Biology subjects

Fischer, E. S.

Publications and source records attributed to Fischer, E. S..

At least 19 recordsLinked to original sources

bioRxiv2026

The mechanism of cysteine sensing by LRRC58

Cysteine is essential for life but toxic in excess. Cells and tissues are exposed to elevated cysteine from endogenous sources and the environment yet maintain low intracellular concentrations to sustain life. Here we identify a mechanism for sensing cysteine to control its abundance and prevent toxicity and premature death. Using cryo-electron microscopy we determine structures of the CRL5L^RRC58 E3 ligase alone and in complex with the cysteine-catabolizing enzyme CDO1. We show that CDO1 interacts with LRRC58 in the vicinity of a zinc-coordinating cysteine motif, within which Cys296 becomes covalently modified upon cysteine exposure. Covalent modification of Cys296 and zinc ejection from this motif drive large structural rearrangements of LRRC58 relayed through Cys319, which destabilize CDO1 binding and position LRRC58 Lys111 for autoubiquitylation. This reorientation of LRRC58 permits CDO1 accumulation, thereby lowering intracellular cysteine. Mutation of Cys319 abrogates inhibitory rearrangement of LRRC58 upon cysteine modification. This renders the ligase constitutively active and prevents CDO1 stabilization, thereby driving systemic cysteine accumulation and premature death in animals. These findings establish the molecular basis of homeostatic cysteine sensing by LRRC58 and demonstrate its essential role in maintaining cysteine concentrations compatible with healthy life.

cell biology↗

bioRxiv2026

Proteasomal surveillance of exposed cysteines

Cysteine residues are critical for both the catalytic activity and structural stability of many proteins, yet their high chemical reactivity threatens proteome integrity. Whether cells have specialized mechanisms to recognize and eliminate proteins with aberrant or exposed cysteines is unknown. By systematically analyzing the stability of thousands of cysteine-containing peptides, here we identify two complementary surveillance mechanisms targeting proteins with exposed cysteine residues. First, a set of E3 ubiquitin ligases, including AMBRA1, TRPC4AP, FBXO3, and FBXO21, detects double cysteines and targets proteins for ubiquitin-dependent proteasomal degradation. Second, the PSMD14/Rpn11 subunit of the 26S proteasome recognizes exposed cysteines in a ubiquitin-independent manner. Both pathways function in quality control to clear misfolded proteins exposing cysteine residues, including zinc finger-containing proteins. Mutational escape from the ubiquitin-independent pathway through dysregulation of ornithine decarboxylase or FOS levels is responsible for two human diseases, Bachmann-Bupp Syndrome and osteoblastoma, respectively. Our findings reveal conserved mechanisms for degrading proteins with exposed cysteines and uncover an alternative mode of substrate recognition by the 26S proteasome.

cell biology↗

bioRxiv2026

Deep-learning-based design of an orthogonal self-labeling protein from K-Ras(G12C)

Self-labeling protein (SLP) tags enable versatile labeling of proteins in live cells, yet only two SLPs are commonly used, limiting multiplexing. Here, we repurposed the oncoprotein K-Ras(G12C) and its covalent inhibitors to create a third, orthogonal SLP system. We used a deep-learning-based approach to radically redesign the sequence of K-Ras(G12C) while preserving its covalent-inhibitor binding pocket. Our top design, LUCI-tag, is a 19 kDa, monomeric, thermostable SLP that rapidly and covalently reacts with commercially available K-Ras(G12C) inhibitors bearing diverse payloads. Unlike existing SLPs, LUCI-tag exhibited payload-agnostic labeling kinetics, outperforming HaloTag7 and SNAP-tag for a negatively charged payload. X-ray crystal structures of drug-bound and drug-free LUCI-tag showed the design was structurally accurate and contained a preorganized inhibitor-binding pocket that could explain its rapid labeling kinetics. Proteomics and cell-signaling experiments confirmed LUCI-tag is biologically inert. LUCI-tag enabled rapid, wash-free, live-cell imaging and simultaneous three-color multiplexed experiments with HaloTag7 and SNAP-tag. This work establishes LUCI-tag as an immediately useful orthogonal SLP and demonstrates that covalent drug-target pairs can be repurposed into a broadly applicable platform for protein labeling.

bioengineering↗

bioRxiv2026

Unbiased, systematic surfaceome profiling of Acute Leukemia to identify novel immunotherapy targets

