Biology subjects

Elledge, S. J.

Publications and source records attributed to Elledge, S. J..

12 recordsLinked to original sources

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

The diverse functions of viral microproteins

A large fraction of the uncharacterized proteome consists of microproteins. Viruses, rich in microproteins with potent activities, provide a unique system for exploring their functions and evolution. Here, we systematically annotate viral microproteins through a phenotype-first approach: construction of a >50,000-element small ORF library spanning the microprotein-coding potential of human-infecting viruses, followed by broad screens that enrich candidates for high-resolution profiling by Perturb-seq, mass spectrometry, AI-guided structure prediction, and mechanistic studies. This atlas identified >2,000 active microproteins that modulate host pathways through diverse mechanisms, including molecular mimicry, hijacking of key regulators, and altering subcellular localization. We further show the oncogenic potential of BNLF2b, an uncharacterized Epstein-Barr virus microprotein linked to nasopharyngeal carcinoma. Three principles emerge: viral microproteins are diverse and often multifunctional, show widespread convergence toward shared functions, and serve as reservoirs of evolutionary innovation. These findings elucidate how viral microproteins occupy a large functional landscape, establish them as versatile tools for host manipulation, and provide a framework for functional annotation of uncharacterized proteins, contributing toward predictive systems virology.

systems biology↗

bioRxiv2026

Systems-Scale Structural Modeling Reveals the Germline Architecture of Immunodominance

The adaptive immune system generates diverse antibodies to protect against infection, yet responses often focus on a limited number of antigenic sites, a phenomenon called immunodominance. Using the SARS-CoV-2 receptor-binding domain as a model, this study combines large-scale antibody sequencing, deep mutational scanning, and AlphaFold 3 structural modeling to investigate the basis of immunodominant epitope selection. The results show that germline-encoded antibody features are a primary driver of immunodominance. Specifically, 76% of RBD-targeting antibodies display conserved gene segment usage and/or germline-encoded HCDR3 motifs. Structural analyses identified recurrent germline-encoded residues within these regions that interact with immunodominant epitopes, and mutating these residues eliminated binding. Mutations in SARS-CoV-2 variants frequently disrupt these interactions and are associated with immune escape; other mutations enable germline-mediated recognition and generate new immunodominant epitopes. These findings indicate that innate features, rather than diverse somatic mutations, determine binding specificity for the large majority of the antibody response and underlie antibody immunodominance.

immunology↗

bioRxiv2026

Changes in the Transcriptome and Synthetic Lethal Dependencies Following KRAS Mutant Expression Reveal Profound Tissue-Specificity

Oncogenic KRAS mutations exhibit a striking tissue-restricted tropism, occurring with high frequency in pancreatic, colorectal, and lung adenocarcinomas while remaining rare in other lineages. The molecular basis for why these specific tissues are uniquely permissive to KRAS transformation, and how this context shapes therapeutic vulnerabilities, remains poorly defined. Here, we utilized CRISPR-mediated genome engineering to generate endogenous, conditional KRAS-mutant isogenic cell line models across three primary permissive lineages (lung, colon, and pancreas) and the non-permissive breast lineage. Integrated genome-wide CRISPR fitness screens and comparative transcriptome analyses revealed that KRAS-driven synthetic lethal (SL) dependencies are profoundly shaped by their tissue of origin. Strikingly, we observed minimal overlap in SL hits across lineages, with only three genes shared among the permissive lines, suggesting that the KRAS oncogene operates through divergent, context-specific genetic networks. Mechanistically, we show that KRAS activation induces a universal MYC-driven metabolic signature, but the specific machinery required to sustain this state is lineage-restricted. We identified a dependency on the diphthamide synthesis pathway to maintain translational fidelity amidst a KRAS-induced hyper-translational state. These findings demonstrate that even when driven by the same oncogene, tumors exhibit distinct regulatory landscapes and unique genetic vulnerabilities. Our results provide a framework for developing lineage-aware therapeutic strategies, moving beyond universal KRAS inhibition toward targeted interventions tailored to a tumors specific tissue context. SIGNIFICANCE STATEMENTWhile KRAS mutations drive a significant portion of human malignancies, their prevalence is strikingly restricted to specific lineages, namely pancreatic, colorectal, and lung tissues. This tissue-restricted tropism suggests that oncogenic KRAS does not operate in a vacuum but requires a permissive, tissue-specific molecular landscape to sustain tumorigenesis. By integrating comparative transcriptome analyses with functional genomics across four isogenic lineages, we demonstrate that KRAS synthetic lethal dependencies are not universal but are hardwired to the cell of origin. This work establishes a framework for tissue lineage-aware oncology, shifting treatment paradigms from targeting the KRAS mutation alone to targeting the specific genetic networks, defined by the tissue of origin, that sustain KRAS-driven growth.

