Biology subjects

Jardine, J.

Publications and source records attributed to Jardine, J..

9 recordsLinked to original sources

bioRxiv2026

Molecular glue degraders of CK1α remodel lysosomes and limit ferroptosis in glioblastoma stem-like cells

Glioblastoma stem-like cells (GSCs) are highly plastic tumor cells that drive initiation, progression and therapeutic resistance. Although lysosomal homeostasis has emerged as a critical determinant of GSC fitness, the molecular mechanisms regulating this compartment remain incompletely understood. Here, using complementary proteomic approaches, we identified a lysosomal signature associated with the kinase CK1 in THP1 monocytic cells. Biochemical analyses revealed that genetic or siRNA-mediated depletion of CK1 altered the overall abundance and maturation of late endosome-lysosome-associated proteins in both THP1 cells and two patient-derived GSCs. This lysosomal remodeling was associated with impaired tumorsphere formation capacity in GSCs. To further explore the therapeutic potential of targeting CK1, we employed selective molecular glue degraders (MGDs). Acute CK1 degradation efficiently recapitulated lysosomal remodeling and impaired tumorsphere formation in GSCs. While CK1-targeted MGDs did not sensitize GSCs to lysosomal membrane permeabilization induced by L-leucyl-L-leucine methyl ester (LLOMe), they protected from GPX4 inhibitor-induced ferroptosis. Together, these findings identify CK1 as a regulator of lysosomal fitness and ferroptotic sensitivity, suggesting a connection between CK1-dependent lysosome remodeling and associated cell death pathways.

cancer biology↗

bioRxiv2026

Affinity Maturation Enables Dual Targeting of Cancer-Associated MUC1-Tn/STn and Drives Potent Tumor Control

A major challenge in solid tumor immunotherapy is identifying tumor-restricted antigens while overcoming intratumoral antigen heterogeneity. The truncated O-glycans Tn (GalNAc-O-Ser/Thr) and STn (Neu5Ac2-6GalNAc-O-Ser/Thr) are aberrantly displayed on the transmembrane glycoprotein MUC1 across a broad spectrum of epithelial malignancies, making MUC1-Tn/STn an attractive shared tumor-associated antigen class. However, heterogeneous intratumoral Tn/STn expression renders single-glycoform-specific antibodies vulnerable to antigen-driven escape. To address this challenge, we used structure-guided directed evolution and yeast surface display to affinity-mature the anti-MUC1-Tn antibody 5E5, generating variants with high-affinity dual recognition of MUC1-Tn and MUC1-STn. Co-crystal structures revealed that heavy-chain CDR loops cooperatively engage the GalNAc moiety primarily through hydrogen-bonding networks, in which HCDR3 residue 98 fine-tunes glycoform preference: Tyr98 favors MUC1-Tn recognition, whereas His98 enhances MUC1-STn engagement, while both support high-affinity recognition of both glycoforms. When reformatted as bispecific T-cell engagers, the affinity-matured variants mediated potent and durable cytolytic activity against cancer cells with heterogeneous glycan profiles, suppressed mixed Tn/STn tumors in vivo where the parental antibody failed, and showed enhanced activity in patient-derived tumor-like clusters. Together, these findings establish functional glycoform coverage as a design principle for antibody engineering, whereby a single paratope is optimized to recognize a defined spectrum of tumor-associated glycoforms while preserving antigen specificity, providing a strategy to overcome glycan heterogeneity in solid tumor immunotherapy.

