Biology subjects

Schulman, B. A.

Publications and source records attributed to Schulman, B. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Global Data Fitting Enables Kinetic Dissection of Cullin-RING Ligase-Catalyzed Ubiquitylation: From Substrate Priming to Poly-ubiquitin Chain Formation

The discovery and characterization of the ubiquitin system was founded on classical enzymology approaches that established fundamental principles of protein ubiquitylation. Cullin-RING ligases (CRLs) represent the largest class of human ubiquitin ligases, yet detailed enzymology of these multi-subunit assemblies has lagged behind other systems such as nucleic acid polymerases. Here, we employed pre-steady state kinetics and global data fitting using KinTek Explorer to systematically investigate the kinetic parameters controlling CRL-dependent ubiquitin transfer. Using a well-characterized in vitro reconstituted system exploring CUL2-dependent CRLs, we modeled substrate priming, showing that the system is highly amenable to global fitting of kinetic data. The results show that product dissociation from the CRL is significantly slower than substrate dissociation, explaining previously reported slow steady-state ubiquitylation rates. Extending this framework to poly-ubiquitin chain formation revealed catalytic efficiencies 15-42-fold greater than substrate priming. Comparative analysis of CUL1-dependent and CUL2-dependent systems showed remarkably similar kinetic parameters, including the Km of ubiquitin-carrying enzyme for CRLs, despite differences in substrate receptor and enzyme architecture. We further examined drug-induced neo-substrate ubiquitylation using the pomalidomide-CRBN-SALL4 system, revealing atypical kinetics characterized by rapid ubiquitin transfer paired with an elevated Km, ultimately yielding catalytic efficiencies comparable to natural substrates. The findings establish pre-steady state kinetics and global data fitting as powerful enzymological tools for dissecting CRL-dependent ubiquitylation across different CRL families and substrate contexts, with important implications for understanding both physiological ubiquitylation and therapeutic targeted protein degradation strategies.

biochemistry↗

bioRxiv2026

Targeting proteasome composition reprograms tumor-intrinsic inflammation through selective degradation of STAT1

Proteasome regulatory particles determine cellular protein degradation capacities. Yet specific ubiquitin-independent degradation targets of individual proteasome regulatory particles, and physiological roles of such pathways, remain largely elusive. Here, through a chemical biology-driven approach, we identify a covalent small molecule that selectively targets a conformationally dynamic pocket in the non-catalytic proteasome regulator PSME4 and modulates tumor cell proteasome targets. Using this chemical probe together with orthogonal genetic perturbations, we show that blocking or reducing PSME4 stabilizes STAT1, restores interferon-associated transcription, enhances antigen presentation, and disrupts IL-6/STAT3 signaling, collectively reprogramming tumor inflammation and suppressing tumor growth in vivo. These findings define a PSME4-STAT1-STAT3 axis that couples proteasomal degradation to antigen presentation and cytokine signaling and provide a framework for pharmacologically targeting non-catalytic proteasome regulators to reprogram tumor immunity. More broadly, our findings reveal that proteasome composition can actively govern the balance between immunostimulatory and immunosuppressive signaling programs.

cancer biology↗

bioRxiv2026

Multimodal substrate recruitment enables CTLH-MKLN1 E3 ligase to target N-, C-, and internal degrons

The GID/CTLH family of E3 ubiquitin ligases employs several substrate receptor subunits that recruit distinct degrons, but the substrate recognition mechanism of the CTLHMKLN1 assembly has largely remained elusive. Here, we reconstitute CTLHMKLN1-dependent ubiquitylation of three biochemically distinct substrates - MKLN1 itself, ZMYND19, and FAM72A-recruited UNG2, and determine cryo-EM structures of each substrate bound to MKLN1. The structures reveal how MKLN1's discoidin and Kelch {beta}-propeller domains engage substrates through multivalent contacts. MKLN1 recruits itself through discoidin - Kelch interactions between MKLN1 dimers, forming an assembly that competes with other substrates. ZMYND19 and FAM72A each bind to MKLN1 through loops engaging the discoidin trench domain while their C-terminal Arg residues engage the MKLN1 Kelch central channel, identifying MKLN1 as an Arg/C-degron receptor. The acetylated N-terminus of UNG2 is positioned within a Y-shaped tunnel of FAM72A, defining an adaptor-mediated Ac/N-degron recognition mechanism. Thus, our data reveal how combinatorial deployment of MKLN1 discoidin and Kelch domains enable a single receptor subunit to recognize a diverse substrate repertoire, adding to a complex picture of degron recognition across the GID/CTLH family.

