Distinct isotopic fractionation in biomolecules, compared with atmospheric values, reflects their biosynthetic origin. Monitoring these fractionation changes offers a valuable approach for probing early metabolic networks. A key question in the study of life’s origins is the role of electronic spin and magnetic surfaces in symmetry breaking and the emergence of homochirality. Here, we used magnetic filters to show that the dynamical interaction with the magnetic surfaces changes the isotope fractions of 13C L-methionine compared with 12C L-methionine. Specifically, mass spectrometry analysis reveals that the isotopic fractionation of both natural and ¹³C-enriched L-methionine is influenced by electron spin-dependent interactions.

In this issue of Molecular Cell, Lee et al.1 address a central structural biology challenge: capturing low-affinity protein interactions in cells and integrating the resulting restraints with cryoelectron microscopy and AlphaFold to localize dynamic interactors on native human proteasomes.

The proteasome is a multisubunit degradation machinery that is essential for maintaining protein homeostasis by breaking down unnecessary or damaged proteins into peptides. While most of these peptides are further processed into amino acids, a subset evades complete degradation and plays key roles in biological processes such as antigen presentation, signaling, and apoptosis. However, the variability in peptide lengths and the diverse composition of proteasomes make their comprehensive identification and characterization particularly challenging. Here, we present a method that enables real-time identification of generated peptides, as well as uncleaved and partially cleaved protein substrates, revealing the processive nature of protein proteasomal degradation. Our intact degradomics workflow is based on intact mass spectrometry measurements and treats the enzymatically produced peptides as if they were generated within the mass spectrometer, akin to top-down products. We applied this approach to determine the kinetic profile of proteasome degradation and compare the real-time activity of proteasomes isolated from different mouse organs, uncovering distinct functionalities of the complex. Overall, this method offers a valuable tool for studying peptide degradation products across various proteasome configurations, while also enabling the investigation of how interacting proteins, inhibitors, and activators influence proteasome activity. Furthermore, its adaptability makes it applicable to a wide range of other proteolytic complexes, broadening its potential impact in the field.

Cooperative regulation of biomolecular function is critical for the ability of all organisms to respond effectively to environmental changes. Such regulation is often manifested in a sigmoidal dependence of enzyme activity on ligand concentration. Various molecular mechanisms have been proposed to underlie such sigmoidal behavior, but they are usually assumed to occur independently of one another. We hypothesized that coexistence of allosteric mechanisms can lead to complex kinetic behavior and higher or lower cooperativity than expected. A mathematical framework that analyses sigmoidal behavior as a function of two cooccurring mechanisms, hysteresis and homotropic binding cooperativity, was developed. The model shows, for example, that i) the observed cooperativity, as measured by the Hill coefficient, can decrease with increasing binding cooperativity, and that ii) unusually high values of the Hill coefficient can be observed. Our mathematical analysis is shown to be relevant for a mutant of Escherichia coli glutathione reductase with an unusually high value of a Hill coefficient for a dimer of about 1.9, in which hysteresis and binding cooperativity coexist. More generally, our findings imply that the repertoire of allosteric regulation is richer than anticipated and suggest that ultrasensitive control in natural or designed systems may arise even in low-order oligomers.

The proteasome is a central proteolytic complex that maintains protein homeostasis by eliminating damaged, misfolded, and regulatory proteins. Beyond this quality control role, it generates bioactive peptides that contribute to immune surveillance, intracellular signaling, neuronal communication, and antimicrobial defense. Proteolysis is mediated by the catalytic β1, β2, and β5 subunits, traditionally defined by caspase-like, trypsin-like, and chymotrypsin-like activities. However, these sites display overlapping and flexible specificities, enabling cleavage after nearly all amino acids. This review focuses on proteasome catalytic activity, with particular emphasis on the biochemical and structural features of the catalytic subunits that define cleavage selectivity. We first provide a historical overview of the discovery of proteolytic activities and trace the evolutionary diversification of subunits that gave rise to specialized variants such as the immunoproteasome, thymoproteasome, intermediate proteasomes, and the spermatoproteasome. We then highlight how advances in computational modeling and structural biology have refined our understanding of cleavage preferences. In addition, we examine how regulatory particles, post-translational modifications, and physiological conditions, including inflammation, oxidative stress, and aging, modulate proteolytic activity. Finally, we discuss the development of selective inhibitors targeting individual catalytic sites, emphasizing their therapeutic potential in cancer, autoimmunity, and infectious disease, and outline future directions for the field.

Proteasomes are essential for protein degradation and maintaining cellular balance, yet their roles in extracellular fluids are not well understood. Our study investigates the freely circulating proteasome in blood, to uncover its unique molecular characteristics, compared to its intracellular counterparts. Using a transgenic mouse model, mass spectrometry, and biochemical tools, we show that the predominant proteasome in serum is the free uncapped 20S particle, which seems to assemble intracellularly before entering the bloodstream. This serum proteasome is composed of constitutive and immuno subunits and exhibits all three catalytic activities. Moreover, the complex displays distinct post-translational modifications, indicating specialization for extracellular roles, as demonstrated by its enhanced caspase-like activity. We also found that physiological stress significantly upregulates serum 20S proteasome levels, paralleling human data. This research highlights the specialized characteristics of circulating proteasomes, offering new insights into protein turnover in the blood with significant implications for understanding proteostasis beyond the intracellular environment.

