Structural insights into the human PA28-20S proteasome enabled by efficient tagging and purification of endogenous proteins.

The ability to produce folded and functional proteins is a necessity for structural biology and many other biological sciences. This task is particularly challenging for numerous biomedically important targets in human cells, including membrane proteins and large macromolecular assemblies, hampering mechanistic studies and drug development efforts. Here we describe a method combining CRISPR-Cas gene editing and fluorescence-activated cell sorting to rapidly tag and purify endogenous proteins in HEK cells for structural characterization. We applied this approach to study the human proteasome from HEK cells and rapidly determined cryogenic electron microscopy structures of major proteasomal complexes, including a high-resolution structure of intact human PA28αß-20S. Our structures reveal that PA28 with a subunit stoichiometry of 3α/4ß engages tightly with the 20S proteasome. Addition of a hydrophilic peptide shows that polypeptides entering through PA28 are held in the antechamber of 20S prior to degradation in the proteolytic chamber. This study provides critical insights into an important proteasome complex and demonstrates key methodologies for the tagging of proteins from endogenous sources.

Purification and Identification of the 20S Proteasome Complex from Zebrafish.

The proteasome is a large polymeric protease complex responsible for degradation of intracellular proteins and generation of peptides. In this study, we purified a native 20S proteasome protein complex from zebrafish (Danio rerio) from the whole body. The cytosolic fraction of zebrafish hydrolyzed Suc-Leu-Leu-Val-Tyr-MCA (Suc-LLVY-MCA), a well-known substrate for the proteasome, in the presence of sodium dodecyl sulfate. From the cytosolic fraction, the 20S proteasome was purified using five column chromatography steps: DEAE cellulose, Q-Sepharose, Sephacryl S-300 gel, hydroxylapatite, and phenyl Sepharose. Electrophoresis and Western blot analyses showed that zebrafish 20S proteasome subunits have molecular masses ranging from 22 to 33 kDa. The subunit composition of the purified 20S proteasome was identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis after two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) separation. Fourteen kinds of 20S subunits were found. As a special characteristic of zebrafish, two proteins of the α1 subunit were identified. In addition, the results suggested that the α8 subunit is in the 20S complex instead of the α4 subunit. In this study, we demonstrated the subunit composition of the 20S proteasome complex present in zebrafish cells.

Extreme parsimony in ATP consumption by 20S complexes in the global disassembly of single SNARE complexes.

Fueled by ATP hydrolysis in N-ethylmaleimide sensitive factor (NSF), the 20S complex disassembles rigid SNARE (soluble NSF attachment protein receptor) complexes in single unraveling step. This global disassembly distinguishes NSF from other molecular motors that make incremental and processive motions, but the molecular underpinnings of its remarkable energy efficiency remain largely unknown. Using multiple single-molecule methods, we found remarkable cooperativity in mechanical connection between NSF and the SNARE complex, which prevents dysfunctional 20S complexes that consume ATP without productive disassembly. We also constructed ATP hydrolysis cycle of the 20S complex, in which NSF largely shows randomness in ATP binding but switches to perfect ATP hydrolysis synchronization to induce global SNARE disassembly, minimizing ATP hydrolysis by non-20S complex-forming NSF molecules. These two mechanisms work in concert to concentrate ATP consumption into functional 20S complexes, suggesting evolutionary adaptations by the 20S complex to the energetically expensive mechanical task of SNARE complex disassembly.

Structure-Based Design of Fluorogenic Substrates Selective for Human Proteasome Subunits.

Proteasomes are established therapeutic targets for hematological cancers and promising targets for autoimmune diseases. In the past, we have designed and synthesized mechanism-based proteasome inhibitors that are selective for the individual catalytic activities of human constitutive proteasomes and immunoproteasomes: ß1c, ß1i, ß2c, ß2i, ß5c and ß5i. We show here that by taking the oligopeptide recognition element and substituting the electrophile for a fluorogenic leaving group, fluorogenic substrates are obtained that report on the proteasome catalytic activity also targeted by the parent inhibitor. Though not generally applicable (ß5c and ß2i substrates showing low activity), effective fluorogenic substrates reporting on the individual activity of ß1c, ß1i, ß2c and ß5i subunits in Raji (human B cell) lysates and purified 20S proteasome were identified in this manner. Our work thus adds to the expanding proteasome research toolbox through the identification of new and/or more effective subunit-selective fluorogenic substrates.