cancer biology↗

bioRxiv2026

Proteome-wide C-degron activity profiling connects conditional regulation of the CTLH E3 ligase complex to ribosome biogenesis

Many E3 ubiquitin ligases recognize cognate degron motifs located at protein termini, but the paucity of bona fide substrates of N-degron and C-degron pathways hampers our understanding of their physiological significance. Here, by devising an expression screening approach to assess the effect of C-terminal "capping" on the stability of thousands of human proteins, we systematically identify a suite of full-length substrates harboring C-terminal degrons. Interrogating one leading candidate, ZMYND19, we characterize a C-degron pathway governed by the Muskelin substrate adaptor of the CTLH E3 ligase complex. Cell-to-cell variability in ZMYND19 stability uncovered conditional regulation, with CTLH-mediated degradation impaired by TNF- stimulation but enhanced by mTOR inhibition. Parallel genetic and proteomic screens identified two poorly characterized proteins, AAMP and AEN, as additional substrates of the CTLHMuskelin C-degron pathway, leading us to define an essential role for AAMP in ribosome maturation through chaperone activity towards ribosomal protein uL16. Altogether, these data define a C-degron pathway through which the Muskelin substrate adaptor connects conditional regulation of the CTLH E3 ligase complex to control of ribosome biogenesis.

molecular biology↗

bioRxiv2025

Structural basis for the midnolin-proteasome pathway and its role in suppressing myeloma

The midnolin-proteasome pathway degrades many nuclear proteins without ubiquitination, but how it operates mechanistically remains unclear. Here, we present structures of the midnolin-proteasome complex, revealing how established proteasomal components are repurposed to enable a unique form of proteolysis. While the proteasomal subunit PSMD2/Rpn1 binds to ubiquitinated or ubiquitin-like proteins, we discover that it also interacts with the midnolin nuclear localization sequence, elucidating how midnolins activity is confined to the nucleus. Likewise, PSMD14/Rpn11, an enzyme that normally cleaves ubiquitin chains, surprisingly functions non-enzymatically as a receptor for the midnolin ubiquitin-like (Ubl) domain, positioning the substrate-binding Catch domain directly above the proteasomal entry site to guide substrates into the proteasome. Moreover, we demonstrate that midnolin downregulation is critical for the survival of myeloma cells by promoting the expression of its transcription factor substrate IRF4. Our findings uncover the mechanisms underlying the midnolin-proteasome pathway and midnolin downregulation as a driver of multiple myeloma.

cell biology↗

bioRxiv2024

Structure of the TXNL1-bound proteasome

Proteasomes degrade diverse proteins in different cellular contexts through incompletely defined regulatory mechanisms. Here, we report the cryo-EM structure of thioredoxin-like protein 1 (TXNL1) bound to the 19S regulatory particle of proteasomes via interactions with PSMD1/Rpn2, PSMD4/Rpn10, and PSMD14/Rpn11. These interactions are necessary for the ubiquitin-independent degradation of TXNL1 upon cellular exposure to metal- or metalloid-containing oxidative agents, thereby establishing a structural basis for the stress-induced degradation of TXNL1.

cell biology↗

bioRxiv2024

An aneuploidy epistasis map reveals metabolic vulnerabilities associated with supernumerary chromosomes in cancer

Despite the general detriment of aneuploidy to cellular fitness, >90% of solid tumors carry an imbalanced karyotype. Regardless of this existing paradox, our understanding of the molecular responses to aneuploidy remains limited. Here, we explore these cellular stresses and unique vulnerabilities in aneuploid human mammary epithelial cells (HMECs) enriched for breast cancer-associated copy number alterations (CNAs). To uncover the genetic dependencies specific to aneuploid cells, we conducted a comprehensive, genome-wide CRISPR knockout screen targeting isogenic diploid and aneuploid HMEC lines. Our study reveals that aneuploid HMECs exhibit an increased reliance on pyrimidine biosynthesis and mitochondrial oxidative phosphorylation genes, and demonstrate heightened fitness advantages upon loss of tumor suppressor genes. Using an integrative multi-omic analysis, we confirm nucleotide pool insufficiency as a key contributor to widespread cellular dysfunction in aneuploid HMECs with net copy number gain. While diploid cells can switch seamlessly between pyrimidine synthesis and salvage, cells with increased chromosomal content exhibit p53 activation and S-phase arrest when relying on salvage alone, and exhibit increased sensitivity to DNA-damaging chemotherapeutics. This work advances our understanding of the consequences of aneuploidy and uncovers potential avenues for patient stratification and therapeutic intervention based on tumor ploidy.

