Human 20S Proteasome Research Articles

Structural basis of human 20S proteasome biogenesis

New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. Here, to understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observe an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.

Structural basis of human 20S proteasome biogenesis.

New proteasomes are produced to accommodate increases in cellular catabolic demand and prevent the accumulation of cytotoxic proteins. Formation of the proteasomal 20S core complex relies on the function of the five chaperones PAC1-4 and POMP. To understand how these chaperones facilitate proteasome assembly, we tagged the endogenous chaperones using CRISPR/Cas gene editing and examined the chaperone-bound complexes by cryo-EM. We observed an early α-ring intermediate subcomplex that is stabilized by PAC1-4, which transitions to β-ring assembly upon dissociation of PAC3/PAC4 and rearrangement of the PAC1 N-terminal tail. Completion of the β-ring and dimerization of half-proteasomes repositions critical lysine K33 to trigger cleavage of the β pro-peptides, leading to the concerted dissociation of POMP and PAC1/PAC2 to yield mature 20S proteasomes. This study reveals structural insights into critical points along the assembly pathway of the human proteasome and provides a molecular blueprint for 20S biogenesis.

Rpt5-Derived Analogs Stimulate Human Proteasome Activity in Cells and Degrade Proteins Forming Toxic Aggregates in Age-Related Diseases.

Aging and age-related diseases are associated with a decline in the capacity of protein turnover. Intrinsically disordered proteins, as well as proteins misfolded and oxidatively damaged, prone to aggregation, are preferentially digested by the ubiquitin-independent proteasome system (UIPS), a major component of which is the 20S proteasome. Therefore, boosting 20S activity constitutes a promising strategy to counteract a decrease in total proteasome activity during aging. One way to enhance the proteolytic removal of unwanted proteins appears to be the use of peptide-based activators of the 20S. In this study, we synthesized a series of peptides and peptidomimetics based on the C-terminus of the Rpt5 subunit of the 19S regulatory particle. Some of them efficiently stimulated human 20S proteasome activity. The attachment of the cell-penetrating peptide TAT allowed them to penetrate the cell membrane and stimulate proteasome activity in HEK293T cells, which was demonstrated using a cell-permeable substrate of the proteasome, TAS3. Furthermore, the best activator enhanced the degradation of aggregation-prone α-synuclein and Tau-441. The obtained compounds may therefore have the potential to compensate for the unbalanced proteostasis found in aging and age-related diseases.

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

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

Protein degradation by human 20S proteasomes elucidates the interplay between peptide hydrolysis and splicing

If and how proteasomes catalyze not only peptide hydrolysis but also peptide splicing is an open question that has divided the scientific community. The debate has so far been based on immunopeptidomics, in vitro digestions of synthetic polypeptides as well as ex vivo and in vivo experiments, which could only indirectly describe proteasome-catalyzed peptide splicing of full-length proteins. Here we develop a workflow—and cognate software - to analyze proteasome-generated non-spliced and spliced peptides produced from entire proteins and apply it to in vitro digestions of 15 proteins, including well-known intrinsically disordered proteins such as human tau and α-Synuclein. The results confirm that 20S proteasomes produce a sizeable variety of cis-spliced peptides, whereas trans-spliced peptides are a minority. Both peptide hydrolysis and splicing produce peptides with well-defined characteristics, which hint toward an intricate regulation of both catalytic activities. At protein level, both non-spliced and spliced peptides are not randomly localized within protein sequences, but rather concentrated in hotspots of peptide products, in part driven by protein sequence motifs and proteasomal preferences. At sequence level, the different peptide sequence preference of peptide hydrolysis and peptide splicing suggests a competition between the two catalytic activities of 20S proteasomes during protein degradation.

Decoding the secrets: how conformational and structural regulators inhibit the human 20S proteasome.

