Preparation of site-specifically fluorophore-labeled polyubiquitin chains for FRET studies of Cdc48 substrate processing.

A critical step in the removal of polyubiquitinated proteins from macromolecular complexes and membranes for subsequent proteasomal degradation is the unfolding of an ubiquitin moiety by the cofactor Ufd1/Npl4 (UN) and its insertion into the Cdc48 ATPase for mechanical translocation. Here, we present a stepwise protocol for the assembly and purification of Lys48-linked ubiquitin chains that are fluorophore labeled at specific ubiquitin moieties and allow monitoring polyubiquitin engagement by the Cdc48-UN complex in a FRET-based assay. For complete details on the use and execution of this protocol, please refer to Williams et al. (2023).1.

The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48.

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.

High-Throughput Assay for Characterizing Rpn11 Deubiquitinase Activity.

Rpn11 is an essential metalloprotease responsible for the en bloc removal of ubiquitin chains from protein substrates that are targeted for degradation by the 26S proteasome. A unique feature of Rpn11 is that its deubiquitinase (DUB) activity is greatly stimulated by the mechanical translocation of the substrate into the proteasomal AAA+ (ATPase Associated with diverse cellular Activities) motor, which delivers the scissile isopeptide bond between a substrate lysine and the proximal moiety of an attached ubiquitin chain to the DUB catalytic active site. As a consequence, Rpn11 cleaves at the base of ubiquitin chains and lacks selectivity towards specific ubiquitin-chain linkage types, which is in contrast to other DUBs, including the related AMSH that selectively cleaves Lys63-linked chains. Prevention of Rpn11's deubiquitinase activity leads to inhibition of proteasomal degradation by stalling substrate translocation. With the proteasome as an approved anticancer target, Rpn11 is therefore an attractive point of attack for the development of new inhibitors, which requires robust biochemical assays to measure DUB activity. Here we describe a method for the purification of the Rpn8/Rpn11 heterodimer and ubiquitin-GC-TAMRA, a model substrate that can be used to characterize the DUB activity of Rpn11 in isolation without the need of purifying 26S proteasomes. This assay thus enables a high-throughput screening platform for Rpn11-targeted small-molecule discovery.

Ubiquitin modulates 26S proteasome conformational dynamics and promotes substrate degradation.

The 26S proteasome recognizes thousands of appropriate protein substrates in eukaryotic cells through attached ubiquitin chains and uses its adenosine triphosphatase (ATPase) motor for mechanical unfolding and translocation into a proteolytic chamber. Here, we used single-molecule Förster resonance energy transfer measurements to monitor the conformational dynamics of the proteasome, observe individual substrates during their progression toward degradation, and elucidate how these processes are regulated by ubiquitin chains. Rapid transitions between engagement- and processing-competent proteasome conformations control substrate access to the ATPase motor. Ubiquitin chain binding functions as an allosteric regulator to slow these transitions, stabilize the engagement-competent state, and aid substrate capture to accelerate degradation initiation. Upon substrate engagement, the proteasome remains in processing-competent states for translocation and unfolding, except for apparent motor slips when encountering stably folded domains. Our studies revealed how ubiquitin chains allosterically regulate degradation initiation, which ensures substrate selectivity in a crowded cellular environment.

Nanoparticulate Drug Delivery Strategies to Address Intestinal Cytochrome P450 CYP3A4 Metabolism towards Personalized Medicine.

Drug dosing in clinical practice, which determines optimal efficacy, toxicity or ineffectiveness, is critical to patients' outcomes. However, many orally administered therapeutic drugs are susceptible to biotransformation by a group of important oxidative enzymes, known as cytochrome P450s (CYPs). In particular, CYP3A4 is a low specificity isoenzyme of the CYPs family, which contributes to the metabolism of approximately 50% of all marketed drugs. Induction or inhibition of CYP3A4 activity results in the varied oral bioavailability and unwanted drug-drug, drug-food, and drug-herb interactions. This review explores the need for addressing intestinal CYP3A4 metabolism and investigates the opportunities to incorporate lipid-based oral drug delivery to enable precise dosing. A variety of lipid- and lipid-polymer hybrid-nanoparticles are highlighted to improve drug bioavailability. These drug carriers are designed to target different intestinal regions, including (1) local saturation or inhibition of CYP3A4 activity at duodenum and proximal jejunum; (2) CYP3A4 bypass via lymphatic absorption; (3) pH-responsive drug release or vitamin-B12 targeted cellular uptake in the distal intestine. Exploitation of lipidic nanosystems not only revives drugs removed from clinical practice due to serious drug-drug interactions, but also provide alternative approaches to reduce pharmacokinetic variability.

