Biology subjects

Hsiao, J. C.

Publications and source records attributed to Hsiao, J. C..

6 recordsLinked to original sources

bioRxiv2026

Bacterial surface display enables lysis-independent joint host-pathogen single-cell profiling

Heterogeneity in host-pathogen interactions arises from variation in both host cell state and pathogen state, yet most single-cell methods capture these features separately. Joint profiling is limited by fundamental technical mismatches between host- and pathogen-derived material, particularly differences in cell wall structure, lysis requirements, and molecular abundance. Here we introduce a lysis-independent strategy that enables unified measurement of intracellular bacterial presence and state alongside host single-cell profiles. We repurpose bacterial surface display to encode promoter activity and bacterial identity as antibody-detectable signals, rendering bacterial features compatible with existing antibody-based single-cell assays. This approach is modular across promoters, epitope tags, display scaffolds, and bacterial species, including Escherichia coli and Mycobacterium tuberculosis. Surface-displayed reporters are detectable during intracellular infection and can be read out by flow cytometry and droplet-based single-cell RNA sequencing without pathogen-specific lysis optimization or pre-sorting on pathogen signal. Applying this method to infected macrophages, we link heterogeneous bacterial uptake to heterogeneous expression of phagocytosis-associated host programs. This strategy enables scalable, joint host-pathogen single-cell measurements and expands the range of pathogens and states accessible to high-throughput single-cell analysis.

bioengineering↗

bioRxiv2025

Deep Mutational Scanning of FDX1 Identifies Key Structural Determinants of Lipoylation and Cuproptosis

Cuproptosis is a recently described form of regulated cell death triggered by ionophore-induced copper (Cu) overload in mitochondria. It is critically dependent on ferredoxin 1 (FDX1), a mitochondrial reductase that facilitates cuproptosis by reducing ionophore-bound Cu(II) to Cu(I) thereby triggering its release, and by promoting mitochondrial protein lipoylation, which is directly targeted by the released Cu to drive cell death. Despite the pivotal role of FDX1 in cuproptosis, the structural determinants underlying its distinct functions remain unclear. To address this, we performed deep mutational scanning (DMS) of FDX1 and identified two conserved, solvent-exposed residues--D136 and D139--on its third alpha helix (-helix 3) that are critical for both FDX1-mediated cuproptosis and lipoylation. Charge-reversal mutations at these positions abolished FDX1s ability to induce cuproptosis and support lipoylation in cells, despite retaining full enzymatic activity in vitro. Guided by structural and genomic analyses, we further identified dihydrolipoamide dehydrogenase (DLD), the E3 subunit of lipoylated complexes as an alternative FDX1 reductase both in cells and in vitro. Together, these findings establish the acidic -helix 3 of FDX1 as a critical interface for its upstream regulation and suggest that FDX1s roles in cuproptosis and in lipoylation are both structurally and functionally linked.

cell biology↗

bioRxiv2023

The interaction between NLRP1 and oxidized TRX1 involves a transient disulfide bond

NLRP1 is an innate immune receptor that detects pathogen-associated signals, assembles into a multiprotein structure called an inflammasome, and triggers a proinflammatory form of cell death called pyroptosis. We previously discovered that the oxidized, but not the reduced, form of thioredoxin-1 directly binds to NLRP1 and represses inflammasome formation. However, the molecular basis for NLRP1s selective association with only the oxidized form of TRX1 has not yet been established. Here, we leveraged Alphafold-Multimer, site-directed mutagenesis, thiol-trapping experiments, and mass spectrometry to reveal that a specific cysteine residue (C427 in humans) on NLRP1 forms a transient disulfide bond with oxidized TRX1. Overall, this work demonstrates how NLRP1 monitors the cellular redox state, further illuminating an unexpected connection between the intracellular redox potential and the innate immune system.

immunology↗

bioRxiv2022

Fc-engineered antibodies leverage neutrophils to drive control of Mycobacterium tuberculosis

