Biology subjects

Peters-Clarke, T. M.

Publications and source records attributed to Peters-Clarke, T. M..

7 recordsLinked to original sources

bioRxiv2026

Targeted extracellular degradation of LRP8 promotes ferroptosis in cancer cells

Tumor reliance on antioxidant defenses creates a vulnerability to ferroptosis, yet strategies to therapeutically disable these systems remain limited. Here, we identify targeted degradation of the selenium uptake receptor LRP8 as an effective approach to decrease the abundance of the ferroptosis-protective enzyme glutathione peroxidase 4 (GPX4). Using bispecific cytokine receptor-targeting chimeras (KineTACs) that couple LRP8 to cytokine receptor internalization pathways, we selectively direct LRP8 to the lysosome for degradation. LRP8 degradation reduces the abundance of several selenoproteins, including GPX4, lowering the cellular threshold for lipid peroxidation and sensitizing cancer cells to ferroptosis. These findings establish receptor-mediated selenium uptake as a critical, targetable node in ferroptosis resistance and demonstrate that extracellular protein degradation can be leveraged to reprogram intracellular translational dependencies in cancer cells. More broadly, this work provides a framework for exploiting nutrient acquisition pathways to overcome therapy resistance.

bioengineering↗

bioRxiv2026

Targeted shedding of extracellular membrane proteins by induced protease recruitment

Extracellular targeted protein degradation has emerged as a promising therapeutic modality to eliminate proteins of interest (POIs) at the cell surface, by using bifunctional molecules to recruit natural recycling receptors or membrane-bound E3 ligases that redirect POIs to the lysosome. Another natural mechanism involves extracellular proteases that cleave and shed extracellular domains. Here, we exploit this endogenous mechanism by engineering bispecific antibody Shedders, that recruit a classic sheddase ADAM10 to POIs, inducing selective ectodomain shedding. We first targeted the immune checkpoint receptor LAG-3 and observed robust depletion of surface LAG-3 accompanied by accumulation of soluble LAG-3 fragments in both engineered cell lines and primary human T cells. Using biochemical and imaging assays, we confirmed that this antibody-induced shedding is restricted to extracellular protease activity and occurs independently of lysosomal trafficking. Notably, induced shedding of LAG-3 on activated primary T cells partially alleviated inhibitory signaling and reinvigorated IFN{gamma} secretion. We extended the scope of induced shedding by developing Shedders that recognize synthetic epitope-tags that enabling rapid assessment of substrate compatibility across diverse targets. Using this platform, we identified multiple immune modulatory cell-surface receptors, including IL6R, CD62L and MIC-A that can be targeted for shedding. In summary, this work establishes a new paradigm for targeted extracellular proteolysis and expands the toolkit for studying extracellular proteolysis with potential therapeutic benefit.

bioengineering↗

bioRxiv2026

Cellular consequences, citrullination substrates, and antigenicity resulting from wild-type and targeted PAD4 on cell surfaces

Protein arginine deiminase-4 (PAD4) catalyzes hydrolysis of arginine to citrulline in proteins that promotes widespread changes in cellular phenotypes through transcriptional regulation that can induce innate immunity and promote cancer. Overexpression and hyperactivity of PAD4 leads to a form of cell death called NETosis that releases PAD4 to the extracellular space. In excess, release of PAD4 is believed to be a major cause of various autoimmune diseases through the generation of anti-citrulline protein antibodies (ACPAs). Little is known about the specific protein substrates that become citrullinated and lead to autoimmunity, but there is growing evidence that PAD4 can be localized to the cell surface in response to inflammation. Here, we further characterize the cellular consequences for exogenous treatment with PAD4 showing that it induces morphological changes that increase cell migration, a hallmark of cancer. We then devised a more simplified and robust proteomics approach to identify PAD4 substrates. We identified some 1000 endogenously citrullinated peptides from 500 proteins, and 3000 citrullinated peptides from 1300 proteins upon exogenous addition of PAD4 both inside and outside of cells. This extracellular set can be further augmented by targeting PAD4 to a cancer target, HER2, using a binding protein conjugate. Finally, we studied how citrullinated cells can induce a robust humoral response in a syngeneic vaccine model to produce ACPAs. We believe these studies further our understanding of cell phenotypic consequences of extracellular PAD4 and new PAD4 substrates both inside and outside of cells that are potential neoepitopes for generation of ACPAs.

cancer biology↗

bioRxiv2025

A cytokine receptor-targeting chimera (kineTAC) toolbox for expanding extracellular targeted protein degradation.