Understanding the 26S proteasome molecular machine from a structural and conformational dynamics perspective.

The 26S proteasome is the essential compartmental protease in eukaryotic cells required for the ubiquitin-dependent clearance of damaged polypeptides and obsolete regulatory proteins. Recently, a combination of high-resolution structural, biochemical, and biophysical studies has provided crucial new insights into the mechanisms of this fascinating molecular machine. A multitude of new cryo-electron microscopy structures provided snapshots of the proteasome during ATP-hydrolysis-driven substrate translocation, and detailed biochemical studies revealed the timing of individual degradation steps, elucidating the mechanisms for substrate selection and the commitment to degradation through conformational transitions. It was uncovered how ubiquitin removal from substrates is mechanically coupled to degradation, and cryo-electron tomography studies gave a glimpse of active proteasomes inside the cell, their subcellular localization, and interactions with protein aggregates. Here, we summarize these advances in our mechanistic understanding of the proteasome, with a particular focus on how its structural features and conformational transitions enable the multi-step degradation process.

The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize Substrate Degradation.

The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.

The Cdc48 unfoldase prepares well-folded protein substrates for degradation by the 26S proteasome.

Cdc48/p97 is an essential and highly conserved AAA+ ATPase that uses its protein-unfoldase activity to extract ubiquitinated polypeptides from macromolecular complexes and membranes. This motor has also been implicated in protein-degradation pathways, yet its exact role in acting upstream of the 26S proteasome remains elusive. Ubiquitinated proteins destined for degradation by the proteasome require an unstructured initiation region to engage with the proteasomal translocation machinery, and Cdc48 was proposed to generate these unfolded segments, yet direct evidence has been missing. Here, we used an in vitro reconstituted system to demonstrate the collaboration of Cdc48 and the 26S proteasome from S. cerevisiae in degrading ubiquitinated, well-folded proteins that lack unstructured segments. Our data indicate that a critical role for Cdc48 in the ubiquitin-proteasome system is to create flexible initiation regions in compact substrates that otherwise would be refractory to engagement and degradation by the proteasome.

Structure and Function of the 26S Proteasome.

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

BRD4 Profiling Identifies Critical Chronic Lymphocytic Leukemia Oncogenic Circuits and Reveals Sensitivity to PLX51107, a Novel Structurally Distinct BET Inhibitor.

Bromodomain and extra-terminal (BET) family proteins are key regulators of gene expression in cancer. Herein, we utilize BRD4 profiling to identify critical pathways involved in pathogenesis of chronic lymphocytic leukemia (CLL). BRD4 is overexpressed in CLL and is enriched proximal to genes upregulated or de novo expressed in CLL with known functions in disease pathogenesis and progression. These genes, including key members of the B-cell receptor (BCR) signaling pathway, provide a rationale for this therapeutic approach to identify new targets in alternative types of cancer. Additionally, we describe PLX51107, a structurally distinct BET inhibitor with novel in vitro and in vivo pharmacologic properties that emulates or exceeds the efficacy of BCR signaling agents in preclinical models of CLL. Herein, the discovery of the involvement of BRD4 in the core CLL transcriptional program provides a compelling rationale for clinical investigation of PLX51107 as epigenetic therapy in CLL and application of BRD4 profiling in other cancers.Significance: To date, functional studies of BRD4 in CLL are lacking. Through integrated genomic, functional, and pharmacologic analyses, we uncover the existence of BRD4-regulated core CLL transcriptional programs and present preclinical proof-of-concept studies validating BET inhibition as an epigenetic approach to target BCR signaling in CLL. Cancer Discov; 8(4); 458-77. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 371.

An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome.

Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active ß hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation.

Structures of neutrophil serine protease 4 reveal an unusual mechanism of substrate recognition by a trypsin-fold protease.

