Biology subjects

Hollingsworth, L. R.

Publications and source records attributed to Hollingsworth, L. R..

9 recordsLinked to original sources

bioRxiv2026

The accessible human surfaceome

Proteins associated with the extracellular face of the plasma membrane ("surface proteins" or the "surfaceome") are important therapeutic targets because they can direct therapeutics to specific cell types and deliver them into cells. However, experimental and computational databases disagree substantially on which proteins make up the human plasma membrane proteome, and none focus specifically on the set of accessible surface proteins that would be useful for therapeutic applications. Here, we used an agentic LLM pipeline to curate the accessible human surfaceome from literature. To assess performance, we built SurfaceBench, a manually curated benchmark of 147 proteins on which five reference databases frequently disagree. Our LLM triage agent classified surface status more accurately than existing databases and recovered 1,108 candidate surface proteins not present in any of them. On held-out clinical and viral-entry surface targets, the pipeline recovered 98-100% of established surface antigens. A literature-anchored deep-dive pipeline run on over 5,000 candidates then returned 1,783 canonical surface proteins and 1,252 with weaker or context-dependent surface evidence. Each queryable record contains 24 LLM-based annotation fields--including confidence, primary localization, and expression breadth--and seven deterministic structural and topological features. Unlike existing databases, our records trace every claim to the agent's reasoning and to exact quotes from the full-text literature it could access. All records can be browsed in an open-source viewer. These results show that LLMs reasoning over unstructured scientific text can build updatable, bespoke databases that outperform expert-curated resources, although performance remains limited by agent access to full-text literature.

bioinformatics↗

bioRxiv2026

Machine Learning enables efficient and effective affinity maturation of nanobodies

Antibodies can bind their targets with exquisite potency and selectivity due in part to large antibody-target protein-protein interaction surface areas. Despite the very large size and diversity of synthetic libraries, in vitro sorting alone tends to yield binders with modest affinities. By analogy to the in vivo affinity maturation in the natural immune system, these initial hits are typically affinity matured in vitro to achieve high affinity binding. However, affinity maturation campaigns can be laborious, often requiring multiple selection rounds and strategies for each clone to be optimized. Here, we investigated whether one could accelerate the discovery of optimized binders using machine learning on sequencing data from single selection sorts of affinity maturation yeast-display campaigns. Our results show that sparse sequencing data from a single sorting round can predict sequences that are enriched after multiple rounds. We also find that linear models outperform deep neural networks and semi-supervised approaches in ranking validated affinity-enhancing substitutions. Linear models are also more interpretable, offering insights into residue preferences that can be leveraged for further engineering. We use our models to design and select optimized nanobody binders to relaxin family peptide receptor 1 (RXFP1), yielding multiple improved binders including 3 sub nanomolar binders with the best exhibiting a [~]2500-fold improvement over WT.

bioinformatics↗

bioRxiv2024

Spatiotemporal proteomic profiling of cellular responses to NLRP3 agonists

Nucleotide-binding domain and leucine-rich repeat pyrin-domain containing protein 3 (NLRP3) is an innate immune sensor that forms an inflammasome in response to various cellular stressors. Gain-of-function mutations in NLRP3 cause autoinflammatory diseases and NLRP3 signalling itself exacerbates the pathogenesis of many other human diseases. Despite considerable therapeutic interest, the primary drivers of NLRP3 activation remain controversial due to the diverse array of signals that are integrated through NLRP3. Here, we mapped subcellular proteome changes to lysosomes, mitochondrion, EEA1-positive endosomes, and Golgi caused by the NLRP3 inflammasome agonists nigericin and CL097. We identified several common disruptions to retrograde trafficking pathways, including COPI and Shiga toxin-related transport, in line with recent studies. We further characterized mouse NLRP3 trafficking throughout its activation using temporal proximity proteomics, which supports a recent model of NLRP3 recruitment to endosomes during inflammasome activation. Collectively, these findings provide additional granularity to our understanding of the molecular events driving NLRP3 activation and serve as a valuable resource for cell biological research. We have made our proteomics data accessible through an open-access Shiny browser to facilitate future research within the community, available at: https://harperlab.connect.hms.harvard.edu/inflame/. We will display anonymous peer review for this manuscript on pubpub.org (https://harperlab.pubpub.org/pub/nlrp3/) rather than a traditional journal. Moreover, we invite community feedback on the pubpub version of this manuscript, and we will address criticisms accordingly.

cell biology↗

bioRxiv2023

Host E3 ubiquitin ligase ITCH mediates Toxoplasma gondii effector GRA35-triggered NLRP1 inflammasome activation and cell-autonomous immunity.