Acquired resistance to drugs that modulate specific protein functions, such as the human proteasome, presents a significant challenge in targeted therapies. This underscores the importance of devising new methodologies to predict drug binding and potential resistance due to specific protein mutations. In this work, we conducted an extensive computational analysis to ascertain the effects of selected mutations (Ala49Thr, Ala50Val, and Cys52Phe) within the active site of the human proteasome. Specifically, we sought to understand how these mutations might disrupt protein function either by altering protein stability or by impeding interactions with a clinical administered drug. Leveraging molecular dynamics simulations and molecular docking calculations, we assessed the effect of these mutations on protein stability and ligand affinity. Notably, our results indicate that the Cys52Phe mutation critically impacts protein-ligand binding, providing valuable insights into potential proteasome inhibitor resistance.

In Silico Discovery and Investigation of Novel Small Molecules Targeting PSMA7 for the Treatment of GBM

The five-year survival rate for glioblastoma (GBM; WHO grade 4) remains only 5%, even with the current standard treatment of maximal safe surgical tumor resection followed by radiation treatment (RT) and concomitant and adjuvant temozolomide (TMZ). Therefore, alternative treatment therapies, such as blood-brain barrier (BBB)-permeable small molecule drugs, are needed to significantly improve patient outcomes by addressing other critical pathways in GBM progression and overcome GBM resistance to the standard treatment. Previously, we identified and validated proteasome subunit alpha type-7 (PSMA7), a subunit of the 20S proteasome, as a druggable, therapeutically vulnerable target in GBM by in vitro and in vivo methods. In this study, we performed in silico molecular docking screenings using a small molecule library to identify BBB-permeable small molecules predicted to bind to PSMA7 and investigated their potential as novel therapeutics for the treatment of GBM. In silico molecular docking screenings were performed using the National Cancer Institute Diversity Set VI small molecule library and the crystal structure of PSMA7 obtained from the native human 20S proteasome (RCSB PDB: 5LE5). Molecules were scored based on their predicted likelihood to bind to PSMA7. Top ranked molecules were screened using the SwissADME webtool, to select molecules predicted to be BBB-permeable and not a substrate for the permeability glycoprotein (P-gp), for testing with in vitro studies. Then, in vitro cell viability assays were used to determine IC50 values for molecules in human GBM PDX and normal human cell lines. The potential for molecules to sensitize GBM cells towards TMZ and/or RT was evaluated in vitro. In vitro proteasome activity assays were used to investigate whether molecules altered the three major protease activities performed by the proteasome. Computational virtual screenings using two docking algorithms (i.e., AutoDock Vina and Glide XP) identified and ranked the top small molecules most likely to bind to PSMA7. In silico screening using the SwissADME webtool predicted 99 of these molecules to be BBB-permeable and not a substrate for the P-gp. Subsequent in vitro cell viability studies determined eight of these molecules have lower IC50 values in human GBM PDX cells compared to normal human cells. Furthermore, we examined the potential of these eight molecules to sensitize GBM cells to TMZ and/or RT in vitro. Moreover, we found that three of these molecules inhibited all three major protease activities performed by the proteasome in GBM cells. Our study identified BBB-permeable small molecules predicted to target PSMA7 that demonstrated greater toxicity to GBM cells than normal cells and inhibited proteasome activities, suggesting a potential mechanism of action. Taken together, these small molecules are potential candidates for preclinical studies and may serve as novel targeted therapies for the treatment of GBM.

New Scaffolds of Proteasome Inhibitors: Boosting Anticancer Potential by Exploiting the Synergy of In Silico and In Vitro Methodologies.

Cancer is a complex multifactorial disease whose pathophysiology involves multiple metabolic pathways, including the ubiquitin-proteasome system, for which several proteasome inhibitors have already been approved for clinical use. However, the resistance to existing therapies and the occurrence of severe adverse effects is still a concern. The purpose of this study was the discovery of novel scaffolds of proteasome inhibitors with anticancer activity, aiming to overcome the limitations of the existing proteasome inhibitors. Thus, a structure-based virtual screening protocol was developed using the structure of the human 20S proteasome, and 246 compounds from virtual databases were selected for in vitro evaluation, namely proteasome inhibition assays and cell viability assays. Compound 4 (JHG58) was shortlisted as the best hit compound based on its potential in terms of proteasome inhibitory activity and its ability to induce cell death (both with IC50 values in the low micromolar range). Molecular docking studies revealed that compound 4 interacts with key residues, namely with the catalytic Thr1, Ala20, Thr21, Lys33, and Asp125 at the chymotrypsin-like catalytic active site. The hit compound is a good candidate for additional optimization through a hit-to-lead campaign.

