scholarly journals Deconvolution of substrate processing by the 26S proteasome reveals a selective kinetic gateway to degradation

2018 ◽  
Author(s):  
J.A.M. Bard ◽  
C. Bashore ◽  
K.C. Dong ◽  
A. Martin
Keyword(s):  
Amino Acid ◽  
Protein Degradation ◽  
Unnatural Amino Acid ◽  
Conformational State ◽  
26S Proteasome ◽  
The Individual ◽  
Detailed Kinetics ◽  
Substrate Processing ◽  
Kinetic Proofreading ◽  
Processing Steps

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

scholarly journals Proteasomal conformation controls unfolding ability

2021 ◽  
Vol 118 (25) ◽  
pp. e2101004118
Author(s):  
Julianna R. Cresti ◽  
Abramo J. Manfredonia ◽  
Christopher E. Bragança ◽  
Joseph A. Boscia ◽  
Christina M. Hurley ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conformational State ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Ubiquitinated Protein ◽  
Equilibrium Conditions ◽  
Substrate Processing

The 26S proteasome is the macromolecular machine responsible for the bulk of protein degradation in eukaryotic cells. As it degrades a ubiquitinated protein, the proteasome transitions from a substrate-accepting conformation (s1) to a set of substrate-processing conformations (s3 like), each stabilized by different intramolecular contacts. Tools to study these conformational changes remain limited, and although several interactions have been proposed to be important for stabilizing the proteasome’s various conformations, it has been difficult to test these directly under equilibrium conditions. Here, we describe a conformationally sensitive Förster resonance energy transfer assay, in which fluorescent proteins are fused to Sem1 and Rpn6, which are nearer each other in substrate-processing conformations than in the substrate-accepting conformation. Using this assay, we find that two sets of interactions, one involving Rpn5 and another involving Rpn2, are both important for stabilizing substrate-processing conformations. Mutations that disrupt these interactions both destabilize substrate-processing conformations relative to the substrate-accepting conformation and diminish the proteasome’s ability to successfully unfold and degrade hard-to-unfold substrates, providing a link between the proteasome’s conformational state and its unfolding ability.

scholarly journals Small-Molecule Inhibitors Targeting Proteasome-Associated Deubiquitinases

2021 ◽  
Vol 22 (12) ◽  
pp. 6213
Author(s):  
Seonghyeon Moon ◽  
Srinivasan Muniyappan ◽  
Sung-Bae Lee ◽  
Byung-Hoon Lee
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Kinetic Studies ◽  
26S Proteasome ◽  
Chemical Screening ◽  
Cellular Context ◽  
Substrate Processing ◽  
Intracellular Proteolysis ◽  
Processing Steps

The 26S proteasome is the principal protease for regulated intracellular proteolysis. This multi-subunit complex is also pivotal for clearance of harmful proteins that are produced throughout the lifetime of eukaryotes. Recent structural and kinetic studies have revealed a multitude of conformational states of the proteasome in substrate-free and substrate-engaged forms. These conformational transitions demonstrate that proteasome is a highly dynamic machinery during substrate processing that can be also controlled by a number of proteasome-associated factors. Essentially, three distinct family of deubiquitinases–USP14, RPN11, and UCH37–are associated with the 19S regulatory particle of human proteasome. USP14 and UCH37 are capable of editing ubiquitin conjugates during the process of their dynamic engagement into the proteasome prior to the catalytic commitment. In contrast, RPN11-mediated deubiquitination is directly coupled to substrate degradation by sensing the proteasome’s conformational switch into the commitment steps. Therefore, proteasome-bound deubiquitinases are likely to tailor the degradation events in accordance with substrate processing steps and for dynamic proteolysis outcomes. Recent chemical screening efforts have yielded highly selective small-molecule inhibitors for targeting proteasomal deubiquitinases, such as USP14 and RPN11. USP14 inhibitors, IU1 and its progeny, were found to promote the degradation of a subset of substrates probably by overriding USP14-imposed checkpoint on the proteasome. On the other hand, capzimin, a RPN11 inhibitor, stabilized the proteasome substrates and showed the anti-proliferative effects on cancer cells. It is highly conceivable that these specific inhibitors will aid to dissect the role of each deubiquitinase on the proteasome. Moreover, customized targeting of proteasome-associated deubiquitinases may also provide versatile therapeutic strategies for induced or repressed protein degradation depending on proteolytic demand and cellular context.

scholarly journals Visualizing ATP-dependent substrate-processing dynamics of the human 26S proteasome at near-atomic resolution

2018 ◽  
Author(s):  
Yuanchen Dong ◽  
Shuwen Zhang ◽  
Zhaolong Wu ◽  
Xuemei Li ◽  
Wei Li Wang ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Eukaryotic Cells ◽  
Power Stroke ◽  
The Third ◽  
Atpase Subunits ◽  
Adp Release ◽  
Substrate Processing ◽  
Ubiquitylated Protein

AbstractThe proteasome is an ATP-dependent 2.5-megadalton machine responsible for ubiquitylated protein degradation in all eukaryotic cells. Here we present cryo-EM structures of the substrate-engaged human 26S proteasome in seven conformational states at 2.8-3.6 Å resolution, captured during polyubiquitylated protein degradation. These structures visualize a continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to processive substrate translocation, during which ATP hydrolysis sequentially navigate through all six ATPase subunits. Three principle modes of coordinated ATP hydrolysis are observed, featuring hydrolytic events in two oppositely positioned ATPases, in two consecutive ATPases, and in one ATPase at a time. They regulate deubiquitylation, translocation initiation and processive unfolding of substrates, respectively. A collective power stroke in the ATPase motor is generated by synchronized ATP binding and ADP release in the substrate-engaging and disengaging ATPases, respectively. It is amplified largely in the substrate-disengaging ATPase, and propagated unidirectionally by coordinated ATP hydrolysis in the third consecutive ATPase.