Toxoplasma gondii is an intracellular parasite that can activate the NLRP1 inflammasome leading to macrophage pyroptosis in Lewis rats, but the underlying mechanism is not well understood. In this study, we performed a genome-wide CRISPR screen and identified the dense granule proteins GRA35, GRA42, and GRA43 as the Toxoplasma effectors mediating cell death in Lewis rat macrophages. GRA35 localizes on the parasitophorous vacuole membrane, where it interacts with the host E3 ubiquitin ligase ITCH. Inhibition of proteasome activity or ITCH knockout prevented pyroptosis in Toxoplasma-infected Lewis rat macrophages, consistent with the "NLRP1 functional degradation model". However, there was no evidence that ITCH directly ubiquitinates or interacts with rat NLRP1. We also found that GRA35-ITCH interaction affected Toxoplasma fitness in IFN{gamma}-activated human fibroblasts, likely due to ITCHs role in recruiting ubiquitin and the parasite-restriction factor RNF213 to the parasitophorous vacuole membrane. These findings identify a new role of host E3 ubiquitin ligase ITCH in mediating effector-triggered immunity, a critical concept that involves recognizing intracellular pathogens and initiating host innate immune responses. IMPORTANCEEffector-triggered immunity represents an innate immune defense mechanism that plays a crucial role in sensing and controlling intracellular pathogen infection. The NLRP1 inflammasome in the Lewis rats can detect Toxoplasma infection, which triggers proptosis in infected macrophages and eliminates the parasites replication niche. The work reported here revealed that host E3 ubiquitin ligase ITCH is able to recognize and interact with Toxoplasma effector protein GRA35 localized on the parasite-host interface, leading to NLRP1 inflammasome activation in Lewis rat macrophages. Furthermore, ITCH-GRA35 interaction contributes to the restriction of Toxoplasma in human fibroblasts stimulated by IFN{gamma}. Thus, this research provides valuable insights into understanding pathogen recognition and restriction mediated by host E3 ubiquitin ligase.

immunology↗

bioRxiv2023

NINJ1 mediates plasma membrane rupture through formation of nanodisc-like rings

The membrane proteins Ninjurin1 (NINJ1) and Ninjurin2 (NINJ2) are upregulated by nerve injury to increase cell adhesion and promote axonal growth in neurons. NINJ1, but not NINJ2, has also been shown to play an essential role in pyroptosis by promoting plasma membrane rupture downstream of gasdermin D (GSDMD) pore formation, as well as in lytic cell death mediated by other pathways. Recombinant NINJ1 and NINJ2 purified in detergent show irregular rings of various diameters as well as curved filaments. While NINJ1 and NINJ2 both formed ring-like structures when mixed with liposomes, strikingly, only NINJ1, but not NINJ2, ruptures liposome membranes, leading to their dissolution. Because of the better feasibility, we determined the cryo-EM structure of NINJ1 ring segments from detergent by segmenting the irregular rings into shorter fragments. Each NINJ1 subunit contains a transmembrane (TM) helical hairpin (3 and 4) that likely mediates NINJ1 membrane localization, as well as the side-by-side interaction between adjacent subunits. There are two extracellular domain amphipathic helices (1 and 2), among which 1 crosses over to the neighboring subunit at the outside facing surface of the ring, to link NINJ1 subunits together into chains. As such, the inner face of the rings is hydrophobic whereas the outer face of the rings is hydrophilic and should repel membranes. Live cell imaging of NINJ1-deficient THP-1 cells reconstituted with NINJ1-eGFP uncovers the pinching off of NINJ1 rings from the cell surface and the loss of NINJ1 to the culture supernatant in oligomerized forms upon inflammasome activation. Formation of rings is also confirmed by super-resolution imaging of endogenous NINJ1 using anti-NINJ1 antibody. These data suggest that membrane insertion of amphipathic helices and formation of rings with a hydrophilic outer surface underlie the mechanism for NINJ1 to pinch off membranes as if it were a nanodisc-forming amphipathic polymer, leading to membrane rupture and lysis during cell death.