Exploration of novel four-membered-heterocycle constructed peptidyl proteasome inhibitors with improved metabolic stability for cancer treatment

Peptides have limitations as active pharmaceutical agents due to rapid hydrolysis by proteases and poor cell permeability. To overcome these limitations, a series of peptidyl proteasome inhibitors embedded with four-membered heterocycles were designed to enhance their metabolic stabilities. All synthesized compounds were screened for their inhibitory activities against human 20S proteasome, and 12 target compounds displayed potent efficacy with IC50 values lower than 20 nM. Additionally, these compounds exhibited strong anti-proliferative activities against multiple myeloma (MM) cell lines (MM1S: 72, IC50 = 4.86 ± 1.34 nM; RPMI-8226: 67, IC50 = 12.32 ± 1.44). Metabolic stability assessments of SGF, SIF, plasma and blood were conducted, and the representative compound 73 revealed long half-lives (Plasma: T1/2 = 533 min; Blood: T1/2 > 1000 min) and good proteasome inhibitory activity in vivo. These results suggest that compound 73 serve as a lead compound for the development of more novel proteasome inhibitors.

A New Support Film for Cryo Electron Microscopy Protein Structure Analysis Based on Covalently Functionalized Graphene.

Protein adsorption at the air-water interface is a serious problem in cryogenic electron microscopy (cryoEM) as it restricts particle orientations in the vitrified ice-film and promotes protein denaturation. To address this issue, the preparation of a graphene-based modified support film for coverage of conventional holey carbon transmission electron microscopy (TEM) grids is presented. The chemical modification of graphene sheets enables the universal covalent anchoring of unmodified proteins via inherent surface-exposed lysine or cysteine residues in a one-step reaction. Langmuir-Blodgett (LB) trough approach is applied for deposition of functionalized graphene sheets onto commercially available holey carbon TEM grids. The application of the modified TEM grids in single particle analysis (SPA) shows high protein binding to the surface of the graphene-based support film. Suitability for high resolution structure determination is confirmed by SPA of apoferritin. Prevention of protein denaturation at the air-water interface and improvement of particle orientations is shown using human 20S proteasome, demonstrating the potential of the support film for structural biology.

Peptidomimetics Based on C-Terminus of Blm10 Stimulate Human 20S Proteasome Activity and Promote Degradation of Proteins

Degradation of misfolded, redundant and oxidatively damaged proteins constitutes one of the cellular processes which are influenced by the 20S proteasome. However, its activity is generally thought to decrease with age which leads to the gradual accumulation of abnormal proteins in cells and their subsequent aggregation. Therefore, increasing proteasomal degradation constitutes a promising strategy to delay the onset of various age-related diseases, including neurodegenerative disorders. In this study we designed and obtained a series of peptidomimetic stimulators of 20S comprising in their sequences the C-terminal fragment of Blm10 activator. Some of the compounds were capable of enhancing the degradation of natively unfolded and oxidatively damaged proteins, such as α-synuclein and enolase, whose applicability as proteasome substrates was evaluated by microscale thermophoresis (MST). Furthermore, they increased the ChT-L activity of the proteasome in HEK293T cell extracts. Our studies indicate that the 20S proteasome-mediated protein substrates hydrolysis may be selectively increased by peptide-based stimulators acting in an allosteric manner. These compounds, after further optimization, may have the potential to counteract proteasome impairment in patients suffering from age-related diseases.

Silybins are stereospecific regulators of the 20S proteasome

A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently “open gate” mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7“ and C-8” of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.

Covalent Inhibition of the Human 20S Proteasome with Homobelactosin C Inquired by QM/MM Studies.