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 µL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.

Characterization of the 20S proteasome of the lepidopteran, Spodoptera frugiperda.

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.

Purification of Large Cytosolic Proteases for In Vitro Assays: 20S and 26S Proteasomes.

Proteasomes are the main cytosolic proteases responsible for generating peptides for antigen processing and presentation in the MHC (major histocompatibility complex) class-I pathway. Purified 20S and 26S proteasomes have been widely used to study both specificity and efficiency of antigen processing. Here, we describe the purification of active human 20S and 26S proteasomes from human erythrocytes by DEAE-ion exchange chromatography, ammonium sulfate precipitation, glycerol density gradient centrifugation, and Superose-6 size exclusion chromatography and their characterization using fluorogenic substrates and specific inhibitors.

Structural mass spectrometry approaches to study the 20S proteasome.

The 20S proteasome is a large multisubunit proteolytic machine that is central to intracellular protein degradation. It is found in all three kingdoms of life and is ubiquitous in archaea and eukaryotes. Since its discovery, much effort employing a diverse array of structural biology methods has been applied to help understand its structure/function relationships. Here, we will specifically focus on the application of native mass spectrometry (MS) approaches for structural investigations of the 20S proteasome. Native MS is a method that examines intact protein assemblies, without disturbing the noncovalent interactions that govern the overall structure. This method is ideally suited to revealing the intrinsic heterogeneity of a given sample and provides insight into the composition, stoichiometry, subunit architecture, and topology of the protein assembly. Initially, we describe native MS-oriented protocols for the isolation of endogenous 20S proteasomes from yeast, rat liver, and human cells. We then highlight the applicability of native MS methodologies, using different instrumental platforms, for structural investigations of the complex. In particular, by means of proteasome biology, we highlight the different approaches used to analyze both intact complexes-their natural heterogeneity and interactions with substrates and regulators-and their individual constituent subunits.

Recombinant Expression, Unnatural Amino Acid Incorporation, and Site-Specific Labeling of 26S Proteasomal Subcomplexes.

The 26S proteasome is the major regulated protease in eukaryotes and is responsible for degrading ubiquitinated substrates. It consists of a barrel-shaped 20S core peptidase and one or two 19S regulatory particles, which recognize, unfold, and translocate substrates into the core. The regulatory particle can be further divided into two multi-subunit complexes: the base and the lid. Here we present protocols for expressing the Saccharomyces cerevisiae base and lid recombinantly in Escherichia coli and purifying the assembled subcomplexes using a tandem affinity purification method. The purified complexes can then be reconstituted with 20S core to form fully functional proteasomes. Furthermore, we describe a method for incorporating the unnatural amino acid p-azido-L-phenylalanine into the recombinant complexes at any residue position, allowing for non-disruptive site-specific modifications of these large assemblies. The use of recombinant proteins allows for complete mutational control over the proteasome regulatory particle, enabling detailed studies of the mechanism by which the proteasome processes its substrates. The ability to then specifically modify residues in the regulatory particle opens the door to a wide range of previously impossible biochemical and biophysical studies. The techniques described below for incorporating unnatural amino acids into the proteasomal subcomplexes should be widely transferable to other recombinant proteins, whether individually purified or in larger multi-subunit assemblies.

Methods to Rapidly Prepare Mammalian 26S Proteasomes for Biochemical Analysis.

