Abstract Cellular responses to amino acid fluctuations often hinge on ubiquitin-mediated control of metabolic enzymes, yet the underlying E3 ligase pathways remain poorly defined. Using quantitative proteomics and active cullin-RING ligase (CRL) profiling, we identify LRRC58 as a cysteine-responsive substrate receptor whose stability increases sharply under cysteine starvation. Proteomics reveals an inverse relationship between LRRC58 and the metabolic enzyme cysteine dioxygenase 1 (CDO1), suggesting a cysteine-linked regulatory axis. Biochemical reconstitution and cryo-EM structures show that LRRC58 forms an active CUL2- or CUL5-based CRL that selectively positions CDO1 for ubiquitylation at Lys8. Disease mutant versions of CDO1 mapping to the LRRC58 interface and impaired for the endogenous ubiquitylation pathway were degraded through orthogonal targeting by a VHL-based degrader. Together, our proteomics-guided discovery pipeline, cellular stability studies, and structural analyses uncover a metabolically-tuned LRRC58-CDO1 pathway that links cysteine availability to selective proteasomal turnover, reveals principles of metabolite-regulated CRL activity, and showcases mechanisms distinguishing endogenous and targeted protein degradation.

Targeted protein degradation modulates protein function beyond the inhibition of enzyme activity or protein–protein interactions. Most degrader drugs function by directly mediating the proximity between a neosubstrate and a hijacked E3 ligase. Here we identify pseudo-natural products derived from (−)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2KLHDC3, which we identified to natively mediate ubiquitin-mediated degradation of IDO1. Therefore, iDegs increase IDO1 turnover using the native proteolytic pathway. In contrast to clinically explored IDO1 inhibitors, iDegs reduce the formation of kynurenine by both inhibition and induced degradation of the enzyme and thus also modulate the non-enzymatic functions of IDO1. This unique mechanism of action may open up alternative therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1. In targeted protein degradation, a degrader molecule brings a neosubstrate protein proximal to a hijacked E3 ligase for its ubiquitination. Here, pseudo-natural products derived from (−)-myrtanol—iDegs—are identified to inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs prime apo-IDO1 ubiquitination and subsequent degradation using its native proteolytic pathway.

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that down-regulate target RNAs, including messenger RNAs from most human genes1-3. Within each complex, the miRNA pairs to target RNAs, and AGO provides effector function while also protecting the miRNA from cellular nucleases2-5. Although much is known about miRNA-directed gene regulation, less is known about how miRNAs themselves are regulated. One pathway that regulates miRNAs involves unusual targets called 'trigger' RNAs, which reverse the canonical regulatory logic and instead down-regulate miRNAs6-9. This target-directed miRNA degradation (TDMD) is thought to require a cullin-RING E3 ligase because it depends on the cullin protein CUL3 and other ubiquitylation components, including the BC-box protein ZSWIM8 (refs. 10,11). ZSWIM8 is required for murine perinatal viability and for destabilization of most short-lived miRNAs, which suggests biological importance of TDMD11-13. Here, biochemical and cellular assays establish AGO binding and polyubiquitylation by the ZSWIM8-CUL3 E3 ligase as the key regulatory steps of TDMD, and thereby define a unique cullin-RING E3 ligase class. Cryogenic electron microscopy analyses show ZSWIM8 recognizing distinct AGO and RNA conformations shaped by pairing of the miRNA to the trigger. Specificity of AGO ubiquitylation is established through generalizable RNA-RNA, RNA-protein and protein-protein interactions. The substrate features recognized by the E3 ligase do not conform to a conventional degron14,15 but instead establish a two-RNA-factor authentication mechanism for specifying a protein ubiquitylation substrate.

Small molecules toggling the ubiquitin-proteasome system (UPS) are powerful regulators of protein degradation. Yet, mechanistic knowledge of how endogenous ligands gate UPS decisions remains rudimentary. Here, we define control of UPS access to Tryptophan-2,3-dioxygenase (TDO2), which converts the essential amino acid tryptophan (Trp) to N-formylkynurenine. When Trp concentrations are limiting, TDO2 is degraded to avert tryptophanemia. Using CRISPRi screening and biochemistry, we identify a CK2-FBXW11 kinase-E3 ligase cascade that generates and recognizes tandem TDO2 phosphodegrons when not protected by Trp. Trp binding to an exosite safeguards TDO2 from phosphorylation-dependent ubiquitylation. Effects of Trp analogs on CK2-FBXW11-dependent ubiquitylation indicated that the indole, amino, and carboxylate groups are necessary for substrate shielding. Cryo-EM reveals how these moieties order a region proximal to the phosphodegrons; without Trp, this segment is flexible, enabling phosphorylation-coupled ubiquitylation. Overall, our data uncovered an endogenous small molecule allosterically stabilizing its own metabolizing enzyme through protection from a phosphorylation-ubiquitylation cascade. ### Competing Interest Statement B.A.S. is a member of the scientific advisory boards of Proxygen and Lyterian. The other authors declare no competing interests. Max Planck Society, https://ror.org/01hhn8329 European Union, ERC AdvG, UPSmeetMet, 101098161 to BAS Boehringer Ingelheim Fonds, https://ror.org/00dkye506