20S proteasome is a main player in the protein degradation pathway in the cytosol, thus intervening in multiple pivotal cellular processes. Over the years the proteasome has emerged as a crucial target for the treatment of many diseases such as neurodegenerative diseases, cancer, autoimmune diseases, developmental disorders, cystic fibrosis, diabetes, cardiac diseases, atherosclerosis, and aging. In this work, the mechanism of proteasome covalent inhibition with bisbenzyl-protected homobelactosin C (hBelC) was explored using quantum mechanics/molecular mechanics (QM/MM) methods. Molecular dynamic simulations were used to describe key interactions established between the hBelC and its unique binding mode in the primed site of the β5 subunit. The free energy surfaces were computed to characterize the kinetics and thermodynamics of the inhibition process. This study revealed that although the final inhibition product for hBelC is formed according to the same molecular mechanism as one described for hSalA, the free energy profile of the reaction pathway differs significantly from the one previously reported for γ-lactam-β-lactone containing inhibitors in terms of the height of the activation barrier as well as the stabilization of the final product. Moreover, it was proved that high stabilization of the covalent adduct formed between β5-subunit and hBelC, together with the presence of aminocarbonyl side chain in the structure of the inhibitor which prevents the hydrolysis of the ester bond from taking place, determines its irreversible character.

Effect of Sperm 26S Proteasome on Embryo Formation and Development in In Vitro Fertilization.

The aim of this study was to evaluate the effects of sperm proteasome activity on fertilization outcome and embryo development after IVF. Following density gradient centrifugation for IVF purpose, the spermatozoa of 84 infertile patients with tubal factor were evaluated by luciferase enzymatic activity to assess the proteasome quantity. The mean age of patients was 33.8years, and the mean concentration of human spermatozoa 26S proteasome was 674.53ng/ml. After IVF, the embryos were scored for morphology. The spermatozoa proteasome activity was both positively correlated with fertilization rate in vitro (P = 0.0003) and 2PN rate (P = 0.0007). Compared to low fertilization rate group, the high fertilization rate group showed a significantly higher level of spermatozoa proteasome activity (P = 0.002). In conclusion, sperm proteasome activity provides additional data on sperm functional capacity in terms of fertilization during IVF.

The Y\u03a6 motif defines the structure-activity relationships of human 20S proteasome activators

The 20S proteasome (20S) facilitates turnover of most eukaryotic proteins. Substrate entry into the 20S first requires opening of gating loops through binding of HbYX motifs that are present at the C-termini of certain proteasome activators (PAs). The HbYX motif has been predominantly characterized in the archaeal 20S, whereas little is known about the sequence preferences of the human 20S (h20S). Here, we synthesize and screen ~120 HbYX-like peptides, revealing unexpected differences from the archaeal system and defining the h20S recognition sequence as the Y-F/Y (YФ) motif. To gain further insight, we create a functional chimera of the optimized sequence, NLSYYT, fused to the model activator, PA26E102A. A cryo-EM structure of PA26E102A-h20S is used to identify key interactions, including non-canonical contacts and gate-opening mechanisms. Finally, we demonstrate that the YФ sequence preferences are tuned by valency, allowing multivalent PAs to sample greater sequence space. These results expand the model for termini-mediated gating and provide a template for the design of h20S activators.

Developing a Targeted Quantitative Strategy for Sulfoxide-Containing MS-Cleavable Cross-Linked Peptides to Probe Conformational Dynamics of Protein Complexes.

