Systematic identification of 20S proteasome substrates.

For years, proteasomal degradation was predominantly attributed to the ubiquitin-26S proteasome pathway. However, it is now evident that the core 20S proteasome can independently target proteins for degradation. With approximately half of the cellular proteasomes comprising free 20S complexes, this degradation mechanism is not rare. Identifying 20S-specific substrates is challenging due to the dual-targeting of some proteins to either 20S or 26S proteasomes and the non-specificity of proteasome inhibitors. Consequently, knowledge of 20S proteasome substrates relies on limited hypothesis-driven studies. To comprehensively explore 20S proteasome substrates, we employed advanced mass spectrometry, along with biochemical and cellular analyses. This systematic approach revealed hundreds of 20S proteasome substrates, including proteins undergoing specific N- or C-terminal cleavage, possibly for regulation. Notably, these substrates were enriched in RNA- and DNA-binding proteins with intrinsically disordered regions, often found in the nucleus and stress granules. Under cellular stress, we observed reduced proteolytic activity in oxidized proteasomes, with oxidized protein substrates exhibiting higher structural disorder compared to unmodified proteins. Overall, our study illuminates the nature of 20S substrates, offering crucial insights into 20S proteasome biology.

C-terminal domain dimerization in yeast Hsp90 is moderately modulated by the other domains.

Heat shock protein 90 (Hsp90) serves as a crucial regulator of cellular proteostasis by stabilizing and regulating the activity of numerous substrates, many of which are oncogenic proteins. Therefore, Hsp90 is a drug target for cancer therapy. Hsp90 comprises three structural domains, a highly conserved amino-terminal domain (NTD), a middle domain (MD), and a carboxyl-terminal domain (CTD). The CTD is responsible for protein dimerization, is crucial for Hsp90's activity, and has therefore been targeted for inhibiting Hsp90. Here we addressed the question of whether the CTD dimerization in Hsp90, in the absence of bound nucleotides, is modulated by allosteric effects from the other domains. We studied full length (FL) and isolated CTD (isoC) yeast Hsp90 spin-labeled with a Gd(III) tag by double electron-electron resonance measurements to track structural differences and to determine the apparent dissociation constant (Kd). We found the distance distributions for both the FL and isoC to be similar, indicating that the removal of the NTD and MD does not significantly affect the structure of the CTD dimer. The low-temperature double electron-electron resonance-derived Kd values, as well as those obtained at room temperature using microscale thermophoresis and native mass spectrometry, collectively suggested the presence of some allosteric effects from the NTDs and MDs on the CTD dimerization stability in the apo state. This was evidenced by a moderate increase in the Kd for the isoC compared with the FL mutants. Our results reveal a fine regulation of the CTD dimerization by allosteric modulation, which may have implications for drug targeting strategies in cancer therapy.

A counter-enzyme complex regulates glutamate metabolism in Bacillus subtilis.

Multi-enzyme assemblies composed of metabolic enzymes catalyzing sequential reactions are being increasingly studied. Here, we report the discovery of a 1.6 megadalton multi-enzyme complex from Bacillus subtilis composed of two enzymes catalyzing opposite ('counter-enzymes') rather than sequential reactions: glutamate synthase (GltAB) and glutamate dehydrogenase (GudB), which make and break glutamate, respectively. In vivo and in vitro studies show that the primary role of complex formation is to inhibit the activity of GudB. Using cryo-electron microscopy, we elucidated the structure of the complex and the molecular basis of inhibition of GudB by GltAB. The complex exhibits unusual oscillatory progress curves and is necessary for both planktonic growth, in glutamate-limiting conditions, and for biofilm growth, in glutamate-rich media. The regulation of a key metabolic enzyme by complexing with its counter enzyme may thus enable cell growth under fluctuating glutamate concentrations.

Characterizing Endogenous Protein Complexes with Biological Mass Spectrometry.

Biological mass spectrometry (MS) encompasses a range of methods for characterizing proteins and other biomolecules. MS is uniquely powerful for the structural analysis of endogenous protein complexes, which are often heterogeneous, poorly abundant, and refractive to characterization by other methods. Here, we focus on how biological MS can contribute to the study of endogenous protein complexes, which we define as complexes expressed in the physiological host and purified intact, as opposed to reconstituted complexes assembled from heterologously expressed components. Biological MS can yield information on complex stoichiometry, heterogeneity, topology, stability, activity, modes of regulation, and even structural dynamics. We begin with a review of methods for isolating endogenous complexes. We then describe the various biological MS approaches, focusing on the type of information that each method yields. We end with future directions and challenges for these MS-based methods.