Extracellular targeted protein degradation (eTPD) is as an important new modality for manipulating the extracellular proteome. However, most eTPD receptors are expressed broadly or are restricted to the liver. Cytokine receptor targeting chimeras (kineTACs) are genetically encoded bispecifics for eTPD that fuse a natural ligand like CXCL12 to an antibody, directing soluble or membrane proteins for lysosomal degradation using the widely expressed chemokine receptor CXCR7 (Pance K, Gramespacher JA., Byrnes, JR, Salangsang F., Serrano JAC, Cotton AD, Steri V, and Wells JA, Nat. Biotechnol. 2023, 41, 273-281). Here, we dramatically expand the kineTAC toolbox by constructing 81 new kineTACs based on an unbiased list of cytokines, chemokines and growth factors. Remarkably, 55 of these expressed at suitable levels for analysis without any optimization. Many of these kineTACs bind receptors that have unique cell-type expression profiles, allowing for eTPD in specific cells and tissues and some were more potent than the original CXCL12-based kineTAC. We further show the internalizing capability of a kineTAC can enhance the performance of antibody drug conjugates. We believe these simple, genetically encoded tools will be useful for expanding the applications for optimized or cell type-selective eTPD.

synthetic biology↗

bioRxiv2025

Low-density lipoprotein receptor-targeting chimeras for membrane protein degradation and enhanced drug delivery

Antibody-based therapeutics encompass diverse modalities for targeting tumor cells. Among these, antibody-drug conjugates (ADCs) and extracellular targeted protein degradation (eTPD) specifically depend on efficient lysosomal trafficking for activity. However, many tumor antigens exhibit poor internalization, limiting ADC effectiveness. To address this, we developed low-density lipoprotein receptor-targeting chimeras (LIPTACs), leveraging the constitutive endocytic and recycling activity of the LDLR to enhance lysosomal delivery. LIPTACs enable efficient and selective degradation of diverse extracellular membrane proteins. Additionally, by coupling LIPTACs with cytotoxic payloads to generate degrader-drug conjugates, we can achieve superior intracellular delivery and enhanced cytotoxicity compared to conventional ADCs. The dual modality addresses key challenges of inadequate internalization in conventional ADCs and cytotoxic potency for current eTPD strategies. Our findings demonstrate that LDLR-mediated trafficking can enhance eTPD and ADCs, providing a hybrid blueprint for developing next-generation antibody therapeutics with broader utility and improved efficacy in cancer treatment.

bioengineering↗

bioRxiv2023

One-Hour Phosphoproteome Analysis with the Orbitrap Astral Mass Spectrometer

Owing to its roles in cellular signal transduction, protein phosphorylation plays critical roles in myriad cell processes. That said, detecting and quantifying protein phosphorylation has remained a challenge. We describe the use of a novel mass spectrometer (Orbitrap Astral) coupled with data-independent acquisition (DIA) to achieve rapid and deep analysis of human and mouse phosphoproteomes. With this method we map approximately 30,000 unique human phosphorylation sites within a half-hour of data collection. The technology was benchmarked to other state-of-the-art MS platforms using both synthetic peptide standards and with EGF-stimulated HeLa cells. We applied this approach to generate a phosphoproteome multi-tissue atlas of the mouse. Altogether, we detected 81,120 unique phosphorylation sites within 12 hours of measurement. With this unique dataset, we examine the sequence, structural, and kinase specificity context of protein phosphorylation. Finally, we highlight the discovery potential of this resource with multiple examples of novel phosphorylation events relevant to mitochondrial and brain biology.

systems biology↗

bioRxiv2022

Highly fucosylated N-glycans at the synaptic vesicle and neuronal plasma membrane

At neuronal synapses, synaptic vesicles (SVs) require glycoproteins for normal trafficking, and N-linked glycosylation is required for delivery of the major SV glycoproteins synaptophysin and SV2A to SVs. The molecular compositions of SV N-glycans, which may drive important neurobiological processes, are largely unknown. In this study, we combined organelle isolation techniques, fluorescence detection of N-glycans, and high-resolution mass spectrometry to characterize N-glycosylation at synapses and SVs from mouse brain. Detecting over 2,500 unique glycopeptides from over 550 glycoproteins, we found that abundant SV proteins harbor N-glycans with fucose on their complex antennae, and we identify a highly fucosylated N-glycan enriched in SVs as compared to synaptosomes. Antennary fucosylation was also characteristic of plasma membrane proteins and cell adhesion molecules with established roles in synaptic function and development. Our results represent the first defined N-glycoproteome of a neuronal organelle and raise new questions in the glycobiology of synaptic pruning and neuroinflammation.

neuroscience↗