Cell surface proteomics provides a direct topological assessment of the outer membrane of cells and enables the capture of low abundance proteins that may be missed by whole cell proteomics. Here we present an unbiased atlas of the whole cell and surface proteomes of 25 commonly used leukemic cell lines, encompassing both lymphoid and myeloid lineages, and a variety of driver mutations. Paired-wise analysis highlights recurrent surface proteins that are not detected by whole cell proteomics. Coupling this dataset to RNA-sequencing, we also discovered genes where protein and RNA abundances are discordant. In KMT2A-rearranged AML, CD70 expression was increased across cell lines and validated in primary patient samples, supporting CD70 as a candidate therapeutic target in this disease. Several proteins are enriched in the surface proteomes but lack surface annotation, adding to the growing list of potential non-canonical cell surface proteins. These findings reveal a substantial pool of proteins absent from conventional surface annotations, including RNA-binding proteins, an emerging class of candidate immunotherapeutic targets.

molecular biology↗

bioRxiv2026

Covalent remodeling of CRBN creates a non-canonical neosubstrate interface with NTAQ1

Molecular glue degrader EM12-FS covalently modifies cereblon (CRBN) His353, enabling selective recruitment of the neosubstrate NTAQ1 to the CRL4CRBN ubiquitin ligase. We determined the cryo-EM structure of the NTAQ1-EM12-FS-CRBN-DDB1 complex, revealing a non-canonical neosubstrate interface created by covalent remodeling of the CRBN sensor loop. Imidazylation repositions His353 to eliminate the steric clash that prevents NTAQ1 engagement by reversible IMiDs, and the engineered interface is stabilized by a distinctive T-shaped C-H/{pi} interaction between sulfated His353 and NTAQ1 Phe126. Biochemical and mutational analyses define the determinants of ternary complex formation and ubiquitination. These findings show that site-specific synthetic modification of CRBN can reprogram induced-proximity pharmacology, expanding specificity beyond the G-loop degron and establishing a framework for covalent engineering of new degrader modalities.

biochemistry↗

bioRxiv2026

Distinct Proteasomal Pathways Drive Oncogenic PPM1D Activation

PPM1D is a serine/threonine phosphatase and DNA damage response (DDR) regulator recurrently activated in cancer through amplification or C-terminal truncating mutations that increase its abundance. Here we show that truncating mutations fundamentally rewire PPM1D proteostasis, unmasking an oncogenic function of an alternative protein degradation pathway. While full-length PPM1D undergoes rapid ubiquitin-independent proteasomal degradation via a C-terminal degron, truncating mutations redirect degradation to a slower UBR5-mediated ubiquitin-dependent pathway. The resulting accumulation of PPM1D suppresses DDR signaling and enhances cellular fitness under genotoxic stress, which is further amplified by UBR5 loss. Consistent with selective pressure on this axis, cancers harboring PPM1D truncating mutations are enriched for UBR5 loss-of-function mutations. Together, these findings identify escape from ubiquitin-independent proteasomal degradation as a mechanism of oncogenic adaptation and establish proteostatic routing as a regulatory layer linking protein degradation, DDR signaling, and cancer evolution.

cell biology↗

bioRxiv2026

Z-TAC enables custom and combinatorial degradation of cell surface proteins

Cell-surface degrader platforms typically require target-specific engineering and have therefore been applied to a relatively small set of protein targets. Here we report Z-TAC, a strategy that enables plug-and-play conversion of existing IgG antibodies into cell-surface protein degraders. Across multiple targets from distinct protein families, Z-TAC induced efficient and sustained degradation of both individual receptors and receptor combinations. For a multi-pass membrane receptor lacking selective antagonists, Z-TAC mediated complete receptor degradation and functional inhibition, demonstrating the ability of this platform to overcome the limitations of conventional pharmacological approaches. This study delineates a generalizable and scalable strategy for functional perturbation of the cell-surface proteome.