Rapid, gentle isolation of 26S proteasomes from cells or tissues is an essential step for studies of the changes in proteasome activity and composition that can occur under different physiological or pathological conditions and in response to pharmacological agents. We present here three different approaches to affinity purify or to prepare proteasome-rich cell fractions. The first method uses affinity tags fused to proteasome subunits and has been useful in several cell lines for studies of proteasome structure by cryo-electron microscopy and composition by mass spectrometry. A second method uses the proteasome's affinity for a ubiquitin-like (UBL) domain and can be used to purify these particles from any cell or tissue. This method does not require expression of a tagged subunit and has proven to be very useful to investigate how proteasomal activity changes in different physiological states (e.g., fasting or aging), with neurodegenerative diseases, and with drugs or hormones that cause subunit phosphorylation. A third, simple method that is based on the 26S proteasome's high molecular weight (about 2.5 MDa) concentrates these particles greatly by differential centrifugation. This method maintains the association of proteasomes with ubiquitin (Ub) conjugates and many other loosely associated regulatory proteins and is useful to study changes in proteasome composition under different conditions.

Measurement of the Multiple Activities of 26S Proteasomes.

Because proteasomes catalyze most of the protein degradation in mammalian cells, and their functioning is essential for cellular homeostasis, proteasome structure, biochemical mechanisms, and regulation in normal and disease states are now widely studied and are of major importance. In addition, inhibitors of the proteasome's peptidase activity have proven to be very valuable as research tools and in the treatment of hematologic malignancies, and a number of newer pharmacological agents that alter proteasome function are being developed. The rapid degradation of ubiquitinated proteins by the 26S proteasome involves multiple enzymatic and non-enzymatic steps, including the binding of ubiquitinated substrates to the 19S particle (Subheading 3.2), opening the gated substrate entry channel into the 20S particle (Subheading 3.3), disassembly of the Ub chain (Subheading 3.4), ATP hydrolysis (Subheading 3.5), substrate unfolding and translocation, and proteolysis within the 20S particle (Subheadings 3.3 and 3.7). Assaying each of these processes is important if we are to fully understand the physiological regulation of proteasome function and the effects of disease or drugs. Here, we describe several methods that we have found useful to measure many of these individual activities using purified proteasomes. Studies using these approaches have already provided valuable new insights into the effects of post-synthetic modifications to 26S subunits, the physiological regulation of the ubiquitin-proteasome system, and the impairment of proteasome activity in neurodegenerative disease. These advances would not have been possible if only the standard assays of peptidase activity were used.

Scalable In Vitro Proteasome Activity Assay.

We developed a degradation assay based on fluorescent protein substrates that are efficiently recognized, unfolded, translocated, and hydrolyzed by the proteasome. The substrates consist of three components: a proteasome-binding tag, a folded domain, and an initiation region. All the components of the model substrate can be changed to modulate degradation, and the assay can be performed in parallel in 384-well plates. These properties allow the assay to be used to explore a wide range of experimental conditions and to screen proteasome modulators.

Dissecting Dynamic and Heterogeneous Proteasome Complexes Using In Vivo Cross-Linking-Assisted Affinity Purification and Mass Spectrometry.

Protein-protein interactions are essential for protein complex formation and function. Affinity purification coupled with mass spectrometry (AP-MS) is the method of choice for studying protein-protein interactions at the systems level under different physiological conditions. Although effective in capturing stable protein interactions, transient, weak, and/or dynamic interactors are often lost due to extended procedures during conventional AP-MS experiments. To circumvent this problem, we have recently developed XAP (in vivo cross-linking (X)-assisted affinity purification)-MS strategy to better preserve dynamic protein complexes under native lysis conditions. In addition, we have developed XBAP (in vivo cross-linking (X)-assisted bimolecular tandem affinity purification)-MS method by incorporating XAP with bimolecular affinity purification to define dynamic and heterogeneous protein subcomplexes. Here we describe general experimental protocols of XAP- and XBAP-MS to study dynamic protein complexes and their subcomplexes, respectively. Specifically, we present their applications in capturing and identifying proteasome dynamic interactors and ubiquitin receptor (UbR)-proteasome subcomplexes.

Naphthoquinone amino acid derivatives, synthesis and biological activity as proteasome inhibitors.