biochemistry↗

bioRxiv2026

Regulation of the proteasome 20S core particle by the Parkinsonism-associated Proteins FBXO7 and PI31

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 FBXO7s C-terminal domain engages multiple subunits within the CP interior, blocking the {beta}5 peptidase activity. In contrast, we visualized PI31 engagement of all three catalytic sites within the 20S CP, revealing the previously unknown structural basis for {beta}1 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 Parkinsons disease.

biochemistry↗

bioRxiv2026

A CK2-FBXW11 kinase-E3 ubiquitin ligase cascade is a metabolic sensor regulating Tryptophan 2,3-dioxygenase stability

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.

biochemistry↗

bioRxiv2026

The E3 ubiquitin ligase mechanism specifying target-directed microRNA degradation

MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to form complexes that down-regulate target RNAs, including mRNAs from most human genes1-3. Within each complex, the miRNA pairs to target mRNAs to specify their repression, and AGO provides effector function while also protecting the miRNA from cellular nucleases2-5. Although much has been learned about this mode of posttranscriptional gene regulation, less is known about how the miRNAs themselves are regulated. In one such regulatory pathway, unusual miRNA targets called "trigger" RNAs reverse the canonical regulatory logic and instead down-regulate microRNAs6-21. This target-directed miRNA degradation (TDMD) is thought to require a cullin-RING E3 ligase (CRL) because it depends on the cullin protein CUL3 and other ubiquitylation components, including the BC-box protein ZSWIM8 (ref. 22,23). ZSWIM8 is required for murine perinatal viability and for destabilization of most short-lived miRNAs, but is otherwise poorly understood23-25. Here, we demonstrate that a human AGO-miRNA- trigger complex selectively binds ZSWIM8 for CUL3-mediated polyubiquitylation of the AGO protein within this complex. Cryogenic electron-microscopy (cryo-EM) analyses show how ZSWIM8 recognizes the distinct AGO2 and miRNA-trigger conformations shaped by pairing of the miRNA to the trigger. For example, this pairing extracts the miRNA from a binding pocket within AGO2, allowing the pocket to be captured by ZSWIM8, and it directs the trigger RNA along a distinct trajectory to be also recognized by ZSWIM8. These results biochemically establish AGO binding and polyubiquitylation as the key regulatory step of TDMD, define a unique CRL class, and reveal generalizable RNA-RNA, RNA-protein, and protein-protein interactions that specify the ubiquitin-mediated degradation of AGO with exquisite selectivity. The substrate features recognized by the E3 ubiquitin ligase do not conform to a conventional degron26-28, but rather establish a two-RNA-factor authentication mechanism specifying a protein ubiquitylation substrate.

biochemistry↗

bioRxiv2025

Cysteine availability tunes ubiquitin signaling via inverse stability of LRRC58 E3 ligase and its substrate CDO1

Cellular responses to amino acid fluctuations often hinge on ubiquitin-mediated control of metabolic enzymes, yet the underlying E3 ligase pathways remain incompletely 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. By contrast, targeted protein degradation via the VHL-based CRL is achieved by recruitment of a distinct surface of CDO1, and more promiscuous ubiquitylation independent of Lys8. 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.

biochemistry↗

bioRxiv2025

Structural basis of an EMC:Spf1 insertase-dislocase complex in the eukaryotic endoplasmic reticulum

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.

biochemistry↗

bioRxiv2025

Employing neutron-encoded monoUbs to study E2/E3 ligase activity and selectivity for assembling Ub chains

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. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/655449v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@130203aorg.highwire.dtl.DTLVardef@93c9adorg.highwire.dtl.DTLVardef@9d9bedorg.highwire.dtl.DTLVardef@167ea0c_HPS_FORMAT_FIGEXP M_FIG C_FIG