bioengineering↗

bioRxiv2026

An epigenetic bifunctional that toggles between transactivation and repression

The targeted modulation of gene expression with bifunctional small molecules enables the precise control of cellular and biological processes. To screen for ligands that could be used to induce gene expression, we conjugated the high affinity FKBP(F36V) binder, AP1867, to known high-affinity binders of activating epigenetic machinery. We tested these bifunctionals in a FKBP(F36V)-tagged transcription factor reporter system and found bifunctional induced transactivation is relatively common, being observed for bifunctionals with BET ligand JQ1, p300/CBP ligand GNE-781, CDK9 ligand SNS-032, and BRD9 ligand iBRD9. aTAG-2 (mAP1867-C8-GNE781) was identified as the strongest and most potent transactivator, possessing single-digit nanomolar activity. When tested in models where oncogenic RNA binding protein-transcription factor fusion proteins have been FKBP(F36V)-tagged, we unexpectedly observed rapid collapse of the fusion transcriptional program. In a tagged Ewing sarcoma model, aTAG-2 exhibits at least three distinct mechanisms of action: i) RIPTAC mediated p300/CBP inhibition, ii) ubiquitination- and ternary complex-dependent EWS/FLI degradation, and iii) replacement of p300 with CBP at EWS/FLI bound chromatin loci. Together, these data establish bifunctionals targeting p300/CBP that toggle between a program of ultra-potent transactivation and repression depending on cellular context. Overall demonstrating that induced proximity with a given ligand does not encode a fixed functional outcome.

biochemistry↗

bioRxiv2025

Development of Degraders and 2-pyridinecarboxyaldehyde (2-PCA) as a recruitment Ligand for FBXO22

Targeted protein degradation (TPD) is a promising therapeutic strategy that requires the discovery of small molecules that induce proximity between E3 ubiquitin ligases and proteins of interest. FBXO22 is an E3 ligase that is overexpressed in many cancers and implicated in tumorigenesis. While FBXO22 was previously identified as capable of recognizing ligands bearing a primary amine degron, further investigation and development of recruitment ligands is required to enable its broader utility for TPD. Here, we describe the discovery of chemical probes that can either selectively degrade FBXO22 or recruit this ligase for TPD applications. First, we describe AHPC(Me)-C6-NH2 as a potent and selective FBXO22 degrader (DC50 = 77 nM, Dmax = 99%) that is suitable for interrogating the effects of FBXO22 loss of function. Further, we discovered that the simple hexane-1,6-diamine acts as a minimal FBXO22 self-degrader, whereas shorter C4 (putrescine) to C5 (cadaverine) analogs, found in mammalian cells, do not induce degradation. Finally, we found that 2-pyridinecarboxaldehyde (2-PCA) functions as a novel electrophilic degron capable of forming a reversible thioketal with cysteine 326 for recruiting FBXO22. Conjugating 2-PCA to various ligands successfully induced FBXO22-dependent degradation of BRD4 and CDK12. Collectively, these chemical probes will facilitate the study of FBXO22 biology and broaden its applicability in TPD.

biochemistry↗

bioRxiv2025

LuxSit Pro and sPro: Next-Generation Designed Luciferases for Bioluminescent Reporting and Complementation-Based Detection

Bioluminescent systems are widely used as molecular reporters in life sciences due to their low background, broad dynamic range, and the key advantage of not requiring external excitation. However, natural luciferases often lack the brightness, stability, and long-lasting light emission needed to support a wide range of high-sensitivity applications. Here, we describe our next-generation luciferase solution: LuxSit Pro and its substrate, Luxterazine (LTZ). LuxSit Pro is a de novo luciferase optimized for steady brightness and structural stability through an iterative combination of computational and experimental methods. Composed of only 117 amino acids, LuxSit Pro is currently the smallest highest-performing known luciferase, exhibiting exceptional brightness and biochemical stability with robust recombinant expression in both mammalian and bacterial systems. Notably, LuxSit Pro was deliberately designed devoid of lysine and cysteine residues which enormously expands its potential applications. This property renders the reporter largely resistant to post-translational modifications such as ubiquitination and also enables site-directed chemical modifications, as lysines or cysteines can be reintroduced at specific sites to serve as chemical handles for augmenting or adapting the enzymes function. The novel synthetic substrate, Luxterazine (LTZ), is a compound that enables LuxSit Pro to deliver bright, steady, blue light emission ([~]490 nm) and offers improved solubility compared to existing Coelenterazine analogs. Finally, we also developed LuxSit sPro, a highly sensitive two-component complementation system for the quantitative detection of molecular interactions. The broad potential applications of LuxSit Pro and sPro, in combination with Luxterazine, are poised to drive the development of next-generation reporters and biosensor systems for the research and in vitro diagnostics fields.

molecular biology↗

bioRxiv2025

Decoding protein-peptide interactions using a large, target-agnostic yeast surface display library