The ubiquitin-proteasome system has been largely investigated for its key role in protein degradation mechanisms that regulate both apoptosis and cell division. Because of their antitumour activity, different classes of proteasome inhibitors have been identified to date. Some of these compounds are currently employed in the clinical treatment of several types of cancer among which multiple myeloma. Here, we describe the design, chemistry, biological activity and modelling studies of a large series of amino acid derivatives linked to a naphthoquinone pharmacophoric group through variable spacers. Some analogues showed interesting inhibitory potency for the ß1 and ß5 subunits of the proteasome with IC50 values in the sub-µm range.

Purification of 26S Proteasomes and Their Subcomplexes from Plants.

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type plants, and another that employs the genetic replacement of individual subunits with epitope-tagged variants combined with affinity purification. In addition to these purification protocols, we describe methods commonly used to analyze the activity and composition of the complex.

THE SHORTENED ISOFORM OF a-TUBULIN IS DETECTED IN COMPLEX WITH PROTEASOMES.

The proteasome is a multi-subunit protein complex that serves as a major pathway for intracellular protein degradation playing important functions in various biological processes. By using MALDI-ICR-mass-spectrometry and Western-blot analysis, we have shown the presence of shortened isoform of a-tubulin in complex with the affinity-purified proteasomes from stable cell lines K562 and HEK293.

Determination of Protein Carbonylation and Proteasome Activity in Seeds.

Reactive oxygen species (ROS) have been shown to be toxic but also function as signaling molecules in a process called redox signaling. In seeds, ROS are produced at different developmental stages including dormancy release and germination. Main targets of oxidation events by ROS in cell are lipids, nucleic acids, and proteins. Protein oxidation has various effects on their function, stability, location, and degradation. Carbonylation represents an irreversible and unrepairable modification that can lead to protein degradation through the action of the 20S proteasome. Here, we present techniques which allow the quantification of protein carbonyls in complex protein samples after derivatization by 2,4-dinitrophenylhydrazine (DNPH) and the determination proteasome activity by an activity-based protein profiling (ABPP) using the probe MV151. These techniques, routinely easy to handle, allow the rapid assessment of protein carbonyls and proteasome activity in seeds in various physiological conditions where ROS may act as signaling or toxic elements.

Structure of the human 26S proteasome at a resolution of 3.9 Å.

Protein degradation in eukaryotic cells is performed by the Ubiquitin-Proteasome System (UPS). The 26S proteasome holocomplex consists of a core particle (CP) that proteolytically degrades polyubiquitylated proteins, and a regulatory particle (RP) containing the AAA-ATPase module. This module controls access to the proteolytic chamber inside the CP and is surrounded by non-ATPase subunits (Rpns) that recognize substrates and deubiquitylate them before unfolding and degradation. The architecture of the 26S holocomplex is highly conserved between yeast and humans. The structure of the human 26S holocomplex described here reveals previously unidentified features of the AAA-ATPase heterohexamer. One subunit, Rpt6, has ADP bound, whereas the other five have ATP in their binding pockets. Rpt6 is structurally distinct from the other five Rpt subunits, most notably in its pore loop region. For Rpns, the map reveals two main, previously undetected, features: the C terminus of Rpn3 protrudes into the mouth of the ATPase ring; and Rpn1 and Rpn2, the largest proteasome subunits, are linked by an extended connection. The structural features of the 26S proteasome observed in this study are likely to be important for coordinating the proteasomal subunits during substrate processing.

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation.

Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbR-proteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a general and useful proteomic tool for isolating and studying dynamic and heterogeneous protein subcomplexes in cells that have not been fully characterized.

26S and PA28-20S Proteasome Activity in Cytosolic Extracts from Embryonic Stem Cells.

The proteasome is a complex multisubunit protease that plays a major role in the degradation of proteins in eukaryotic cells. Proteasome function is one of the key players regulating the proteome and it is vital for many cellular processes. The method described here makes it possible to assay the proteolytic capacities of proteasome complexes separately in crude cytosolic extracts from ES cells. The method is based on hydrolysis of a fluorogenic peptide substrate in lysates prepared under conditions that favor the interactions of the 20S proteasomal catalytical core with either the 19S or the PA28αß proteasome regulator.