Novel vaccination and therapeutic strategies are urgently needed to mitigate the tuberculosis (TB) epidemic. While extensive efforts have focused on potentiating cell-mediated immunity to control Mycobacterium tuberculosis (Mtb) infection, less effort has been invested in exploiting the humoral immune system to combat Mtb. Emerging data point to a role for antibodies in microbial control of Mtb, however the precise mechanism(s) of this control remain incompletely understood. Here we took an antibody Fc-engineering approach to determine whether Fc-modifications could improve the ability of antibodies to restrict Mtb, and to define Fc-mediated mechanism(s) antibodies leverage for this restriction. Using an antibody specific to the capsular polysaccharide -glucan, we engineer a panel of Fc variants to augment or dampen select antibody effector functions, rationally building antibodies with enhanced capacity to promote Mtb restriction in a human whole blood model of infection. Surprisingly, restrictive Fc-engineered antibodies drive Mtb control in a neutrophil, not monocyte, dependent manner. Using single cell RNA sequencing, we show that restrictive antibodies promote neutrophil survival and expression of cell intrinsic antimicrobial programs. These data provide a roadmap for exploiting Fc-engineered antibodies as a novel class of TB therapeutics able to harness the protective functions of neutrophils to achieve disease control.

immunology↗

bioRxiv2022

A ubiquitin-independent proteasome pathway controls the CARD8 inflammasome

CARD8 is a pattern-recognition receptor that forms a caspase-1-activating inflammasome. CARD8 undergoes autoproteolysis, generating an N-terminal (NT) fragment with a disordered region and a ZU5 domain and a C-terminal (CT) fragment with UPA and CARD domains. DPP8 and DPP9 (DPP8/9) inhibitors, including Val-boroPro (VbP), accelerate the degradation of the NT fragment via a poorly characterized proteasome-mediated pathway, thereby releasing the inflammatory CT fragment from autoinhibition. Here, we show that the core 20S proteasome, which degrades disordered and misfolded proteins independent of ubiquitin, controls CARD8 activation. In unstressed cells, the 20S proteasome degrades just the NT disordered region, leaving behind the folded ZU5, UPA, and CARD domains to act as an inhibitor of inflammasome assembly. In VbP-stressed cells, the 20S proteasome degrades the entire NT fragment, perhaps due to ZU5 domain unfolding, freeing the CT fragment from autoinhibition. Overall, this work shows that CARD8 NTs susceptibility to 20S proteasome-mediated degradation controls inflammasome activation.

immunology↗

bioRxiv2021

Structural mechanism of CARD8 regulation by DPP9

CARD8 is a germline-encoded pattern recognition receptor that detects intracellular danger signals. Like the related inflammasome sensor NLRP1, CARD8 undergoes constitutive autoprocessing within its function-to-find domain (FIIND), generating two polypeptides that stay associated and autoinhibited. Certain pathogen- and danger-associated activities, including the inhibition of the serine dipeptidases DPP8 and DPP9 (DPP8/9), induce the proteasome-mediated degradation of the N-terminal (NT) fragment, releasing the C-terminal (CT) fragment to form a caspase-1 activating inflammasome. DPP8/9 also bind directly to the CARD8 FIIND, but the role that this interaction plays in CARD8 inflammasome regulation is not yet understood. Here, we solved several cryo-EM structures of CARD8 bound to DPP9, with or without the DPP inhibitor Val-boroPro (VbP), which revealed a ternary complex composed of one DPP9, the full-length CARD8, and one CARD8-CT. Through structure-guided biochemical and cellular experiments, we demonstrated that DPP9s structure restrains CARD8-CT after proteasomal degradation. Moreover, although DPP inhibitors do not directly displace CARD8 from DPP9 in vitro, we show that they can nevertheless destabilize this complex in cells. Overall, these results demonstrate that DPP8/9 inhibitors cause CARD8 inflammasome activation via at least two distinct mechanisms, one upstream and one downstream of the proteasome.

immunology↗