Protein-peptide interactions underlie key biological processes and are commonly utilized in biomedical research and therapeutic discovery. It is often desirable to identify peptide sequence properties that confer high-affinity binding to a target protein. However, common approaches to such characterization are typically low throughput and only sample regions of sequence space near an initial hit. To overcome these challenges, we built a yeast surface displayed library representing [~]6.1 x 109 unique peptides. We then performed screens against diverse protein targets, including two antibodies, an E3 ubiquitin ligase, and an essential membrane-bound bacterial enzyme. In each case, we observed motifs that appear to drive peptide binding and we identified multiple novel, high-affinity clones. These results highlight the librarys utility as a robust and versatile tool for discovering peptide ligands and for characterizing protein-peptide binding interactions more generally. To enable further studies, we will make the library freely available upon request.

bioengineering↗

bioRxiv2024

SMAC mimetics induce human macrophages to phagocytose live cancer cells

Macrophages engulf apoptotic bodies and cellular debris as part of homeostasis, but they can also phagocytose live cells such as aged red blood cells. Pharmacologic reprogramming with the SMAC mimetic LCL161 in combination with T cell-derived cytokines can induce macrophages to phagocytose live cancer cells in mouse models. Here we extend these findings to encompass a wide range of monovalent and bivalent SMAC mimetic compounds, demonstrating that live cell phagocytosis is a class effect of these agents. We demonstrate robust phagocytosis of live pancreatic and breast cancer cells by primary human macrophages across a range of healthy donors. Unlike mouse macrophages where combination of SMAC mimetics with lymphotoxin enhanced phagocytosis, human macrophages were more efficiently polarized to phagocytose live cells by the combination of SMAC mimetics and IFN{psi}. We profiled phagocytic macrophages by transcriptional and proteomic methodologies, uncovering a positive feedback loop of autocrine TNF production.

immunology↗

bioRxiv2024

Structural ubiquitin contributes to K48 linkage-specificity of the HECT ligase Tom1

Homologous to E6AP C-Terminus (HECT) ubiquitin ligases play key roles in essential pathways such as DNA repair, cell cycle control or protein quality control. Tom1 is one of five HECT ubiquitin E3 ligases encoded in the S. cerevisiae genome and prototypical for a ligase with pleiotropic functions such as ubiquitin chain amplification, orphan quality control and DNA damage response. Structures of full-length HECT ligases, including the Tom1 ortholog HUWE1, have been reported, but how domains beyond the conserved catalytic module contribute to catalysis remains largely elusive. Here, through cryogenic electron microscopy (cryo-EM) of Tom1 during an active ubiquitylation cycle, we demonstrate that the extended domain architecture directly contributes to activity. We identify a Tom1-ubiquitin architecture during ubiquitylation involving a non-canonical ubiquitin binding site in the solenoid shape of Tom1. We demonstrate that this ubiquitin binding site coordinates a structural ubiquitin contributing to the fidelity of K48 poly-ubiquitin chain assembly.

biochemistry↗

bioRxiv2024

Ternary complex formation of VCP, VCPIP1 and p47 facilitates Golgi reassembly

VCP/p97 regulates a wide range of cellular processes, including post-mitotic Golgi reassembly. In this context, VCP is assisted by p47, an adapter protein, and VCPIP1, a deubiquitinase (DUB). However, how they organize into a functionally competent complex and promote reassembly remains unknown. Here, we use cryo-EM to characterize VCP-VCPIP1 and VCP-VCPIP1-p47 complexes. We show that VCPIP1 engages VCP through two interfaces: one involving the N-domain of VCP and the UBX domain of VCPIP1, and the other involving the VCP D2 domains and a region of VCPIP1 we refer to as VCPID. The p47 UBX domain competitively binds to the VCP N-domain, while not affecting VCPID binding. We show that VCPID is critical for VCP-mediated enhancement of DUB activity and Golgi reassembly.

biochemistry↗

bioRxiv2024

Development of potent and selective CK1α Molecular Glue Degraders

Molecular glue degraders (MGDs) are small molecules that facilitate proximity between a target protein and an E3 ubiquitin ligase thereby inducing target protein degradation. Glutarimide-containing compounds are MGDs that bind to cereblon (CRBN) and recruit neosubstrates. Through explorative synthesis of a glutarimide-based library, we discovered a series of molecules that induce casein kinase 1 alpha (CK1) degradation. By scaffold hopping and rational modification of the chemical scaffold, we identified an imidazo[1,2-a]pyrimidine compound that induces potent and selective CK1 degradation. A structure-activity relationship study of the lead compound, QXG-6442, identified the structural features that contribute to degradation potency and selectivity compared to other frequently observed neosubstrates. The glutarimide library screening and structure activity relationship medicinal chemistry approach we employed is generally useful for developing new molecular glue degraders towards new targets of interest.