cancer biology↗

bioRxiv2024

Identification of MYC synthetic lethal genes and networks

MYC is a potent oncogene that is frequently overexpressed in human tumors arising in different tissues. To date there are no approved therapies to directly antagonize oncogenic MYC and its role in driving tumorigenesis. As an alternative approach we employed genetic screens using CRISPR and shRNA to identify the genes that are required for the survival and growth of cells harboring high levels of MYC expression. We find that cells with elevated MYC require the expression of many pro-growth and metabolic pathways including genes involved in mitochondrial citrate production and transport. This citrate producing pathway is critical for cells with elevated MYC to generate the necessary acetyl-CoA to drive the lipid synthesis required for increased proliferation. Inhibition of this pathway results in reduced proliferation and in vivo tumor growth providing a potential therapeutic strategy to target MYC-driven cancers. HIGHLIGHTS- CRISPR and shRNA screens identify synthetic lethal interactions with overexpressed MYC - MYC overexpressing cells are more sensitive to disruption of citrate production and transport - Inhibition of SLC25A1 reduces growth of MYC driven tumors

cancer biology↗

bioRxiv2024

Cholesterol biosynthesis inhibition synergizes with AKT inhibitors in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is responsible for a disproportionate number of breast cancer deaths due to its molecular heterogeneity, high recurrence rate and lack of targeted therapies. Dysregulation of the phosphoinositide 3-kinase (PI3K)/AKT pathway occurs in approximately 50% of TNBC patients. We performed a genome-wide CRISPR/Cas9 screen with PI3K and AKT inhibitors to find targetable synthetic lethalities in TNBC. We identified cholesterol homeostasis as a collateral vulnerability with AKT inhibition. Disruption of cholesterol homeostasis with pitavastatin synergized with AKT inhibition to induce TNBC cytotoxicity in vitro, in mouse TNBC xenografts and in patient-derived, estrogen receptor (ER)-negative breast cancer organoids. Neither ER-positive breast cancer cell lines nor ER-positive organoids were sensitive to combined AKT inhibitor and pitavastatin. Mechanistically, TNBC cells showed impaired sterol regulatory element-binding protein 2 (SREBP-2) activation in response to single agent or combination treatment with AKT inhibitor and pitavastatin. This was rescued by inhibition of the cholesterol trafficking protein Niemann-Pick C1 (NPC1). NPC1 loss caused lysosomal cholesterol accumulation, decreased endoplasmic reticulum cholesterol levels and promoted SREBP-2 activation. Taken together, these data identify a TNBC-specific vulnerability to the combination of AKT inhibitors and pitavastatin mediated by dysregulated cholesterol trafficking. Our work motivates combining AKT inhibitors with pitavastatin as a therapeutic modality in TNBC.

cancer biology↗

bioRxiv2020

KIAA1841, a novel SANT and BTB domain-containing protein, inhibits class switch recombination

Class switch recombination (CSR) enables B cells to produce different immunoglobulin isotypes and mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using a shRNA library screen in a mouse B cell line, we have identified the novel protein KIAA1841 (NM_027860) as a negative regulator of CSR. KIAA1841 is an uncharacterized protein of 82kD containing SANT and BTB domains. The BTB domain of KIAA1841 exhibited characteristic properties such as self-dimerization and interaction with co-repressor proteins. Overexpression of KIAA1841 inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR.

molecular biology↗

bioRxiv2018

Natural selection contributed to immunological differences between human hunter-gatherers and agriculturalists

The shift from a hunter-gatherer (HG) to an agricultural (AG) mode of subsistence is believed to have been associated with profound changes in the burden and diversity of pathogens across human populations. Yet, the extent to which the advent of agriculture may have impacted the evolution of the human immune system remains unknown. Here we present a comparative study of variation in the transcriptional responses of peripheral blood mononuclear cells to bacterial and viral stimuli between Batwa rainforest hunter-gatherers and Bakiga agriculturalists from Uganda. We observed increased divergence between hunter-gatherers and agriculturalists in the transcriptional response to viruses compared to that for bacterial stimuli. We demonstrate that a significant fraction of these transcriptional differences are under genetic control, and we show that positive natural selection has helped to shape population differences in immune regulation. Across the set of genetic variants underlying inter-population immune response differences, however, the signatures of positive selection were disproportionately observed in the rainforest hunter-gatherers. This result is counter to expectations based on the popularized notion that shifts in pathogen exposure due to the advent of agriculture imposed radically heightened selective pressures in agriculturalist populations.

evolutionary biology↗