cancer biology↗

bioRxiv2026

The membrane distal domain of CD16a allosterically regulates NK cell ADCC

Antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells is mediated by the activating IgG receptor CD16a (Fc{gamma}RIIIa), yet the molecular mechanisms governing receptor activation remain poorly understood. We demonstrate that the membrane-distal domain 1 (D1) of CD16a functions as an allosteric checkpoint that controls ADCC independently of IgG-Fc binding. A nanobody, C28, that binds an electronegative patch in D1 dose-dependently blocks NK cell ADCC against multiple therapeutic antibodies without affecting direct cytotoxicity. A second nanobody, C21, binding an adjacent D1 epitope has no such effect. Cryo-EM structures of the CD16a-IgG-nanobody complex reveal that C28 allosterically competes with core-fucosylated IgG and stabilizes a closed D1 conformation resembling unliganded receptor, even when Fc is bound. Molecular dynamics simulations show that occupation of the D1 epitope rigidifies the IgG-binding site, stabilizing CD16a overall in contrast with IgG binding alone. The nanobody C28 restricts CD3{zeta} phosphorylation in both resting and ADCC-activated NK cells, revealing tonic inhibitory control upstream of the signaling cascade. Using MINFLUX nanoscopy, we also show that CD16a forms dimers of [~]9 nm spacing on the NK cell surface, a geometry unaltered by the ADCC-enhancing L48H polymorphism. Drawing on structural parallels with the IgE receptor Fc{varepsilon}RI, which is held inactive as a cholesterol-stabilized dimer, we propose that CD16a dimerization through D1 contacts represents a conserved autoinhibitory mechanism among Fc receptors. Consistent with this model, structure-guided disruption of the C28 epitope in NK-92 cells enhances ADCC potency and killing kinetics, providing a blueprint for engineering improved cellular immunotherapeutics.

immunology↗

bioRxiv2025

The Kinase CK1α coordinates Initiation and Termination of the cGAS-STING Pathway

The cGAS-STING pathway is an evolutionarily conserved antimicrobial defense mechanism that senses cytosolic DNA to trigger innate immune responses. cGAS and STING play dual roles in tumorigenesis, promoting antitumor immunity and cell death while fueling tumor growth and metastasis. However, the mechanisms fine-tuning this pathway remain elusive. Using proteomic approaches, we report that Casein Kinase 1 alpha (CK1) operates as a bimodal regulator of the cGAS-STING pathway. CK1 supports optimal DNA sensing by preventing the proteasomal degradation of cGAS driven by the cullin-RING ubiquitin ligase 3 (CRL3). Conversely, CK1 facilitates STING degradation and signaling termination in response to STING agonists, tempering IRF3 activation. Exploiting these counterposing functions, we show that selective degradation of CK1 with molecular-glue degraders impaired the survival of a triple-negative breast cancer cell line with chronic cGAS-STING activation and synergized with a STING agonist to kill acute myeloid leukemia cells. Thus, CK1s dual regulatory role in the cGAS-STING pathway presents a promising target for therapeutic development. TEASERThis study unveils CK1 as a bimodal regulator of the cGAS-STING pathway.

immunology↗

bioRxiv2024

Synthetic integrin antibodies discovered by yeast display reveal αV subunit pairing preferences with β subunits

Eight of the 24 integrin heterodimers bind to the tripeptide Arg-Gly-Asp (RGD) motif in their extracellular ligands, and play essential roles in cell adhesion, migration, and homeostasis. Despite similarity in recognizing the RGD motif and some redundancy, these integrins can selectively recognize RGD-containing ligands including fibronectin, vitronectin, fibrinogen, nephronectin and the prodomain of the transforming growth factors to fulfill specific functions in cellular processes. Subtype-specific antibodies against RGD-binding integrins are desirable for investigating their specific functions. In this study, we discovered 11 antibodies that exhibit high specificity and affinity towards integrins V{beta}3, V{beta}5, V{beta}6, V{beta}8, and 5{beta}1 from a synthetic yeast-displayed Fab library. Of these, 6 are function-blocking antibodies containing an R(G/L/T) D motif in their CDR3 sequences. We report antibody binding specificity, kinetics, and binding affinity for purified integrin ectodomains as well as intact integrins on the cell surface. We further employed these antibodies to reveal binding preferences of the V subunit for its 5 {beta}-subunit partners: {beta}6={beta}8>{beta}3>{beta}1={beta}5.

cell biology↗

bioRxiv2023

Necrosulfonamide causes ubiquitination of PCM1 and impairs Ciliogenesis and Autophagy

Centriolar satellites are high-order assemblies, scaffolded by the protein PCM1, that gravitate as particles around the centrosome and play pivotal roles in fundamental cellular processes notably ciliogenesis and autophagy. Despite stringent control mechanisms involving phosphorylation and ubiquitination, the landscape of post-translational modifications shaping these structures remains elusive. Here, we report that necrosulfonamide (NSA), a small molecule known for binding and inactivating the pivotal effector of cell death by necroptosis MLKL, intersects with centriolar satellites, ciliogenesis, and autophagy independently of MLKL. NSA functions as a potent redox cycler and triggers the oxidation and aggregation of PCM1 alongside select partners, while minimally impacting the overall distribution of centriolar satellites. Additionally, NSA-mediated ROS production disrupts ciliogenesis and leads to the accumulation of autophagy markers, partially alleviated by PCM1 deletion. Together, these results identify PCM1 as a redox sensor protein and provide new insights into the interplay between centriolar satellites and autophagy.