Helicase-like functions in phosphate loop containing beta-alpha polypeptides.

The P-loop Walker A motif underlies hundreds of essential enzyme families that bind nucleotide triphosphates (NTPs) and mediate phosphoryl transfer (P-loop NTPases), including the earliest DNA/RNA helicases, translocases, and recombinases. What were the primordial precursors of these enzymes? Could these large and complex proteins emerge from simple polypeptides? Previously, we showed that P-loops embedded in simple ßα repeat proteins bind NTPs but also, unexpectedly so, ssDNA and RNA. Here, we extend beyond the purely biophysical function of ligand binding to demonstrate rudimentary helicase-like activities. We further constructed simple 40-residue polypeptides comprising just one ß-(P-loop)-α element. Despite their simplicity, these P-loop prototypes confer functions such as strand separation and exchange. Foremost, these polypeptides unwind dsDNA, and upon addition of NTPs, or inorganic polyphosphates, release the bound ssDNA strands to allow reformation of dsDNA. Binding kinetics and low-resolution structural analyses indicate that activity is mediated by oligomeric forms spanning from dimers to high-order assemblies. The latter are reminiscent of extant P-loop recombinases such as RecA. Overall, these P-loop prototypes compose a plausible description of the sequence, structure, and function of the earliest P-loop NTPases. They also indicate that multifunctionality and dynamic assembly were key in endowing short polypeptides with elaborate, evolutionarily relevant functions.

Influence of Asp Isomerization on Trypsin and Trypsin-like Proteolysis.

Long-lived proteins (LLPs), although less common than their short-lived counterparts, are increasingly recognized to play important roles in age-related diseases such as Alzheimer's. In particular, spontaneous chemical modifications can accrue over time that serve as both indicators of and contributors to disrupted autophagy. For example, isomerization in LLPs is common and occurs in the absence of protein turnover while simultaneously interfering with the protein turnover by impeding proteolysis. In addition to the biological implications this creates, isomerization may also interfere with its own analysis. To clarify, bottom-up proteomics experiments rely on protein digestion by proteases, most commonly trypsin, but the extent to which isomerization might interfere with trypsin digestion is unknown. Here, we use a combination of liquid chromatography and mass spectrometry to examine the effect of isomerization on proteolysis by trypsin and chymotrypsin. Isomerized aspartic acid and serine residues (which represent the most common sites of isomerization in LLPs) were placed at various locations relative to the preferred protease cleavage point to evaluate the influence on digestion efficiency. Trypsin was found to be relatively tolerant of isomerization, except when present at the residue immediately C-terminal to Arg/Lys. For chymotrypsin, the influence of isomerization on digestion was less predictable, resulting in long-range interference for some isomer/peptide combinations. Given the trypsin- and chymotrypsin-like behaviors of the 20S proteasome, and to further establish the biological relevance of isomerization in LLPs, substrates with isomerized sites were also tested against proteasomal degradation. Significant disruption of 20S proteolysis was observed, suggesting that if LLPs persist long enough to isomerize, it will be difficult for the cells to digest them.

Direct-MS analysis of antibody-antigen complexes.

In recent decades, antibodies (Abs) have attracted the attention of academia and the biopharmaceutical industry due to their therapeutic properties and versatility in binding a vast spectrum of antigens. Different engineering strategies have been developed for optimizing Ab specificity, efficacy, affinity, stability and production, enabling systematic screening and analysis procedures for selecting lead candidates. This quality assessment is critical but usually demands time-consuming and labor-intensive purification procedures. Here, we harnessed the direct-mass spectrometry (direct-MS) approach, in which the analysis is carried out directly from the crude growth media, for the rapid, structural characterization of designed Abs. We demonstrate that properties such as stability, specificity and interactions with antigens can be defined, without the need for prior purification.

Quinone Methide-Based Organophosphate Hydrolases Inhibitors: Trans Proximity Labelers versus Cis Labeling Activity-Based Probes.