Trypsin-fold proteases, the largest mammalian protease family, are classified by their primary substrate specificity into one of three categories, trypsin-like, chymotrypsin-like, and elastase-like, based on key structural features of their active site. However, the recently discovered neutrophil serine protease 4 (NSP4, also known as PRSS57) presents a paradox: NSP4 exhibits a trypsin-like specificity for cleaving substrates after arginine residues, but it bears elastase-like specificity determining residues in the active site. Here we show that NSP4 has a fully occluded S1 pocket and that the substrate P1-arginine adopts a noncanonical "up" conformation stabilized by a solvent-exposed H-bond network. This uncommon arrangement, conserved in all NSP4 orthologs, enables NSP4 to process substrates after both arginine as well as post-translationally modified arginine residues, such as methylarginine and citrulline. These findings establish a distinct paradigm for substrate recognition by a trypsin-fold protease and provide insights into the function of NSP4.

The role of DNA bending in type IIA topoisomerase function.

Type IIA topoisomerases control DNA supercoiling and separate newly replicated chromosomes using a complex DNA strand cleavage and passage mechanism. Structural and biochemical studies have shown that these enzymes sharply bend DNA by as much as 150°; an invariant isoleucine, which has been seen structurally to intercalate between two base pairs outside of the DNA cleavage site, has been suggested to promote deformation. To test this assumption, we examined the role of isoleucine on DNA binding, bending and catalytic activity for a bacterial type IIA topoisomerase, Escherichia coli topoisomerase IV (topo IV), using a combination of site-directed mutagenesis and biochemical assays. Our data show that alteration of the isoleucine (Ile172) did not affect the basal ATPase activity of topo IV or its affinity for DNA. However, the amino acid was important for DNA bending, DNA cleavage and supercoil relaxation. Moreover, an ability to bend DNA correlated with efficacy with which nucleic acid substrates stimulate ATP hydrolysis. These data show that DNA binding and bending by topo IV can be uncoupled, and indicate that the stabilization of a highly curved DNA geometry is critical to the type IIA topoisomerase catalytic cycle.

Structural analysis of a therapeutic monoclonal antibody dimer by hydroxyl radical footprinting.

Hydroxyl radical footprinting is a covalent labeling strategy used to probe the conformational properties of proteins in solution. We describe the first application of this high resolution technique for characterizing the structure of a therapeutic monoclonal antibody (mAb) dimer. As monitored by size-exclusion chromatography (SEC), therapeutic mAbs typically contain small amounts of a dimer species relative to the primary monomeric form in its drug substance or drug product. To determine its structural orientation, a sample enriched in an IgG1 mAb dimer was oxidized by hydroxyl radicals generated by exposure of the aqueous solution to synchrotron X-rays in millisecond timescales. The antibody monomer that served as a control was oxidized in a similar fashion. The oxidized samples were digested with trypsin and analyzed by RP-UHPLC-MS. The footprinting data show that peptides displaying decreased rates of oxidation (i.e., regions of increased protection) in the dimer are localized in the light and heavy chains of the Fab domain. The interface region for the monomers comprising the dimer was thus inferred to be between their Fab arms, allowing us to model two possible theoretical dimer orientations: a head-to-head, single arm-bound Fab-to-Fab dimer, and a head-to-head, double arm-bound Fab (') 2-to-Fab (') 2 dimer. Lower resolution fragment-SEC analysis of the dimer and monomer samples treated with papain or FabRICATOR enzyme provided complimentary evidence to support the Fab/Fab orientation of the IgG1 dimer.

Loss of the tumor suppressor BAP1 causes myeloid transformation.

De-ubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with increased risk of mesothelioma and uveal melanoma. Somatic BAP1 mutations occur in various malignancies. We show that mouse Bap1 gene deletion is lethal during embryogenesis, but systemic or hematopoietic-restricted deletion in adults recapitulates features of human myelodysplastic syndrome (MDS). Knockin mice expressing BAP1 with a 3xFlag tag revealed that BAP1 interacts with host cell factor-1 (HCF-1), O-linked N-acetylglucosamine transferase (OGT), and the polycomb group proteins ASXL1 and ASXL2 in vivo. OGT and HCF-1 levels were decreased by Bap1 deletion, indicating a critical role for BAP1 in stabilizing these epigenetic regulators. Human ASXL1 is mutated frequently in chronic myelomonocytic leukemia (CMML) so an ASXL/BAP1 complex may suppress CMML. A BAP1 catalytic mutation found in a MDS patient implies that BAP1 loss of function has similar consequences in mice and humans.

Engineering and structural characterization of a linear polyubiquitin-specific antibody.

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.

Preparation of distinct ubiquitin chain reagents of high purity and yield.