cell biology↗

bioRxiv2022

HIV skews the SARS-CoV-2 B cell response toward an extrafollicular maturation pathway

BackgroundHIV infection dysregulates the B cell compartment, affecting memory B cell formation and the antibody response to infection and vaccination. Understanding the B cell response to SARS-CoV-2 in people living with HIV (PLWH) may explain the increased morbidity, reduced vaccine efficacy, reduced clearance, and intra-host evolution of SARS-CoV-2 observed in some HIV-1 coinfections. MethodsWe compared B cell responses to COVID-19 in PLWH and HIV negative (HIV-ve) patients in a cohort recruited in Durban, South Africa, during the first pandemic wave in July 2020 using detailed flow cytometry phenotyping of longitudinal samples with markers of B cell maturation, homing and regulatory features. ResultsThis revealed a coordinated B cell response to COVID-19 that differed significantly between HIV-ve and PLWH. Memory B cells in PLWH displayed evidence of reduced germinal center (GC) activity, homing capacity and class-switching responses, with increased PD-L1 expression, and decreased Tfh frequency. This was mirrored by increased extrafollicular (EF) activity, with dynamic changes in activated double negative (DN2) and activated naive B cells, which correlated with anti-RBD-titres in these individuals. An elevated SARS-CoV-2 specific EF response in PLWH was confirmed using viral spike and RBD bait proteins. ConclusionsDespite similar disease severity, these trends were highest in participants with uncontrolled HIV, implicating HIV in driving these changes. EF B cell responses are rapid but give rise to lower affinity antibodies, less durable long-term memory, and reduced capacity to adapt to new variants. Further work is needed to determine the long-term effects of HIV on SARS-CoV-2 immunity, particularly as new variants emerge. FundingThis work was supported by a grant from the Wellcome Trust to the Africa Health Research Institute (Wellcome Trust Strategic Core Award [grant number 201433/Z/16/Z]). Additional funding was received from the South African Department of Science and Innovation through the National Research Foundation (South African Research Chairs Initiative, [grant number 64809]), and the Victor Daitz Foundation.

immunology↗

bioRxiv2021

One-shot identification of SARS-CoV-2 S RBD escape mutants using yeast screening

The potential emergence of SARS-CoV-2 Spike (S) escape mutants is a threat to reduce the efficacy of existing vaccines and neutralizing antibody (nAb) therapies. An understanding of the antibody/S escape mutations landscape is urgently needed to preemptively address this threat. Here we describe a rapid method to identify escape mutants for nAbs targeting the S receptor binding site. We identified escape mutants for five nAbs, including three from the public germline class VH3-53 elicited by natural COVID-19 infection. Escape mutations predominantly mapped to the periphery of the ACE2 recognition site on the RBD with K417, D420, Y421, F486, and Q493 as notable hotspots. We provide libraries, methods, and software as an openly available community resource to accelerate new therapeutic strategies against SARS-CoV-2. One Sentence SummaryWe present a facile method to identify antibody escape mutants on SARS-CoV-2 S RBD.

biochemistry↗

bioRxiv2020

A novel USP30 inhibitor recapitulates genetic loss of USP30 and sets the trigger for PINK1-PARKIN amplification of mitochondrial ubiquitylation.

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. It has been proposed as an actionable target to alleviate the loss of function of the mitophagy pathway governed by the Parkinsons Disease associated genes PINK1 and PRKN. We present the characterisation of a N-cyano pyrrolidine derived compound, FT3967385, with high selectivity for USP30. The compound is well tolerated with no loss of total mitochondrial mass. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery that directly interacts with USP30, represents a robust biomarker for both USP30 loss and inhibition. We have conducted proteomics analyses on a SHSY5Y neuroblastoma cell line model to directly compare the effects of genetic loss of USP30 with selective inhibition in an unbiased fashion. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are most prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. In this model, USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 Ubiquitin proceeds more rapidly and to a greater extent in cells either lacking USP30 or subject to USP30 inhibition.

cell biology↗