Quinone methide (QM) chemistry is widely applied including in enzyme inhibitors. Typically, enzyme-mediated bond breaking releases a phenol product that rearranges into an electrophilic QM that in turn covalently modifies protein side chains. However, the factors that govern the reactivity of QM-based inhibitors and their mode of inhibition have not been systematically explored. Foremost, enzyme inactivation might occur in cis, whereby a QM molecule inactivates the very same enzyme molecule that released it, or by trans if the released QMs diffuse away and inactivate other enzyme molecules. We examined QM-based inhibitors for enzymes exhibiting phosphoester hydrolase activity. We tested different phenolic substituents and benzylic leaving groups, thereby modulating the rates of enzymatic hydrolysis, phenolate-to-QM rearrangement, and the electrophilicity of the resulting QM. By developing assays that distinguish between cis and trans inhibition, we have identified certain combinations of leaving groups and phenyl substituents that lead to inhibition in the cis mode, while other combinations gave trans inhibition. Our results suggest that cis-acting QM-based substrates could be used as activity-based probes to identify various phospho- and phosphono-ester hydrolases, and potentially other hydrolases.

Correction: Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces.

[This corrects the article DOI: 10.1371/journal.pcbi.1007207.].

Simple yet functional phosphate-loop proteins.

Abundant and essential motifs, such as phosphate-binding loops (P-loops), are presumed to be the seeds of modern enzymes. The Walker-A P-loop is absolutely essential in modern NTPase enzymes, in mediating binding, and transfer of the terminal phosphate groups of NTPs. However, NTPase function depends on many additional active-site residues placed throughout the protein's scaffold. Can motifs such as P-loops confer function in a simpler context? We applied a phylogenetic analysis that yielded a sequence logo of the putative ancestral Walker-A P-loop element: a ß-strand connected to an α-helix via the P-loop. Computational design incorporated this element into de novo designed ß-α repeat proteins with relatively few sequence modifications. We obtained soluble, stable proteins that unlike modern P-loop NTPases bound ATP in a magnesium-independent manner. Foremost, these simple P-loop proteins avidly bound polynucleotides, RNA, and single-strand DNA, and mutations in the P-loop's key residues abolished binding. Binding appears to be facilitated by the structural plasticity of these proteins, including quaternary structure polymorphism that promotes a combined action of multiple P-loops. Accordingly, oligomerization enabled a 55-aa protein carrying a single P-loop to confer avid polynucleotide binding. Overall, our results show that the P-loop Walker-A motif can be implemented in small and simple ß-α repeat proteins, primarily as a polynucleotide binding motif.

Intracellular Protein-Drug Interactions Probed by Direct Mass Spectrometry of Cell Lysates.

Understanding protein-ligand interactions in a cellular context is an important goal in molecular biology and biochemistry, and particularly for drug development. Investigators must demonstrate that drugs penetrate cells and specifically bind their targets. Towards that end, we present a native mass spectrometry (MS)-based method for analyzing drug uptake and target engagement in eukaryotic cells. This method is based on our previously introduced direct-MS method for rapid analysis of proteins directly from crude samples. Here, direct-MS enables label-free studies of protein-drug binding in human cells and is used to determine binding affinities of lead compounds in crude samples. We anticipate that this method will enable the application of native MS to a range of problems where cellular context is important, including protein-protein interactions, drug uptake and binding, and characterization of therapeutic proteins.

Higher-order oligomerization promotes localization of SPOP to liquid nuclear speckles.

Membrane-less organelles in cells are large, dynamic protein/protein or protein/RNA assemblies that have been reported in some cases to have liquid droplet properties. However, the molecular interactions underlying the recruitment of components are not well understood. Herein, we study how the ability to form higher-order assemblies influences the recruitment of the speckle-type POZ protein (SPOP) to nuclear speckles. SPOP, a cullin-3-RING ubiquitin ligase (CRL3) substrate adaptor, self-associates into higher-order oligomers; that is, the number of monomers in an oligomer is broadly distributed and can be large. While wild-type SPOP localizes to liquid nuclear speckles, self-association-deficient SPOP mutants have a diffuse distribution in the nucleus. SPOP oligomerizes through its BTB and BACK domains. We show that BTB-mediated SPOP dimers form linear oligomers via BACK domain dimerization, and we determine the concentration-dependent populations of the resulting oligomeric species. Higher-order oligomerization of SPOP stimulates CRL3(SPOP) ubiquitination efficiency for its physiological substrate Gli3, suggesting that nuclear speckles are hotspots of ubiquitination. Dynamic, higher-order protein self-association may be a general mechanism to concentrate functional components in membrane-less cellular bodies.

Mass Spectrometry Analysis of Intact Proteins from Crude Samples.