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↗

bioRxiv2020

DPP9 directly sequesters the NLRP1 C-terminus to repress inflammasome activation

NLRP1 is a cytosolic inflammasome sensor that mediates activation of caspase-1, which in turn induces cytokine maturation and pyroptotic cell death1-6. Gain-of-function NLPR1 mutations cause skin inflammatory diseases including carcinoma, keratosis, and papillomatosis7-14. NLRP1 contains a unique function-to-find domain (FIIND) that autoproteolyzes into noncovalently associated subdomains15-18. Proteasomal degradation of the autoinhibitory N-terminal fragment (NT) activates NLRP1 by releasing the inflammatory C-terminal fragment (CT)19,20. Cytosolic dipeptidyl peptidases 8 and 9 (DPP8/9) interact with NLRP1, and small-molecule DPP8/9 inhibitors activate NLRP1 by poorly characterized mechanisms11,19,21. Here, we report cryo-EM structures of the human NLRP1-DPP9 complex, alone and in complex with the DPP8/9 inhibitor Val-boroPro (VbP). Surprisingly, the NLRP1-DPP9 complex is a ternary complex comprised of DPP9, one intact FIIND of a non-degraded full-length NLRP1 (NLRP1-FL) and one NLRP1-CT freed by NT degradation. The N-terminus of the NLRP1-CT unfolds and inserts into the DPP9 active site but is not cleaved by DPP9, and this binding is disrupted by VbP. Structure-based mutagenesis reveals that the binding of NLRP1-CT to DPP9 requires NLRP1-FL and vice versa, and inflammasome activation by ectopic NLRP1-CT expression is rescued by co-expressing autoproteolysis-deficient NLRP1-FL. Collectively, these data indicate that DPP9 functions as a "bomb-diffuser" to prevent NLRP1-CTs from inducing inflammation during homeostatic protein turnover.

immunology↗

bioRxiv2020

Novel mode of filament formation in UPA-promoted CARD8 and NLRP1 Inflammasomes

NLRP1 and CARD8 are related cytosolic sensors that upon activation form supramolecular signalling complexes known as canonical inflammasomes, resulting in caspase-1 activation, cytokine maturation and/or pyroptotic cell death. NLRP1 and CARD8 use their C-terminal (CT) fragments containing a caspase recruitment domain (CARD) and the UPA subdomain of a function-to-find domain (FIIND) for self-oligomerization and recruitment of the inflammasome adaptor ASC and/or caspase-1. Here, we report cryo-EM structures of NLRP1-CT and CARD8-CT assemblies, in which the respective CARDs form central helical filaments that are promoted by oligomerized, but flexibly linked UPAs surrounding the filaments. We discover that subunits in the central NLRP1CARD filament dimerize with additional exterior CARDs, which roughly doubles its thickness and is unique among all known CARD filaments. The thick NLRP1 filament only forms with the presence of UPA, which we hypothesize drives the intrinsic propensity for NLRP1CARD dimerization. Structural analyses provide insights on the requirement of ASC for NLRP1-CT signalling and the contrasting direct recruitment of caspase-1 by CARD8-CT. Additionally, we present a low-resolution 4 ASCCARD–4 caspase-1CARD octamer structure, illustrating that ASC uses opposing surfaces for NLRP1, versus caspase-1, recruitment. These structures capture the architecture and specificity of CARD inflammasome polymerization in NLRP1 and CARD8.Competing Interest StatementH.W. is a co-founder of Ventus Therapeutics. The other authors declare no competing financial interests.View Full Text

immunology↗

bioRxiv2018

HIV-1 Env gp41 Transmembrane Domain Dynamics are Modulated by Lipid, Water, and Ion Interactions

The gp41 transmembrane domain (TMD) of the envelope glycoprotein (Env) of the human immunodeficiency virus (HIV) modulates the conformation of the viral envelope spike, the only druggable target on the surface of the virion. Understanding of TMD dynamics is needed to better probe and target Env with small molecule and antibody therapies. However, little is known about TMD dynamics due to difficulties in describing native membrane properties. Here, we performed atomistic molecular dynamics simulations of a trimeric, prefusion TMD in a model, asymmetric viral membrane that mimics the native viral envelope. We found that water and chloride ions permeated the membrane and interacted with the highly conserved arginine bundle, (R696)3, at the center of the membrane and influenced TMD stability by creating a network of hydrogen bonds and electrostatic interactions. We propose that this (R696)3 - water - anion network plays an important role in viral fusion with the host cell by modulating protein conformational changes within the membrane. Additionally, R683 and R707 at the exofacial and cytofacial membrane-water interfaces, respectively, are anchored in the lipid headgroup region and serve as a junction point for stabilization of the termini. The membrane thins as a result of the tilting of the TMD trimer, with nearby lipids increasing in volume, leading to an entropic driving force for TMD conformational change. These results provide additional detail and perspective on the influence of certain lipid types on TMD dynamics and rationale for targeting key residues of the TMD for therapeutic design. These insights into the molecular details of TMD membrane anchoring will build towards a greater understanding of dynamics that lead to viral fusion with the host cell.

biophysics↗