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↗

bioRxiv2024

Lysosomal storage disease proteo/lipidomic profiling using nMOST links ferritinophagy with mitochondrial iron deficiencies in cells lacking NPC2

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 multi-omic 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 lyso-phosphatidylcholine species and multi-lamellar 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.

cell biology↗

bioRxiv2024

Visualizing chaperone-mediated multistep assembly of the human 20S proteasome

Dedicated assembly factors orchestrate stepwise production of many molecular machines, including the 28-subunit proteasome core particle (CP) that mediates protein degradation. Here, we report cryo-EM reconstructions of seven recombinant human subcomplexes that visualize all five chaperones and the three active site propeptides across a wide swath of the assembly pathway. Comparison of these chaperone-bound intermediates and a matching mature CP reveals molecular mechanisms determining the order of successive subunit additions, and how proteasome subcomplexes and assembly factors structurally adapt upon progressive subunit incorporation to stabilize intermediates, facilitate the formation of subsequent intermediates, and ultimately rearrange to coordinate proteolytic activation with gated access to active sites. The structural findings reported here explain many previous biochemical and genetic observations. This work establishes a methodologic approach for structural analysis of multiprotein complex assembly intermediates, illuminates specific functions of assembly factors, and reveals conceptual principles underlying human proteasome biogenesis.

biochemistry↗

bioRxiv2023

Structural snapshots along K48-linked ubiquitin chain formation by the HECT E3 UBR5

Ubiquitin chain formation by HECT catalytic domain-containing E3 ligases regulates vast biology, yet the structural mechanisms remain unknown. We employed chemistry and cryo-EM to visualize stable mimics of the intermediates along K48-linked ubiquitin chain formation by the human E3, UBR5. The structural data reveal a {approx}620 kDa UBR5 dimer as the functional unit, comprising a scaffold with flexibly-tethered ubiquitin-binding UBA domains, and elaborately arranged HECT domains. Chains are forged by a UBA domain capturing an acceptor ubiquitin, with its K48 lured into the active site by numerous interactions between the acceptor ubiquitin, manifold UBR5 elements, and the donor ubiquitin. The cryo-EM reconstructions allow defining conserved HECT domain conformations catalyzing ubiquitin transfer from E2 to E3, and from E3. Our data show how a full-length E3, ubiquitins to be adjoined, E2, and intermediary products guide a feed-forward HECT domain conformational cycle establishing a highly efficient, broadly targeting, K48-linked ubiquitin chain forging machine.

biochemistry↗

bioRxiv2023

Insights into the ISG15 transfer cascade by the UBE1L activating enzyme

The attachment of the ubiquitin-like protein ISG15 to substrates is a well-established antiviral signalling mechanism of the innate immune response. However, despite the identification of thousands of substrates and clear roles in antiviral immunity, a molecular understanding of ISG15 selection and transfer through its cognate E1-E2- E3 enzyme cascade is largely unknown. Here, we present a 3.45 [A] cryo-EM structure of a chemically trapped UBE1L-UBE2L6 complex bound to activated ISG15. This structure reveals the details of the first steps of ISG15 recognition and UBE2L6 recruitment by UBE1L. Taking advantage of viral effector proteins from severe acute respiratory coronavirus 2 (SARS-CoV-2) and influenza B virus (IBV), we validated the structure and confirmed the importance of the ISG15 C-terminal ubiquitin-like domain in the adenylation reaction. Moreover, biochemical characterization of the UBE1L-ISG15 and UBE1L-UBE2L6 interactions enabled the design of ISG15 and UBE2L6 mutants with altered selectively for the ISG15 and ubiquitin conjugation pathways. Together, our study provides much needed insight into the specificity determinants that ensure the fidelity of ISG15 signalling during the antiviral response.

biochemistry↗

bioRxiv2023

Activity-based profiling of cullin-RING ligase networks by conformation-specific probes