PROTACs are commonly developed by linking E3 ligase-recruiting ligands to established inhibitors of a protein target, often resulting in degraders that retain enzymatic inhibition. Type II inhibition of cyclin-dependent kinases (CDKs) has been challenging, as reported compounds generally exhibit weak biochemical potency and limited cellular activity. Consistent with these limitations, most reported CDK degraders have been derived from type I ATP-competitive inhibitors. Here, we explored whether targeted protein degradation could enable functional CDK targeting from a type II kinase scaffold. Using the multikinase inhibitor regorafenib as a starting scaffold, we generated a focused library of CRL4CRBN-recruiting bifunctional molecules and profiled their degradation activity using quantitative mass spectrometry-based proteomics. This analysis unexpectedly revealed CDK5 and CDK6, kinases not inhibited by the parent scaffold, as degradation targets. Optimization of this series led to JHK-02-108-2, a selective CDK6 degrader that does not display a hook effect and promotes potent CDK6 degradation despite weak CDK6 binding and negligible CDK6 inhibition. In cellular models of acute myeloid leukemia (AML) and glioblastoma, JHK-02-108-2 induced sustained G1 arrest and reduced phosphorylation of the retinoblastoma protein. Interestingly, subtle modifications in PROTAC architecture redirected degradation selectivity, yielding JHK-02-102-1 as a selective type II CDK5 degrader derived from the same scaffold. Together, these findings establish the first type II inhibitor-derived selective CDK6 degrader and demonstrate that targeted protein degradation can enable functional CDK targeting from type II kinase scaffolds.

FBXO7 and PI31 variants are linked to rare Parkinsonian syndromes, implicating their dysfunction in neurodegeneration. We define how both engage each other and regulate the proteasome 20S core particle (CP). In cells, each can associate independently with the proteasome, with multiple domains in FBXO7 contributing. Utilizing cryo-EM we visualized how FBXO7's C-terminal domain engages multiple subunits within the CP interior, blocking the β5 peptidase activity. In contrast, we visualized PI31 engagement of all three catalytic sites within the 20S CP, revealing the previously unknown structural basis for β1 inhibition. Furthermore, we establish how disease-associated variants impact both FBXO7 and PI31 function, including disruption of proteasome inhibition and SKP1-FBXO7-PI31 complex assembly. These results establish an unexpected function for FBXO7, providing a mechanistic basis for investigation of its role in proteasome regulation in Parkinson's disease.

Many proteins localize in membraneless organelles. However, understanding the steps along membraneless organelle formation-and the structural impact on granule constituents-has been hindered by limited resolution of intracellular data. We address these challenges through in situ cryo-electron tomography (cryo-ET) along with formation of yeast proteasome storage granules (PSGs). During the transition from proliferation to quiescence, doubly capped 26S proteasomes arrested in an inactive state arrange into ∼7.5 MDa trimeric units, dispersed in the nucleoplasm and congregated along the nuclear envelope near the nuclear pore. 9-Å-resolution cryo-ET structures reveal that cytoplasmic PSGs formed in various energy-limiting conditions are paracrystalline arrays of bundled fibers, assembled from stacking of proteasome trimers. The paracrystalline arrangement maintains a pool of fully assembled inactive 26S proteasomes that are released in energy-rich conditions. Overall, our data reveal structural steps along the assembly of an intracellular membraneless organelle in situ and quinary structure formation controlling a major eukaryotic regulatory machine.

Small molecules that induce protein interactions hold tremendous potential as new medicines, probes for molecular pathways and tools for agriculture. Explosive growth of targeted protein degradation drug development has spurred renewed interest in proximity-inducing molecules, especially molecular glue degraders (MGDs). These compounds catalyze the destruction of disease-causing proteins by reshaping protein surfaces and promoting cooperative binding between ubiquitylating enzymes and target proteins. MGD discovery for predefined targets is a major challenge in contemporary drug discovery. Here, we solve this important chemical challenge through 'chemocentric' MGD discovery of ZZ1, a BET-family protein degrader and a prodrug of a negatively charged glue. ZZ1 activation unmasks a sulfinic acid that binds the modular CTLH ubiquitin ligase complex through a basic pocket in its YPEL5 subunit. These findings demonstrate a previously unrecognized capacity of YPEL5 to recruit CTLH substrates and enable the discovery of MGDs for exceedingly common acidic and basic degrons.