An Unnatural Amino Acid that Mimics a Tripeptide β-Strand and Forms β-Sheetlike Hydrogen-Bonded Dimers [J. Am. Chem. Soc.2000,122, 7654-7661]. 

2001 ◽  
Vol 123 (7) ◽  
pp. 1545-1546
Author(s):  
James S. Nowick ◽  
De Michael Chung ◽  
Kalyani Maitra ◽  
Santanu Maitra ◽  
Kimberly D. Stigers ◽  
...  
Keyword(s):  
Amino Acid ◽  
Unnatural Amino Acid ◽  
Hydrogen Bonded

Purification and Characterization of Lupus Anticoagulant Like Protein from Agkistrodon Halys Brevicaudus Venom

1996 ◽  
Vol 76 (06) ◽  
pp. 0993-0997
Author(s):  
Zhao-Yan Li ◽  
Xiao-Wei Wu ◽  
Tie-Fu Yu ◽  
Eric C-Y Lian
Keyword(s):  
Amino Acid ◽  
Lupus Anticoagulant ◽  
Inhibitory Effect ◽  
Clotting Factor ◽  
Acid Oxidase ◽  
Crude Venom ◽  
Amino Acid Oxidase ◽  
Sds Page ◽  
The Individual ◽  
Reducing Condition

SummaryBy means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)Q(Q/K). It was devoid of phospho-lipaseA, fibrino(geno)lytic, 5′-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell’s viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.

scholarly journals RAPID COMMUNICATION: Residual feed intake in beef cattle is associated with differences in protein turnover and nutrient transporters in ruminal epithelium

2019 ◽  
Vol 97 (5) ◽  
pp. 2181-2187
Author(s):  
Ahmed A Elolimy ◽  
Emad Abdel-Hamied ◽  
Liangyu Hu ◽  
Joshua C McCann ◽  
Daniel W Shike ◽  
...  
Keyword(s):  
Amino Acid ◽  
Beef Cattle ◽  
Protein Degradation ◽  
Insulin Signaling ◽  
Feed Intake ◽  
Feed Efficiency ◽  
Protein Turnover ◽  
Residual Feed Intake ◽  
Ubiquitin Proteasome ◽  
Ruminal Epithelium

Abstract Residual feed intake (RFI) is a widely used measure of feed efficiency in cattle. Although the precise biologic mechanisms associated with improved feed efficiency are not well-known, most-efficient steers (i.e., with low RFI coefficient) downregulate abundance of proteins controlling protein degradation in skeletal muscle. Whether cellular mechanisms controlling protein turnover in ruminal tissue differ by RFI classification is unknown. The aim was to investigate associations between RFI and signaling through the mechanistic target of rapamycin (MTOR) and ubiquitin-proteasome pathways in ruminal epithelium. One hundred and forty-nine Red Angus cattle were allocated to 3 contemporary groups according to sex and herd origin. Animals were offered a finishing diet for 70 d to calculate the RFI coefficient for each. Within each group, the 2 most-efficient (n = 6) and least-efficient animals (n = 6) were selected. Compared with least-efficient animals, the most-efficient animals consumed less feed (P < 0.05; 18.36 vs. 23.39 kg/d DMI). At day 70, plasma samples were collected for insulin concentration analysis. Ruminal epithelium was collected immediately after slaughter to determine abundance and phosphorylation status of 29 proteins associated with MTOR, ubiquitin-proteasome, insulin signaling, and glucose and amino acid transport. Among the proteins involved in cellular protein synthesis, most-efficient animals had lower (P ≤ 0.05) abundance of MTOR, p-MTOR, RPS6KB1, EIF2A, EEF2K, AKT1, and RPS6KB1, whereas MAPK3 tended (P = 0.07) to be lower. In contrast, abundance of p-EEF2K, p-EEF2K:EEF2K, and p-EIF2A:EIF2A in most-efficient animals was greater (P ≤ 0.05). Among proteins catalyzing steps required for protein degradation, the abundance of UBA1, NEDD4, and STUB1 was lower (P ≤ 0.05) and MDM2 tended (P = 0.06) to be lower in most-efficient cattle. Plasma insulin and ruminal epithelium insulin signaling proteins did not differ (P > 0.05) between RFI groups. However, abundance of the insulin-responsive glucose transporter SLC2A4 and the amino acid transporters SLC1A3 and SLC1A5 also was lower (P ≤ 0.05) in most-efficient cattle. Overall, the data indicate that differences in signaling mechanisms controlling protein turnover and nutrient transport in ruminal epithelium are components of feed efficiency in beef cattle.

Nγ‐Hydroxyasparagine: a Multifunctional Unnatural Amino Acid That is a Good P1 Substrate of Asparaginyl Peptide Ligases

2021 ◽  
Author(s):  
Chuan-Fa Liu ◽  
Yiyin Xia ◽  
Janet To ◽  
Ning-Yu Chan ◽  
Side Hu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Unnatural Amino Acid

scholarly journals Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽  
2020 ◽  
Author(s):  
Thomas L. Williams ◽  
Debra J. Iskandar ◽  
Alexander R. Nödling ◽  
Yurong Tan ◽  
Louis Y. P. Luk ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Genetic Code ◽  
Unnatural Amino Acids ◽  
Trna Synthetase ◽  
Unnatural Amino Acid ◽  
Amino Acid Incorporation ◽  
Acid Incorporation ◽  
Genetic Code Expansion ◽  
Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

Sign in / Sign up

Export Citation Format

Share Document