scholarly journals Structure, Dynamics and Function of the 26S Proteasome

2020 ◽  
pp. 1-151
Author(s):  
Youdong Mao
Keyword(s):  
Atp Hydrolysis ◽  
Three Dimensional ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Large Network ◽  
Processing Efficiency ◽  
Aaa Atpase ◽  
Substrate Degradation ◽  
Dynamics And Function ◽  
Almost All

AbstractThe 26S proteasome is the most complex ATP-dependent protease machinery, of ~2.5 MDa mass, ubiquitously found in all eukaryotes. It selectively degrades ubiquitin-conjugated proteins and plays fundamentally indispensable roles in regulating almost all major aspects of cellular activities. To serve as the sole terminal “processor” for myriad ubiquitylation pathways, the proteasome evolved exceptional adaptability in dynamically organizing a large network of proteins, including ubiquitin receptors, shuttle factors, deubiquitinases, AAA-ATPase unfoldases, and ubiquitin ligases, to enable substrate selectivity and processing efficiency and to achieve regulation precision of a vast diversity of substrates. The inner working of the 26S proteasome is among the most sophisticated, enigmatic mechanisms of enzyme machinery in eukaryotic cells. Recent breakthroughs in three-dimensional atomic-level visualization of the 26S proteasome dynamics during polyubiquitylated substrate degradation elucidated an extensively detailed picture of its functional mechanisms, owing to progressive methodological advances associated with cryogenic electron microscopy (cryo-EM). Multiple sites of ubiquitin binding in the proteasome revealed a canonical mode of ubiquitin-dependent substrate engagement. The proteasome conformation in the act of substrate deubiquitylation provided insights into how the deubiquitylating activity of RPN11 is enhanced in the holoenzyme and is coupled to substrate translocation. Intriguingly, three principal modes of coordinated ATP hydrolysis in the heterohexameric AAA-ATPase motor  were discovered to regulate intermediate functional steps of the proteasome, including ubiquitin-substrate engagement, deubiquitylation, initiation of substrate translocation and processive substrate degradation. The atomic dissection of the innermost working of the 26S proteasome opens up a new era in our understanding of the ubiquitin-proteasome system and has far-reaching implications in health and disease.

scholarly journals Ccr4 is a novel shuttle factor required for ubiquitin-dependent proteolysis by the 26S proteasome

2020 ◽  
Author(s):  
Ganapathi Kandasamy ◽  
Ashis Kumar Pradhan ◽  
R Palanimurugan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Degradation ◽  
Ubiquitin Proteasome System ◽  
Proteasomal Degradation ◽  
Rna Degradation ◽  
26S Proteasome ◽  
Cellular Proteins ◽  
Cellular Homeostasis ◽  
Substrate Degradation ◽  
Ubiquitin Proteasome

AbstractDegradation of short-lived and abnormal proteins are essential for normal cellular homeostasis. In eukaryotes, such unstable cellular proteins are selectively degraded by the ubiquitin proteasome system (UPS). Furthermore, abnormalities in protein degradation by the UPS have been linked to several human diseases. Ccr4 protein is a known component of the Ccr4-Not complex, which has established roles in transcription, mRNA de-adenylation and RNA degradation etc. Excitingly in this study, we show that Ccr4 protein has a novel function as a shuttle factor that promotes ubiquitin-dependent degradation of short-lived proteins by the 26S proteasome. Using a substrate of the well-studied ubiquitin fusion degradation (UFD) pathway, we found that its UPS-mediated degradation was severely impaired upon deletion of CCR4 in Saccharomyces cerevisiae. Additionally, we show that Ccr4 binds to cellular ubiquitin conjugates and the proteasome. In contrast to Ccr4, most other subunits of the Ccr4-Not complex proteins are dispensable for UFD substrate degradation. From our findings we conclude that Ccr4 functions in the UPS as a shuttle factor targeting ubiquitylated substrates for proteasomal degradation.

scholarly journals Structural insights into the functional cycle of the ATPase module of the 26S proteasome

2017 ◽  
Vol 114 (6) ◽  
pp. 1305-1310 ◽  
Author(s):  
Marc Wehmer ◽  
Till Rudack ◽  
Florian Beck ◽  
Antje Aufderheide ◽  
Günter Pfeifer ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
Core Particle ◽  
Nucleotide Analogs ◽  
The Core ◽  
Cryo Electron Microscopy ◽  
Intracellular Proteins ◽  
Ubiquitin Proteasome ◽  
Structural Insights

In eukaryotic cells, the ubiquitin–proteasome system (UPS) is responsible for the regulated degradation of intracellular proteins. The 26S holocomplex comprises the core particle (CP), where proteolysis takes place, and one or two regulatory particles (RPs). The base of the RP is formed by a heterohexameric AAA+ ATPase module, which unfolds and translocates substrates into the CP. