Together, these findings reveal a mechanistic link between oxysterol accumulation and neuronal dysfunction, supporting the hypothesis that elevated levels of 24S-HC and 27-HC, commonly observed in Parkinsons and Alzheimers disease, may actively drive neurodegenerative processes. Targeting oxysterol metabolism may therefore represent a promising therapeutic avenue for intervention in neurodegenerative disorders.

neuroscience↗

bioRxiv2025

Themis dominates T cell exhaustion by regulation of TCR and PD-1 signaling

T cell exhaustion is important to protect the host from immunopathology during chronic viral infection, but it also impairs T cell anti-tumor immunity1-5. A fundamental unresolved question is whether and how T cell exhaustion is determined at the onset of TCR signaling6-8. Here we report an unexpected role of Themis, a TCR-proximal signaling molecule9, in T cell exhaustion. Chronic viral infection in mice usually leads to T cell exhaustion and survival of the host. Surprisingly, Themis T-cell conditional knockout mice died from severe CD8+-dependent lung immunopathology in chronic viral infection, showing Themis importance in establishing T cell exhaustion. We found that Themis-deficient CD8+ T cells were hyperactivated at the single-cell level - producing more TNF and IFN{gamma} compared to wild-type counterparts - but defective in population-level expansion. Moreover, TCF-1 and TOX expression were inhibited in Themis-deficient CD8+ T cells, thereby impairing differentiation of exhausted T cell precursors (T-pex) and maintenance of terminally exhausted T cells (T-ex), respectively. Mechanistically, Themis initially promotes TCR signaling to induce PD-1 expression and subsequently mediates PD-1 signaling. In the latter, Themis binds to PD-1 and promotes PD-1 phosphorylation and its recruitment of SHP2, thereby acting as a negative regulator to inhibit T cell effector functions. Without Themis, the orderly regulation of TCR and PD-1 signaling, and therefore exhaustion, is disrupted. Thus, our results unequivocally demonstrate that Themis-mediated early TCR signaling plays a decisive role in T cell exhaustion and provide a novel mechanism of PD-1 signaling through Themis.

immunology↗

bioRxiv2024

Evolutionary fingerprint in rodent PD1 confers weakened activity and enhanced tumor immunity compared to human PD1

Mechanistic understanding of the immune checkpoint receptor PD1 is largely based on mouse models, but human and mouse PD1 orthologs exhibit only 59.6% identity in amino acid sequences. Here we show that human PD1 is more inhibitory than mouse PD1 due to stronger interactions with the ligands PDL1 and PDL2 and with the effector phosphatase Shp2. A novel motif highly conserved among PD1 orthologs in vertebrates except in rodents is primarily responsible for the differential Shp2 recruitment. Evolutionary analysis suggested that rodent PD1 orthologs uniquely underwent functional relaxation, particularly during the K-Pg boundary. Humanization of the PD1 intracellular domain disrupted the anti-tumor activity of mouse T cells while increasing the magnitude of anti-PD1 response. Together, our study uncovers species-specific features of the PD1 pathway, with implications to PD1 evolution and differential anti-PD(L)1 responses in mouse models and human patients.

immunology↗

bioRxiv2024

Graphene microelectrode arrays, 4D structured illumination microscopy, and a machine learning-based spike sorting algorithm permit the analysis of ultrastructural neuronal changes during neuronal signalling in a model of Niemann-Pick disease type C

Simultaneously recording network activity and ultrastructural changes of the synapse is essential for advancing our understanding of the basis of neuronal functions. However, the rapid millisecond-scale fluctuations in neuronal activity and the subtle sub-diffraction resolution changes of synaptic morphology pose significant challenges to this endeavour. Here, we use graphene microelectrode arrays (G-MEAs) to address these challenges, as they are compatible with high spatial resolution imaging across various scales as well as high temporal resolution electrophysiological recordings. Furthermore, alongside G-MEAs, we deploy an easy-to-implement machine learning-based algorithm to efficiently process the large datasets collected from MEA recordings. We demonstrate that the combined use of G-MEAs, machine learning (ML)-based spike analysis, and four-dimensional (4D) structured illumination microscopy (SIM) enables the monitoring of the impact of disease progression on hippocampal neurons which have been treated with an intracellular cholesterol transport inhibitor mimicking Niemann-Pick disease type C (NPC) and show that synaptic boutons, compared to untreated controls, significantly increase in size, which leads to a loss in neuronal signalling capacity.

neuroscience↗

bioRxiv2023

Innate extracellular Hsp70 inflammatory properties are mediated by the interaction of Siglec-E and LOX-1 receptors

Innate immune responses to cell damage-associated molecular patterns induce a controlled degree of inflammation, ideally avoiding the promotion of intense unwanted inflammatory adverse events. When released by damaged cells, Hsp70 can stimulate different responses that range from immune activation to immune suppression. The effects of Hsp70 are mediated through innate receptors expressed primarily by myeloid cells, such as dendritic cells (DCs). The regulatory innate receptors that bind to extracellular mouse Hsp70 (mHsp70) are not fully characterized, and neither are their potential interactions with activating innate receptors. Here, we describe that extracellular mHsp70 interacts with a receptor complex formed by inhibitory Siglec-E and activating LOX-1 on DCs. We also find that this interaction takes place within lipid microdomains, and Siglec-E acts as a negative regulator of LOX-1-mediated innate activation upon mHsp70 or oxidized LDL binding. Thus, HSP70 can both bind to and modulate the interaction of inhibitory and activating innate receptors on the cell surface. These findings add another dimension of regulatory mechanism to how self-molecules contribute to dampening of exacerbated inflammatory responses.

immunology↗

bioRxiv2022

A Binary RNA and DNA Self-Amplifying Platform for Next Generation Vaccines and Therapeutics

Conventional mRNA-based vaccines were instrumental in lowering the burden of the pandemic on healthcare systems and in reducing mortality. However, such first-generation vaccines have significant weaknesses. Here, we describe a high-performance binary recombinant vectoral platform offering the flexibility to be used as a self-amplifying mRNA or a self-amplifying DNA. Both formats drive long-lasting expression and actuate robust antibody responses against SAR-CoV-2 spike, and neither format require encapsulation with lipid nanoparticles (LNP) in the generation immune responses. The platform combines the power of conventional mRNA with the low-dosage of self-amplifying vectors together with the simplicity, rapid creation, ease of storage, and convenience of distribution of plasmid DNA vectors. This platform promises to pave the way for more effective, less expensive, and truly democratized vaccines and therapeutics. One-Sentence SummaryGemini: a versatile platform that improves on existing vaccine formats in terms of effectiveness, manufacturing, distribution, and cost.

immunology↗

bioRxiv2021

Semisynthesis of functional transmembrane proteins in GUVs

Cellular transmembrane (TM) proteins are essential sentries of the cell facilitating cell-cell communication, internal signaling, and solute transport. Reconstituting functional TM proteins into model membranes remains a challenge due to the difficulty of expressing hydrophobic TM domains and the required use of detergents. Herein, we use a intein-mediated ligation strategy to semisynthesize bitopic TM proteins in synthetic membranes. We have adapted the trans splicing capabilities of split inteins for a native peptide ligation between a synthetic TM peptide embedded in the membrane of giant unilamellar vesicles (GUVs) and an expressed soluble protein. We demonstrate that the extracellular domain of programmed cell death protein 1 (PD-1), a mammalian transmembrane immune checkpoint receptor, retains its function for binding its ligand PD-L1 at a reconstituted membrane interface after ligation to a synthetic TM peptide in GUV membranes. We envision that the construction of full-length TM proteins using orthogonal split intein-mediated semisynthetic protein ligations will expand applications of membrane protein reconstitution in pharmacology, biochemistry, biophysics, and artificial cell development.

biochemistry↗

bioRxiv2021

Molecular Features Underlying Shp1/Shp2 Discrimination by Immune Checkpoint Receptors

Numerous inhibitory immunoreceptors operate by recruiting phosphatase effectors Shp1 and Shp2 through conserved motifs ITIM and ITSM. Despite the similarity, these receptors exhibit distinct effector binding specificities, as exemplified by PD-1 and BTLA, which preferentially recruit Shp2 and Shp1 respectively. The molecular basis of Shp1/Shp2 discrimination is unclear. Here, we provide evidence that optimal PD-1 and BTLA binding to both Shp1 and Shp2 occurs via a bivalent, parallel mode that involves both SH2 domains of Shp1/Shp2. Moreover, PD-1 mainly uses its ITSM to discriminate Shp2 from Shp1 via their C-terminal SH2 domains. Supportive of this model, swapping the Shp1-cSH2 with Shp2-cSH2 enabled PD-1:Shp1 association in T cells. In contrast, BTLA primarily utilizes its ITIM to discriminate Shp1 from Shp2 via their N-terminal SH2 domains. Substitution of glycine at pY+1 position of the PD-1-ITIM with alanine, a residue conserved in several Shp1-recruiting receptors, was sufficient to induce PD-1:Shp1 interaction in T cells. Finally, mutagenesis screening shows that Shp1 recruitment exhibits a bell-shaped dependence on the side chain volume of the pY+1 residue of ITIM. Collectively, we provide a molecular interpretation of the Shp1/Shp2-binding specificities of PD-1 and BTLA, with general implications for the mechanism of effector discrimination by inhibitory receptors.

immunology↗

bioRxiv2021

The combined influence of viscoelastic and adhesive cues on fibroblast spreading and focal adhesion formation

Tissue fibrosis is characterized by progressive extracellular matrix (ECM) stiffening and loss of viscoelasticity that ultimately results in reduced organ functionality. Cells bind to the ECM through integrins, where av integrin engagement in particular has been correlated with fibroblast activation into contractile myofibroblasts that drive fibrosis progression. There is a significant unmet need for in vitro hydrogel systems that deconstruct the complexity of native tissues to better understand the individual and combined effects of stiffness, viscoelasticity, and integrin engagement on fibroblast behavior. Here, we developed hyaluronic acid hydrogels with independently tunable cell-instructive properties (stiffness, viscoelasticity, ligand presentation) to address this challenge. Hydrogels with mechanics matching normal or fibrotic lung tissue were synthesized using a combination of covalent crosslinks and supramolecular interactions to tune viscoelasticity. Cell adhesion was mediated through incorporation of either RGD peptide or engineered fibronectin fragments promoting preferential integrin engagement via v{beta}3 or 5{beta}1. We showed that preferential v{beta}3 engagement enabled human lung fibroblasts to assume a myofibroblast-like phenotype on fibrosis-mimicking stiff elastic hydrogels with increased spreading, actin stress fiber organization, and focal adhesion maturation as indicated by paxillin organization. In contrast, preferential 5{beta}1 binding suppressed these metrics. Viscoelasticity, mimicking the mechanics of healthy tissue, largely curtailed fibroblast spreading and focal adhesion organization independent of adhesive ligand type, highlighting its role in preventing fibroblast activation. Together these results provide new insights into how mechanical and adhesive cues collectively guide disease-relevant cell behaviors.

bioengineering↗

bioRxiv2021

Adult porcine (Sus scrofa) derived inner ear cells possessing multipotent stem/progenitor cell characteristics in in vitro cultures

The human inner ear compared with that of other mammalian species is very complex. Although the mouses cochlea is frequently studied the mouses inner ear continues to develop postnatally whilst the human inner ear is fully developed by the third month of gestation which leads one to question the applicability of findings based on research on mice to human regenerative therapies. Here, we report a novel in vitro culture of adult porcine (Sus scrofa) inner ear cells developed from post-mortem labyrinth specimens. Anatomical findings based on maximal transverse and vertical axial diameters and the length of the cochlear duct suggest that the pigs cochlea is similar to the human cochlea. In vitro cultures of porcine cochlear and vestibular cells showed the persistence of both inner ear hair cell (HC), supporting cell (SC) and stem/progenitor cell characteristics across passages up to 6 based on scanning electron microscopy, fluorescence immunocytochemistry and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Our findings showed that porcine cochlear and vestibular epithelia maintained multipotent stem/progenitor cell populations into adulthood although their regenerative capacities differed across the passages. The development of a viable and reproducible method to culture porcine inner ear cells provides an important investigative tool that can be utilized to study and evaluate the pathophysiological causes and cellular consequences of human inner ear disorders.

cell biology↗

bioRxiv2019

Spatial control of viscoelasticity in phototunable hyaluronic acid hydrogels

Viscoelasticity has emerged as a critical regulator of cell behavior. However, there is an unmet need to develop biomaterials where viscoelasticity can be spatiotemporally tuned to mimic the dynamic and heterogeneous nature of tissue microenvironments. Toward this objective, we developed a modular hyaluronic acid hydrogel system combining light-mediated covalent and supramolecular crosslinking to afford spatiotemporal control of network viscoelastic properties. Covalently crosslinked elastic hydrogels or viscoelastic hydrogels combining covalent and supramolecular interactions were fabricated to match healthy and fibrotic liver stiffness. LX-2 human hepatic stellate cells cultured on viscoelastic substrates displayed reduced spreading, less actin stress fiber organization, and lower myocardin-related transcription factor A (MRTF-A) nuclear localization compared to cells on elastic hydrogels. We further demonstrated the dynamic capabilities of our hydrogel system through photomediated secondary incorporation of either covalent or supramolecular crosslinks to modulate viscoelastic properties. We used photopatterning to create hydrogel models with well-controlled patterned regions of stiff elastic mechanics representing fibrotic tissue nodules surrounded by regions of soft viscoelastic hydrogel mimicking healthy tissue. Cells responded to the local mechanics of the patterned substrate with increased spreading in fibrosis-mimicking regions. Together, this work represents an important step forward toward the creation of hydrogel models with spatiotemporal control of both stiffness and viscoelastic cell-instructive cues.