biochemistry↗

bioRxiv2024

High-throughput diversification of protein-ligand surfaces to discover chemical inducers of proximity

Chemical inducers of proximity (CIPs) stabilize biomolecular interactions, often causing an emergent rewiring of cellular biochemistry1,2. While rational design strategies can expedite the discovery of heterobifunctional CIPs, monovalent, molecular glue-like CIPs have relied predominantly on serendipity3. Envisioning a prospective approach to discover molecular glues for a pre-selected target, we hypothesized that pre-existing ligands could be systematically decorated with chemical modifications to empirically discover protein-ligand surfaces that are tuned to cooperatively engage another protein interface. Here, we used sulfur(VI)-fluoride exchange (SuFEx)-based high-throughput chemistry (HTC) to install 3,163 structurally diverse chemical building blocks onto ENL and BRD4 ligands and then screened the crude products for degrader activity. This revealed dHTC1, a potent, selective, and stereochemistry-dependent degrader of ENL. It recruits CRL4CRBN to ENL through an extended interface of protein-protein and protein-ligand contacts, but only after pre-forming the ENL:dHTC1 complex. We also characterized two structurally distinct BRD4 degraders, including dHTC3, a molecular glue that selectively dimerizes the first bromodomain of BRD4 to SCFFBXO3, an E3 ligase not previously accessible for chemical rewiring. Altogether, this study introduces HTC as a facile tool to discover new CIPs and actionable cellular effectors of proximity pharmacology.

biochemistry↗

bioRxiv2024

Discovery of electrophilic degraders that exploit SNAr chemistry

Targeted covalent inhibition (TCI) and targeted protein degradation (TPD) have proven effective in pharmacologically addressing formerly undruggable targets. Integration of both methodologies has resulted in the development of electrophilic degraders where recruitment of a suitable E3 ubiquitin ligase is achieved through formation of a covalent bond with a cysteine nucleophile. Expanding the scope of electrophilic degraders requires the development of electrophiles with tempered reactivity that enable selective ligase recruitment and reduce cross-reactivity with other cellular nucleophiles. In this study, we report the use of chemical moieties that enable nucleophilic aromatic substitution (SNAr) reactions in the rational design of electrophilic protein degraders. Appending an SNAr covalent warhead to several preexisting small molecule inhibitors transformed them into degraders, obviating the need for a defined E3 ligase recruiter. The SNAr covalent warhead is versatile; it can recruit various E3 ligases, including DDB1 and CUL4 associated factor 11 (DCAF11), DDB1 and CUL4 associated factor 16 (DCAF16), and possibly others. The incorporation of an SNAr covalent warhead into the BRD4 inhibitor led to the discovery of degraders with low picomolar degradation potency. Furthermore, we demonstrate the broad applicability of this approach through rational functional switching from kinase inhibitors into potent degraders.

biochemistry↗

bioRxiv2024

Charged Molecular Glue Discovery Enabled by Targeted Degron Display

Small molecules that induce protein interactions hold tremendous potential as new medicines, as probes for molecular pathways, and as tools for agriculture. Explosive growth of targeted protein degradation (TPD) drug development has spurred renewed interest in proximity-inducing molecules and especially Molecular Glue Degraders (MGDs). These compounds catalyze destruction of disease-causing proteins by reshaping protein surfaces and promoting cooperative binding between ubiquitylating enzymes and target proteins. MGD discovery for pre-defined targets is a major challenge in contemporary drug discovery. The field is limited by a lack of approaches that can exploit charged ligand-binding pockets, thus excluding a major fraction of ubiquitin ligases (E3s) that evolved to recognize exceedingly common acidic and basic degrons. Here we solve these important chemical challenges through "chemocentric" MGD discovery of ZZ1, a BET-family protein degrader and a prodrug of a negatively charged glue (c-Glue). ZZ1 activation unmasks a sulfinic acid moiety that binds the modular GID/CTLH ubiquitin ligase complex via a basic pocket in its YPEL5 subunit. YPEL5 is a CRBN structural homolog and an essential non-Cullin ubiquitin ligase cofactor expressed in cancers of the bone marrow. These findings demonstrate a previously unrecognized capacity of YPEL5 to recruit GID/CTLH substrates, and they provide a powerful strategy to discover c-Glues that induce proximity to ubiquitin ligases with similarly desirable properties.

biochemistry↗