E3 ligases partner with E2 enzymes to regulate vast eukaryotic biology. The hierarchical nature of these pairings, with >600 E3s and ~40 E2s in humans, necessitates that E2s cofunction with numerous different E3s. Here, focusing on E3s in the RING-between-RING (RBR) family and their partner UBE2L3 and UBE2D-family E2s, we report an approach to interrogate selected pathways. We screened phage-displayed libraries of structure-based E2 variants (E2Vs) to discover enzymes with enhanced affinity and specificity toward half of all RBR E3 ligases (ARIH1, ARIH2, ANKIB1, CUL9, HOIL1, HOIP, and RNF14). Collectively, these E2Vs allowed distinguishing actions of different cofunctioning E3s, obtaining high-resolution cryogenic Electron Microscopy (cryo-EM) structures of an RBR E3 in the context of a substrate-bound multiprotein complex, and profiling an endogenous RBR E3 response to an extracellular stimulus. Overall, we anticipate that E2V technology will be a generalizable tool to enable in-depth mechanistic and structural analysis of E3 ligase functions, and mapping their activity states and protein partners in cellular signaling cascades.

To define and systematically characterize the human E3 ubiquitin ligase (E3) landscape, we generated the E3-ome, a compendium of E3s encoded by the human genome. The E3-ome integrates experimental data, bioinformatics, and published research, revealing 672 high-confidence E3s. We standardized E3 classifications to create a unified framework for annotation and comparative analysis. The E3-ome identified several previously unrecognized domains, motifs, E3 candidates, and relationships, expanding the diversity of E3s. Furthermore, the E3-ome mapped the spatial and physiological organization of E3s across human tissues and cell types, revealing context-dependent E3s. Genetic analyses identified disease-associated variants across the E3-ome, linking E3s to diverse human pathologies. Together, these analyses define the human E3 landscape at high resolution and deliver a foundational resource to drive mechanistic and therapeutic discovery.

During normal cellular homeostasis, unfolded and mislocalized proteins are recognized and removed, preventing the build-up of toxic byproducts1. When protein homeostasis is perturbed during ageing, neurodegeneration or cellular stress, proteins can accumulate several forms of chemical damage through reactive metabolites2,3. Such modifications have been proposed to trigger the selective removal of chemically marked proteins3-6; however, identifying modifications that are sufficient to induce protein degradation has remained challenging. Here, using a semi-synthetic chemical biology approach coupled to cellular assays, we found that C-terminal amide-bearing proteins (CTAPs) are rapidly cleared from human cells. A CRISPR screen identified FBXO31 as a reader of C-terminal amides. FBXO31 is a substrate receptor for the SKP1-CUL1-F-box protein (SCF) ubiquitin ligase SCF-FBXO31, which ubiquitylates CTAPs for subsequent proteasomal degradation. A conserved binding pocket enables FBXO31 to bind to almost any C-terminal peptide bearing an amide while retaining exquisite selectivity over non-modified clients. This mechanism facilitates binding and turnover of endogenous CTAPs that are formed after oxidative stress. A dominant human mutation found in neurodevelopmental disorders reverses CTAP recognition, such that non-amidated neosubstrates are now degraded and FBXO31 becomes markedly toxic. We propose that CTAPs may represent the vanguard of a largely unexplored class of modified amino acid degrons that could provide a general strategy for selective yet broad surveillance of chemically damaged proteins.

Lysosomal storage diseases (LSDs) comprise ~50 monogenic disorders marked by the buildup of cellular material in lysosomes, yet systematic global molecular phenotyping of proteins and lipids is lacking. We present a nanoflow-based multiomic single-shot technology (nMOST) workflow that quantifies HeLa cell proteomes and lipidomes from over two dozen LSD mutants. Global cross-correlation analysis between lipids and proteins identified autophagy defects, notably the accumulation of ferritinophagy substrates and receptors, especially in NPC1-/- and NPC2-/- mutants, where lysosomes accumulate cholesterol. Autophagic and endocytic cargo delivery failures correlated with elevated lysophosphatidylcholine species and multilamellar structures visualized by cryo-electron tomography. Loss of mitochondrial cristae, MICOS complex components, and OXPHOS components rich in iron-sulfur cluster proteins in NPC2-/- cells was largely alleviated when iron was provided through the transferrin system. This study reveals how lysosomal dysfunction affects mitochondrial homeostasis and underscores nMOST as a valuable discovery tool for identifying molecular phenotypes across LSDs.