Abstract 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.

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.

Fluorinated aromatic amino acids (FAAs) are promising tools when studying protein structure and dynamics by NMR spectroscopy. The incorporation FAAs in mammalian expression systems has been introduced only recently. Here, we investigate the effects of FAAs incorporation in proteins expressed in human cells, focusing on the probability of incorporation and its consequences on the 19 F NMR spectra. By combining 19 F NMR, direct MS and x-ray crystallography, we demonstrate that the probability of FAA incorporation is only a function of the FAA concentration in the expression medium and is a pure stochastic phenomenon. In contrast with the MS data, the x-ray structures of carbonic anhydrase II reveal that while the 3D structure is not affected, certain positions lack fluorine, suggesting that crystallization selectively excludes protein molecules featuring subtle conformational modifications. This study offers a predictive model of the FAA incorporation efficiency and provides a framework for controlling protein fluorination in mammalian expression systems.

Controlled degradation of proteins is necessary for ensuring their abundance and sustaining a healthy and accurately functioning proteome. One of the degradation routes involves the uncapped 20S proteasome, which cleaves proteins with a partially unfolded region, including those that are damaged or contain intrinsically disordered regions. This degradation route is tightly controlled by a recently discovered family of proteins named Catalytic Core Regulators (CCRs). Here, we show that CCRs function through an allosteric mechanism, coupling the physical binding of the PSMB4 β-subunit with attenuation of the complex's three proteolytic activities. In addition, by dissecting the structural properties that are required for CCR-like function, we could recapitulate this activity using a designed protein that is half the size of natural CCRs. These data uncover an allosteric path that does not involve the proteasome's enzymatic subunits but rather propagates through the non-catalytic subunit PSMB4. This way of 20S proteasome-specific attenuation opens avenues for decoupling the 20S and 26S proteasome degradation pathways as well as for developing selective 20S proteasome inhibitors.

Protein degradation is one of the essential mechanisms that enables reshaping of the proteome landscape in response to various stimuli. The largest E3 ubiquitin ligase family that targets proteins to degradation by catalyzing ubiquitination is the cullin-RING ligases (CRLs). Many of the proteins that are regulated by CRLs are central to tumorigenesis and tumor progression, and dysregulation of the CRL family is frequently associated with cancer. The CRL family comprises ∼300 complexes, all of which are regulated by the COP9 signalosome complex (CSN). Therefore, CSN is considered an attractive target for therapeutic intervention. Research efforts for targeted CSN inhibition have been directed towards inhibition of the complex enzymatic subunit, CSN5. Here, we have taken a fresh approach focusing on CSNAP, the smallest CSN subunit. Our results show that the C-terminal region of CSNAP is tightly packed within the CSN complex, in a groove formed by CSN3 and CSN8. We show that a 16 amino acid C-terminal peptide, derived from this CSN-interacting region, can displace the endogenous CSNAP subunit from the complex. This, in turn, leads to a CSNAP null phenotype that attenuates CSN activity and consequently CRLs function. Overall, our findings emphasize the potential of a CSNAP-based peptide for CSN inhibition as a new therapeutic avenue.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

Native mass spectrometry (nMS) enables intact non-covalent complexes to be studied in the gas phase. nMS can provide information on composition, stoichiometry, topology, and, when coupled with surface-induced dissociation (SID), subunit connectivity. Here we describe the characterization of protein complexes by nMS and SID. Substructural information obtained using this method is consistent with the solved complex structure, when a structure exists. This provides confidence that the method can also be used to obtain substructural information for unknowns, providing insight into subunit connectivity and arrangements. High-energy SID can also provide information on proteoforms present. Previously SID has been limited to a few in-house modified instruments and here we focus on SID implemented within an in-house-modified Q Exactive UHMR. However, SID is currently commercially available within the Waters Select Series Cyclic IMS instrument. Projects are underway that involve the NIH-funded native MS resource (nativems.osu.edu), instrument vendors, and third-party vendors, with the hope of bringing the technology to more platforms and labs in the near future. Currently, nMS resource staff can perform SID experiments for interested research groups.

Proteasomes are traditionally considered intracellular complexes that play a critical role in maintaining proteostasis by degrading short-lived regulatory proteins and removing damaged proteins. Remarkably, in addition to these well-studied intracellular roles, accumulating data indicate that proteasomes are also present in extracellular body fluids. Not much is known about the origin, biological role, mode(s) of regulation or mechanisms of extracellular transport of these complexes. Nevertheless, emerging evidence indicates that the presence of proteasomes in the extracellular milieu is not a random phenomenon, but rather a regulated, coordinated physiological process. In this review, we provide an overview of the current understanding of extracellular proteasomes. To this end, we examine 143 proteomic datasets, leading us to the realization that 20S proteasome subunits are present in at least 25 different body fluids. Our analysis also indicates that while 19S subunits exist in some of those fluids, the dominant proteasome activator in these compartments is the PA28α/β complex. We also elaborate on the positive correlations that have been identified in plasma and extracellular vesicles, between 20S proteasome and activity levels to disease severity and treatment efficacy, suggesting the involvement of this understudied complex in pathophysiology. In addition, we address the considerations and practical experimental methods that should be taken when investigating extracellular proteasomes. Overall, we hope this review will stimulate new opportunities for investigation and thoughtful discussions on this exciting topic that will contribute to the maturation of the field.