In recent years, cross-linking mass spectrometry (XL-MS) has made enormous strides as a technology for probing protein-protein interactions (PPIs) and elucidating architectures of multisubunit assemblies. To define conformational and interaction dynamics of protein complexes under different physiological conditions, various quantitative cross-linking mass spectrometry (QXL-MS) strategies based on stable isotope labeling have been developed. These QXL-MS approaches have effectively allowed comparative analysis of cross-links to determine their relative abundance changes at global scales. Although successful, it remains challenging to consistently obtain quantitative measurements on low-abundant cross-links. Therefore, targeted QXL-MS is needed to enable MS "Western" analysis of cross-links to enhance sensitivity and reliability in quantitation. To this end, we have established a robust parallel reaction monitoring (PRM)-based targeted QXL-MS platform using sulfoxide-containing MS-cleavable cross-linker disuccinimidyl sulfoxide (DSSO), permitting label-free comparative analysis of selected cross-links across multiple samples. In addition, we have applied this methodology to study phosphorylation-dependent conformational dynamics of the human 26S proteasome. The PRM-based targeted QXL-MS analytical platform described here is applicable for all sulfoxide-containing MS-cleavable cross-linkers and can be directly adopted for comparative studies of protein-protein interactions in various cellular contexts.

Metabolism guided optimization of peptidomimetics as non-covalent proteasome inhibitors for cancer treatment

A series of novel non-covalent peptidomimetic proteasome inhibitors possessing bulky group at the C-terminus and N-alkylation at the N-terminus were designed with the aim to increase metabolic stabilities in vivo. All the target compounds were screened for their inhibitory activities against human 20S proteasome, and most analogs exhibited notable potency compared with the positive control bortezomib with IC50 values lower than 10 nM, which also displayed potent cytotoxic activities against multiple myeloma (MM) cell lines and human acute myeloid leukemia (AML) cells. Furthermore, whole blood stability and in vivo proteasome inhibitory activity experiments of selected compounds were conducted for further evaluation, and the representative compound 43 (IC50 = 8.39 ± 2.32 nM, RPMI-8226: IC50 = 15.290 ± 2.281 nM, MM-1S: IC50 = 9.067 ± 3.103 nM, MV-4-11: IC50 = 2.464 ± 0.713 nM) revealed a half-life extension of greater than 9-fold (329.21 min VS 36.79 min) and potent proteasome inhibitory activity in vivo. The positive results confirmed the reliability of the metabolism guided optimization strategy, and the analogs discovered are potential leads for exploring new anti-MM drugs.

On the Origin of the Different Reversible Characters of Salinosporamide A and Homosalinosporamide A in the Covalent Inhibition of the Human 20S Proteasome

On the Origin of the Different Reversible Characters of Salinosporamide A and Homosalinosporamide A in the Covalent Inhibition of the <i>Human</i> 20S Proteasome

Design, synthesis and biological evaluation of dipeptides as novel non-covalent 20S proteasome inhibitors

Based on the interaction modes of the natural 20S proteasome inhibitors TMC-95A, we have previously discovered a dipeptide 1. To explore the SAR around compound 1, we designed and synthesized a series of dipeptides (8–38) with a fragment-based strategy. Among them, nine compounds showed significant inhibitory activities against the chymotrypsin-like activity of human 20S proteasome with IC50 values at the submicromolar level, which were comparable or even superior to the parent compound 1. Meanwhile, they displayed no significant inhibition against trypsin-like and caspase-like activities of 20S proteasome. The results suggested the feasibility to design dipeptides as novel and potent 20S proteasome inhibitors.

Theoretical Studies of the Acid-Base Equilibria in a Model Active Site of the Human 20S Proteasome.

The 20S proteasome is a macromolecule responsible for the chemical step in the ubiquitin-proteasome system of degrading unnecessary and unused proteins of the cell. It plays a central role both in the rapid growth of cancer cells and in viral infection cycles. Herein, we present a computational study of the acid-base equilibria in an active site of the human proteasome (caspase-like), an aspect which is often neglected despite the crucial role protons play in the catalysis. As example substrates, we take the inhibition by epoxy- and boronic acid-containing warheads. We have combined cluster quantum mechanical calculations, replica exchange molecular dynamics, and Bayesian optimization of nonbonded potential terms in the inhibitors. In relation to the latter, we propose an easily scalable approach for the reevaluation of nonbonded potentials making use of the hybrid quantum mechanics molecular mechanics dynamics information. Our results show that coupled acid-base equilibria need to be considered when modeling the inhibition mechanism. The coupling between a neighboring lysine and the reacting threonine is not affected by the presence of the studied inhibitors.