Analysis of intact proteins by native mass spectrometry has emerged as a powerful tool for obtaining insight into subunit diversity, post-translational modifications, stoichiometry, structural arrangement, stability, and overall architecture. Typically, such an analysis is performed following protein purification procedures, which are time consuming, costly, and labor intensive. As this technology continues to move forward, advances in sample handling and instrumentation have enabled the investigation of intact proteins in situ and in crude samples, offering rapid analysis and improved conservation of the biological context. This emerging field, which involves various ion source platforms such as matrix-assisted laser desorption ionization (MALDI) and electrospray ionization (ESI) for both spatial imaging and solution-based analysis, is expected to impact many scientific fields, including biotechnology, pharmaceuticals, and clinical sciences. In this Perspective, we discuss the information that can be retrieved by such experiments as well as the current advantages and technical challenges associated with the different sampling strategies. Furthermore, we present future directions of these MS-based methods, including current limitations and efforts that should be made to make these approaches more accessible. Considering the vast progress we have witnessed in recent years, we anticipate that the advent of further innovations enabling minimal handling of MS samples will make this field more robust, user friendly, and widespread.

Computational Strategies and Challenges for Using Native Ion Mobility Mass Spectrometry in Biophysics and Structural Biology.

Native mass spectrometry (MS) allows the interrogation of structural aspects of macromolecules in the gas phase, under the premise of having initially maintained their solution-phase noncovalent interactions intact. In the more than 25 years since the first reports, the utility of native MS has become well established in the structural biology community. The experimental and technological advances during this time have been rapid, resulting in dramatic increases in sensitivity, mass range, resolution, and complexity of possible experiments. As experimental methods have improved, there have been accompanying developments in computational approaches for analyzing and exploiting the profusion of MS data in a structural and biophysical context. In this perspective, we consider the computational strategies currently being employed by the community, aspects of best practice, and the challenges that remain to be addressed. Our perspective is based on discussions within the European Cooperation in Science and Technology Action on Native Mass Spectrometry and Related Methods for Structural Biology (EU COST Action BM1403), which involved participants from across Europe and North America. It is intended not as an in-depth review but instead to provide an accessible introduction to and overview of the topic-to inform newcomers to the field and stimulate discussions in the community about addressing existing challenges. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05792) focuses on software tools available to help researchers tackle some of the challenges enumerated here.

Software Requirements for the Analysis and Interpretation of Native Ion Mobility Mass Spectrometry Data.

The past few years have seen a dramatic increase in applications of native mass and ion mobility spectrometry, especially for the study of proteins and protein complexes. This increase has been catalyzed by the availability of commercial instrumentation capable of carrying out such analyses. As in most fields, however, the software to process the data generated from new instrumentation lags behind. Recently, a number of research groups have started addressing this by developing software, but further improvements are still required in order to realize the full potential of the data sets generated. In this perspective, we describe practical aspects as well as challenges in processing native mass spectrometry (MS) and ion mobility-MS data sets and provide a brief overview of currently available tools. We then set out our vision of future developments that would bring the community together and lead to the development of a common platform to expedite future computational developments, provide standardized processing approaches, and serve as a location for the deposition of data for this emerging field. This perspective has been written by members of the European Cooperation in Science and Technology Action on Native MS and Related Methods for Structural Biology (EU COST Action BM1403) as an introduction to the software tools available in this area. It is intended to serve as an overview for newcomers and to stimulate discussions in the community on further developments in this field, rather than being an in-depth review. Our complementary perspective (http://dx.doi.org/10.1021/acs.analchem.9b05791) focuses on computational approaches used in this field.

Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces.

Antibodies developed for research and clinical applications may exhibit suboptimal stability, expressibility, or affinity. Existing optimization strategies focus on surface mutations, whereas natural affinity maturation also introduces mutations in the antibody core, simultaneously improving stability and affinity. To systematically map the mutational tolerance of an antibody variable fragment (Fv), we performed yeast display and applied deep mutational scanning to an anti-lysozyme antibody and found that many of the affinity-enhancing mutations clustered at the variable light-heavy chain interface, within the antibody core. Rosetta design combined enhancing mutations, yielding a variant with tenfold higher affinity and substantially improved stability. To make this approach broadly accessible, we developed AbLIFT, an automated web server that designs multipoint core mutations to improve contacts between specific Fv light and heavy chains (http://AbLIFT.weizmann.ac.il). We applied AbLIFT to two unrelated antibodies targeting the human antigens VEGF and QSOX1. Strikingly, the designs improved stability, affinity, and expression yields. The results provide proof-of-principle for bypassing laborious cycles of antibody engineering through automated computational affinity and stability design.