Most eukaryotic membrane proteins are inserted into the membrane at the endoplasmic reticulum (ER). This essential but error-prone process relies on molecular quality control machineries to prevent mistargeting and incorrect structure formation. Here we show that the ER membrane protein complex (EMC) forms an evolutionarily conserved supercomplex with the P5A-ATPase Spf1/ATP13A1. This supercomplex combines the transmembrane domain (TMD) insertase function of the EMC and the TMD dislocase activity of Spf1 in a single entity. Our cryo-EM structure of this supercomplex shows that EMC and Spf1 form a shared intramembrane cavity for substrate engagement and reveals that the ATPase cycle of Spf1 regulates access to this cavity. Together, our study suggests that proteins with opposing biochemical activities in membrane protein biogenesis – insertion versus dislocation – form an integrated molecular machine in eukaryotes to proofread membrane protein insertion and topogenesis. ### Competing Interest Statement BAS is a co-inventor of intellectual property related to DCUND1 inhibitors licensed to Cinsano. BAS is a member of the scientific advisory boards of Proxygen and Lyterian. The other authors declare no competing interests. Deutsche Forschungsgemeinschaft, FE 1581/5-1, SCHU 3196/1-1, FE 2386/2-1 European Research Council, https://ror.org/0472cxd90, 101088970, 864068, 101098161 Max Planck Society, https://ror.org/01hhn8329 Center for Molecular Medicine Cologne (CMMC), CAP-37 Studienstiftung des deutschen Volkes Boehringer Ingelheim Fonds, https://ror.org/00dkye506

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degrader drugs function by directly mediating proximity between a neosubstrate and hijacked E3 ligase. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2KLHDC3, which we identified to natively mediate ubiquitin-mediated degradation of IDO1. Therefore, iDegs increase IDO1 turnover using the native proteolytic pathway. In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and, thus, would also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

While protein ubiquitination is an extensively studied post-translational modification, many aspects of this process remain unclear. Ubiquitin conjugation involves the action of three different types of enzymes working in concert to install ubiquitin onto substrate proteins. Despite efforts, an in vitro mid/high-throughput screen to quickly determine which enzymes work together to build ubiquitin chains and directly analyze the type(s) of chains formed does not exist. In this study, we developed a new multiplexed mass spectrometry-based E1-E2-E3 assay that enables the analysis of whether E2/E3 pairs work together to form ubiquitin chains and concomitantly reports on the nature of the formed ubiquitin chain type(s). The assay employs synthetic modified neutron-encoded monoUb substrates with a distinct molecular weight, enabling the simultaneous analysis of these substrates. Overall, various E2-E3 pairs were screened for their ability to build Ub chains, which furnished a three-dimensional overview of linkage selectivity over time and enzyme concentration. ![Figure][1] ### Competing Interest Statement The authors have declared no competing interest. Institute for Chemical Immunology, ICI00026 European Union’s Horizon 2020 Research and Innovation Programme, 813599 (TRIM-NET) Innovative Medicines Initiative 2 (IMI2) Joint Undertaking, 875510 (EUbOPEN project) The Netherlands Organization for Scientific Research (NWO), 722.014.002, VI.Vidi.192.011 [1]: pending:yes

The GID/C-terminal to LisH (CTLH) E3 is an emerging family of evolutionarily conserved multiprotein E3 ligase complexes implicated in various biological processes including metabolic rewiring, stress-responsive regulation, cellular differentiation, and immunity. Pioneering biochemical reconstitution, cryo-EM, and cell-based studies have illuminated many aspects of the compositional and structural dynamics of GID/CTLH E3 complexes. GID/CTLH E3 undergoes sophisticated regulation through incorporation of interchangeable substrate receptors and association with supramolecular assembly factors enabling higher-order complex formation. Furthermore, paralogous subunits vary and may modulate function across cell types. Additionally, an assortment of regulatory factors fine-tune substrate selection, underscoring the adaptability of this E3 ligase system. Here, we review these distinct ubiquitin ligase features, examine the mechanistic implications of GID/CTLH E3 regulation and the exquisite targeting of oligomeric substrates, and discuss potential for therapeutic application in targeted protein degradation.

Ubiquitin chains define the fates of their modified proteins, often mediating proteasomal degradation in eukaryotes. Yet heterogeneity of intracellular ubiquitination has precluded systematically comparing the degradation capacities of different ubiquitin chains. We developed ubiquitinated reporter evaluation after intracellular delivery (UbiREAD), a technology that monitors cellular degradation and deubiquitination at high temporal resolution after bespoke ubiquitinated proteins are delivered into human cells. Comparing the degradation of a model substrate modified with various K48, K63, or K48/K63-branched ubiquitin chains revealed fundamental differences in their intracellular degradation capacities. K48 chains with three or more ubiquitins triggered degradation within minutes. K63-ubiquitinated substrate was rapidly deubiquitinated rather than degraded. Surprisingly, in K48/K63-branched chains, substrate-anchored chain identity determined the degradation and deubiquitination behavior, establishing that branched chains are not the sum of their parts. UbiREAD reveals a degradation code for ubiquitin chains varying by linkage, length, and topology and a functional hierarchy within branched ubiquitin chains.