The complexity of protein ubiquitination signals derives largely from the variety of polyubiquitin linkage types that can modify a target protein, each imparting distinct functional consequences. Free ubiquitin chains of uniform linkages and length are important tools in understanding how ubiquitin-binding proteins specifically recognize these different polyubiquitin modifications. While some free ubiquitin chain species are commercially available, mutational analyses and labeling schemes are limited to select, marketed stocks. Furthermore, the multimilligram quantities of material required for detailed biophysical and/or structural studies often makes these reagents cost prohibitive. To address these limitations, we have optimized known methods for the synthesis and purification of linear, K11-, K48-, and K63-linked ubiquitin dimers, trimers, and tetramers on a preparative scale. The high purity and relatively high yield of these proteins readily enables material-intensive experiments and provides flexibility for engineering specialized ubiquitin chain reagents, such as fluorescently labeled chains of discrete lengths.

Recognition of UbcH5c and the nucleosome by the Bmi1/Ring1b ubiquitin ligase complex.

The Polycomb repressive complex 1 (PRC1) mediates gene silencing, in part by monoubiquitination of histone H2A on lysine 119 (uH2A). Bmi1 and Ring1b are critical components of PRC1 that heterodimerize via their N-terminal RING domains to form an active E3 ubiquitin ligase. We have determined the crystal structure of a complex between the Bmi1/Ring1b RING-RING heterodimer and the E2 enzyme UbcH5c and find that UbcH5c interacts with Ring1b only, in a manner fairly typical of E2-E3 interactions. However, we further show that the Bmi1/Ring1b RING domains bind directly to duplex DNA through a basic surface patch unique to the Bmi1/Ring1b RING-RING dimer. Mutation of residues on this interaction surface leads to a loss of H2A ubiquitination activity. Computational modelling of the interface between Bmi1/Ring1b-UbcH5c and the nucleosome suggests that Bmi1/Ring1b interacts with both nucleosomal DNA and an acidic patch on histone H4 to achieve specific monoubiquitination of H2A. Our results point to a novel mechanism of substrate recognition, and control of product formation, by Bmi1/Ring1b.

K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody.

Polyubiquitination is a posttranslational modification where ubiquitin chains containing isopeptide bonds linking one of seven ubiquitin lysines with the C terminus of an adjoining ubiquitin are covalently attached to proteins. While functions of K48- and K63-linked polyubiquitin are understood, the role(s) of noncanonical K11-linked chains is less clear. A crystal structure of K11-linked diubiquitin demonstrates a distinct conformation from K48- or K63-linked diubiquitin. We engineered a K11 linkage-specific antibody and use it to demonstrate that K11 chains are highly upregulated in mitotic human cells precisely when substrates of the ubiquitin ligase anaphase-promoting complex (APC/C) are degraded. These chains increased with proteasomal inhibition, suggesting they act as degradation signals in vivo. Inhibition of the APC/C strongly impeded the formation of K11-linked chains, suggesting that a single ubiquitin ligase is the major source of mitotic K11-linked chains. Our results underscore the importance of K11-linked ubiquitin chains as critical regulators of mitotic protein degradation.

The protective antigen component of anthrax toxin forms functional octameric complexes.

The assembly of bacterial toxins and virulence factors is critical to their function, but the regulation of assembly during infection has not been studied. We begin to address this question using anthrax toxin as a model. The protective antigen (PA) component of the toxin assembles into ring-shaped homooligomers that bind the two other enzyme components of the toxin, lethal factor (LF) and edema factor (EF), to form toxic complexes. To disrupt the host, these toxic complexes are endocytosed, such that the PA oligomer forms a membrane-spanning channel that LF and EF translocate through to enter the cytosol. Using single-channel electrophysiology, we show that PA channels contain two populations of conductance states, which correspond to two different PA pre-channel oligomers observed by electron microscopy-the well-described heptamer and a novel octamer. Mass spectrometry demonstrates that the PA octamer binds four LFs, and assembly routes leading to the octamer are populated with even-numbered, dimeric and tetrameric, PA intermediates. Both heptameric and octameric PA complexes can translocate LF and EF with similar rates and efficiencies. Here, we report a 3.2-A crystal structure of the PA octamer. The octamer comprises approximately 20-30% of the oligomers on cells, but outside of the cell, the octamer is more stable than the heptamer under physiological pH. Thus, the PA octamer is a physiological, stable, and active assembly state capable of forming lethal toxins that may withstand the hostile conditions encountered in the bloodstream. This assembly mechanism may provide a novel means to control cytotoxicity.