A mutually inhibitory feedback loop between the 20S proteasome and its regulator, NQO1.

NAD(P)H:quinone-oxidoreductase-1 (NQO1) is a cytosolic enzyme that catalyzes the reduction of various quinones using flavin adenine dinucleotide (FAD) as a cofactor. NQO1 has been also shown to rescue proteins containing intrinsically unstructured domains, such as p53 and p73, from degradation by the 20S proteasome through an unknown mechanism. Here, we studied the nature of interaction between NQO1 and the 20S proteasome. Our study revealed a double negative feedback loop between NQO1 and the 20S proteasome, whereby NQO1 prevents the proteolytic activity of the 20S proteasome and the 20S proteasome degrades the apo form of NQO1. Furthermore, we demonstrate, both in vivo and in vitro, that NQO1 levels are highly dependent on FAD concentration. These observations suggest a link between 20S proteolysis and the metabolic cellular state. More generally, the results may represent a regulatory mechanism by which associated cofactors dictate the stability of proteins, thus coordinating protein levels with the metabolic status.

Specific phospholipid binding to Na,K-ATPase at two distinct sites.

Membrane protein function can be affected by the physical state of the lipid bilayer and specific lipid-protein interactions. For Na,K-ATPase, bilayer properties can modulate pump activity, and, as observed in crystal structures, several lipids are bound within the transmembrane domain. Furthermore, Na,K-ATPase activity depends on phosphatidylserine (PS) and cholesterol, which stabilize the protein, and polyunsaturated phosphatidylcholine (PC) or phosphatidylethanolamine (PE), known to stimulate Na,K-ATPase activity. Based on lipid structural specificity and kinetic mechanisms, specific interactions of both PS and PC/PE have been inferred. Nevertheless, specific binding sites have not been identified definitively. We address this question with native mass spectrometry (MS) and site-directed mutagenesis. Native MS shows directly that one molecule each of 18:0/18:1 PS and 18:0/20:4 PC can bind specifically to purified human Na,K-ATPase (α1ß1). By replacing lysine residues at proposed phospholipid-binding sites with glutamines, the two sites have been identified. Mutations in the cytoplasmic αL8-9 loop destabilize the protein but do not affect Na,K-ATPase activity, whereas mutations in transmembrane helices (TM), αTM2 and αTM4, abolish the stimulation of activity by 18:0/20:4 PC but do not affect stability. When these data are linked to crystal structures, the underlying mechanism of PS and PC/PE effects emerges. PS (and cholesterol) bind between αTM 8, 9, 10, near the FXYD subunit, and maintain topological integrity of the labile C terminus of the α subunit (site A). PC/PE binds between αTM2, 4, 6, and 9 and accelerates the rate-limiting E1P-E2P conformational transition (site B). We discuss the potential physiological implications.

Time-Resolved NMR Analysis of Proteolytic α-Synuclein Processing in vitro and in cellulo.

Targeted proteolysis of the disordered Parkinson's disease protein alpha-synuclein (αSyn) constitutes an important event under physiological and pathological cell conditions. In this work, site-specific αSyn cleavage by different endopeptidases in vitro and by endogenous proteases in extracts of challenged and unchallenged cells was studied by time-resolved NMR spectroscopy. Specifically, proteolytic processing was monitored under neutral and low pH conditions and in response to Rotenone-induced oxidative stress. Further, time-dependent degradation of electroporation-delivered αSyn in intact SH-SY5Y and A2780 cells was analyzed. Results presented here delineate a general framework for NMR-based proteolysis studies in vitro and in cellulo, and confirm earlier reports pertaining to the exceptional proteolytic stability of αSyn under physiological cell conditions. However, experimental findings also reveal altered protease susceptibilities in selected mammalian cell lines and upon induced cell stress.

Mass Spectrometry: A Technique of Many Faces.

Protein complexes form the critical foundation for a wide range of biological process, however understanding the intricate details of their activities is often challenging. In this review we describe how mass spectrometry plays a key role in the analysis of protein assemblies and the cellular pathways which they are involved in. Specifically, we discuss how the versatility of mass spectrometric approaches provides unprecedented information on multiple levels. We demonstrate this on the ubiquitin-proteasome proteolytic pathway, a process that is responsible for protein turnover. We follow the various steps of this degradation route and illustrate the different mass spectrometry workflows that were applied for elucidating molecular information. Overall, this review aims to stimulate the integrated use of multiple mass spectrometry approaches for analyzing complex biological systems.