bioengineering↗

bioRxiv2019

PD-L1:CD80 Heterodimer Triggers CD28 While Repressing Both PD-1 and CTLA4 Pathways

Combined immunotherapy with anti-PD-1/PD-L1 and anti-CTLA4 has resulted in superior clinical responses compared to single agent therapy. The underlying mechanisms for this synergy have yet to be elucidated and investigations have largely focused on cellular interactions. Herein, we report a molecular crosstalk in which the PD-1 ligand PD-L1 and the CTLA4 ligand CD80 heterodimerize in cis. This heterodimerization blocks both PD-L1:PD-1 and CD80:CTLA4 interactions, but preserves the ability of CD80 to activate the T cell costimulatory receptor CD28. Remarkably, PD-L1 expression on antigen presenting cells (APCs) protects CD80 from CTLA4 mediated trans-endocytosis, and the therapeutic PD-L1 blockade antibody atezolizumab paradoxically downregulates CD80 on APCs, presumably reducing its co-stimulatory ability. Importantly, this effect can be negated by co-blockade of CTLA4 with ipilimumab. Our study reveals an unexpected immune stimulatory role of cis-acting PD-L1 and a mechanism of anti-PD-L1/anti-CTLA4 crosstalk, providing a therapeutic rationale for combination blockade of PD-L1 and CTLA4.

immunology↗

bioRxiv2018

TCR-pMHC bond length controls TCR ligand discrimination

T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-pMHCs to trigger antigen-specific adaptive immune responses1-4. Despite intense efforts, the mechanism underlying TCR ligand discrimination remains a major unanswered question in immunology. Here we show that a TCR discriminates between closely related peptides by forming TCR-pMHC bonds with different lengths, which precisely control the accessibility of CD3{zeta} immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. Using in situ fluorescence resonance energy transfer (FRET)3,5, we measured the intermolecular length of single TCR-pMHC bonds and the intramolecular distance of individual TCR-CD3{zeta} complexes at the membrane of live primary T cells. We found that an agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3{zeta} off the inner leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond with the TCR, resulting in partial dissociation of CD3{zeta} from the membrane and weak phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the entire process of signal reception, transduction and regulation. Thus, our data reveal the fundamental mechanism by which a TCR deciphers the structural differences between foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR signaling for ligand discrimination.

immunology↗

bioRxiv2016

In vitro reconstitution of T cell receptor-mediated segregation of the CD45 phosphatase

T cell signaling initiates upon binding of peptide-major histocompatibility complex (pMHC) on an antigen-presenting cell (APC) to the T cell receptor (TCR) on a T cell. TCR phosphorylation in response to pMHC binding is accompanied by segregation of the transmembrane phosphatase CD45 away from TCR-pMHC complexes. The kinetic segregation hypothesis proposes that CD45 exclusion shifts the local kinase-phosphatase balance to favor TCR phosphorylation. Spatial partitioning may arise from the size difference between the large CD45 extracellular domain and the smaller TCR-pMHC complex, although parsing potential contributions of extracellular protein size, actin activity, and lipid domains is difficult in living cells. Here, we reconstitute segregation of CD45 from bound receptor-ligand pairs using purified proteins on model membranes. Using a model receptor-ligand pair (FRB-FKBP), we first test physical and computational predictions for protein organization at membrane interfaces. We then show that the TCR-pMHC interaction causes partial exclusion of CD45. Comparing two developmentally-regulated isoforms of CD45, the larger RABC variant is excluded more rapidly and efficiently (~50%) than the smaller R0 isoform (~20%), suggesting that CD45 isotypes could regulate signaling thresholds in different T cell subtypes. Similar to the sensitivity of T cell signaling, TCR-pMHC interactions with Kds of [≤]15 M were needed to exclude CD45. We further show that the co-receptor PD-1 with its ligand PD-L1, immunotherapy targets that inhibit T cell signaling, also exclude CD45. These results demonstrate that the binding energies of physiological receptor-ligand pairs on the T cell are sufficient to create spatial organization at membrane-membrane interfaces.\n\nSIGNIFICANCE STATEMENTThe interface between a T cell and an antigen-presenting cell (APC) results in the formation of biochemically distinct plasma membrane domains that initiate signaling cascades. Here, using biochemical reconstitution and microscopy, we show that the binding energies of the TCRpMHC and PD-1-PD-L1 complexes are sufficient to create spatial organization at a model membrane-membrane interface. We show that spatial organization depends upon receptor-ligand binding affinity and the relative sizes of the extracellular domains. These biophysical parameters may be used to fine-tune signaling cascades in T cells.

immunology↗