A parasite is a unicellular ormulticellular organism that is dependent on another organism (host) for its survival and proliferation. The parasite benefits from a prolonged association with its host (Loker & Hofkin, 2015), as without it, the parasite cannot grow and multiply. Thus, parasites must keep their host alive for as long as possible without killing it, yet their infection may cause diseases and, in some cases, like malaria, even mortality. Threemain classes of parasites can cause disease in humans: protozoa, helminths and ectoparasites. The former are unicellular eukaryotic parasites (e.g., Plasmodium falciparum and Trypanosoma brucei), whereas the helminths are parasitic worms (e.g., Schistosoma haematobium and Fasciola hepatica), and the ectoparasites, organisms that infest the host skin (e.g., Haemaphysalis longicornis and Sarcoptes scabiei). These classes of parasites are master manipulators whose life cycles feature unique adaptions, involving sophisticated strategies to alter the host environment, including the secretion of multipurpose extracellular vesicles (EVs) (Coakley et al., 2015; Ofir-Birin & Regev-Rudzki, 2019). EVs are heterogeneous in terms of size (30–500 nm in diameter) and transfer functional signals to target cells by carrying a cornucopia of different molecules, such as proteins, glycans, lipids, RNA and DNA (Schorey et al., 2015; Tkach & Théry, 2016). Since the release of EVs is an integral part of a parasite’s life cycle and course of the infection, it stands to reason that these organelles are essential for their survival. Indeed, these shuttling vesicles provide a robust delivery system to facilitate parasitic growth and development, the transfer of virulence factors, adherence to host tissues, and evasion of immune responses (Mardahl et al., 2019; Ofir-Birin et al., 2017). They effectively manipulate the host’s immune system by inhibiting or activating responses as well as by affecting a variety of other (non-immune) target human cells (Marcilla et al., 2014; PMID: 29577413 by HosseiniBeheshti, E : Ofir-Birin & Regev-Rudzki, 2019). A case in point is the malaria parasite Plasmodium (P) falciparum, which secretes EVs while residing in human red blood cells (RBCs) that affect a range of target host cells (Mantel et al., 2016;Mantel et al., 2013; Regev-Rudzki et al., 2013; Sisquella et al., 2017; Ye et al., 2018). Intriguingly, our recent investigation of the protein content of these secreted EVs revealed great enrichment in parasitic and host subunits of the proteasome degradation complex (Dekel et al., 2021). Our results indicate that these proteasome subunits are assembled within the EVs into intact, functional 20S proteasomes, and that the activities of these encapsulated complexes promote parasitic growth. In particular, we found that followingP. falciparum-derived EV introduction, two sequential steps take place. First, RBC host proteins undergo specific phosphorylation events, including in several cytoskeletal proteins. Second, the delivered 20S proteasomes mediate the degradation of the phosphorylated cytoskeleton proteins by a ubiquitinindependent process (Dekel et al., 2021). The result is a reduction in the stiffness of the naïve RBCs’ membrane, thus, priming them for parasitic invasion, with direct implications for the parasite’s growth capacity. The proteasome is a conserved degradation machinery that is vital for maintaining proteostasis by irreversibly removing misfolded, damaged or short-lived regulatory proteins (Baumeister et al., 1998). Two alternative proteasomal degradation mechanisms, which are notmutually exclusive, exists in cells, involving the 26S and 20S proteasome complexes (Goldberg, 2003; Kumar Deshmukh et al., 2019). The 26S proteasome comprises a 19S regulatory particle, which recognises ubiquitin-tagged substrates, and a 20S catalytic core particle, where substrates are degraded via the breakage of peptide bonds. Degradation by the 26S proteasome, which is the major cellular degradation route, is an ATP-dependent process that is coordinated by three different types of enzymes (E1, E2, and E3) that ubiquitinate the substrate and sensitize it to degradation (Hershko &Ciechanover, 1998). In contrast, the 20S proteasome, on its own, can degrade protein substrates in a ubiquitinand ATP-independent manner, by cleaving unfolded or unstructured regions within its substrates (Ben-Nissan & Sharon, 2014; Kumar Deshmukh et al., 2019). Hence, the preference of P. falciparum-derived EVs for 20S-proteasome-mediated degradation over the 26S proteasome pathway may arise from the simplicity and self-reliance of the 20S system. Moreover, the size of the malaria-derived EVs, which mostly ranges between 50 and 200 nm in diameter, likely restricts the encapsulation of 26S proteasomes (∼45 × 20 nm) to a greater degree than that of 20S particles (∼15 × 12 nm).

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.

AbstractUnderstanding 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.

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 βα 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 β-(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.