The cullin-RING E3 ligase (CRL) network comprises over 300 unique complexes that switch from inactive to activated conformations upon site-specific cullin modification by the ubiquitin-like protein NEDD8. Assessing cellular repertoires of activated CRL complexes is critical for understanding eukaryotic regulation. However, probes surveying networks controlled by site-specific ubiquitin-like protein modifications are lacking. We report development of a synthetic antibody recognizing the active conformation of a NEDD8-linked cullin. We established a pipeline probing cellular networks of activated CUL1-, CUL2-, CUL3- and CUL4-containing CRLs, revealing the CRL complexes responding to stimuli. Profiling several cell types showed their baseline neddylated CRL repertoires vary, prime efficiency of targeted protein degradation, and are differentially rewired across distinct primary cell activation pathways. Thus, conformation-specific probes can permit nonenzymatic activity-based profiling across a system of numerous multiprotein complexes, which in the case of neddylated CRLs reveals widespread regulation and could facilitate development of degrader drugs.

biochemistry↗

bioRxiv2022

The structural context of PTMs at a proteome wide scale

The recent revolution in computational protein structure prediction provides folding models for entire proteomes, which can now be integrated with large-scale experimental data. Mass spectrometry (MS)-based proteomics has identified and quantified tens of thousands of post-translational modifications (PTMs), most of them of uncertain functional relevance. In this study, we determine the structural context of these PTMs and investigate how this information can be leveraged to pinpoint potential regulatory sites. Our analysis uncovers global patterns of PTM occurrence across folded and intrinsically disordered regions. We found that this information can help to distinguish regulatory PTMs from those marking improperly folded proteins. Interestingly, the human proteome contains thousands of proteins that have large folded domains linked by short, unstructured regions that are strongly enriched in regulatory phosphosites. These include well-known kinase activation loops that induce protein conformational changes upon phosphorylation. This regulatory mechanism appears to be widespread in kinases but also occurs in other protein families such as solute carriers. It is not limited to phosphorylation but includes ubiquitination and acetylation sites as well. Furthermore, we performed three-dimensional proximity analysis which revealed examples of spatial co-regulation of different PTM types and potential PTM crosstalk. To enable the community to build upon these first analyses, we provide tools for 3D visualization of proteomics data and PTMs as well as python libraries for data accession and processing.

systems biology↗

bioRxiv2021

Multifaceted N-degron recognition and ubiquitylation by GID/CTLH E3 ligases

N-degron E3 ubiquitin ligases recognize specific residues at the N-termini of substrates. Although molecular details of N-degron recognition are known for several E3 ligases, the range of N-terminal motifs that can bind a given E3 substrate binding domain remains unclear. Here, studying the Gid4 and Gid10 substrate receptor subunits of yeast "GID"/human "CTLH" multiprotein E3 ligases, whose known substrates bear N-terminal prolines, we discovered capacity for high-affinity binding to diverse N-terminal sequences determined in part by context. Screening of phage displaying peptide libraries with exposed N-termini identified novel consensus motifs with non-Pro N-terminal residues distinctly binding Gid4 or Gid10 with high affinity. Structural data reveal that flexible loops in Gid4 and Gid10 conform to complementary folds of diverse interacting peptide sequences. Together with analysis of endogenous substrate degrons, the data show that degron identity, substrate domains harboring targeted lysines, and varying E3 ligase higher-order assemblies combinatorially determine efficiency of ubiquitylation and degradation.

biochemistry↗

bioRxiv2021

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

To achieve precise cellular regulation, E3 ubiquitin ligases must be configured to match substrate quaternary structures. Here, by studying the yeast GID complex, mutation of which is Glucose-Induced Degradation deficient, we discover supramolecular chelate assembly as an E3 ligase strategy for targeting an oligomeric substrate. Cryo EM structures show that to bind the tetrameric substrate fructose-1,6-bisphosphatase (Fbp1), two otherwise functional GID E3s assemble into a 20-protein Chelator-GIDSR4, which resembles an organometallic supramolecular chelate. The Chelator-GIDSR4 assembly avidly binds multiple Fbp1 degrons and positions Fbp1 so that its protomers are simultaneously ubiquitylated at lysines near its allosteric and substrate binding sites. Significantly, key structural and biochemical features -including capacity for supramolecular assembly - are preserved in the human ortholog, the CTLH E3. Based on our integrative structural, biochemical and cell biological data, we propose that higher-order E3 ligase assembly generally underlies multipronged targeting, capable of simultaneously incapacitating multiple protomers and functionalities of oligomeric substrates.

biochemistry↗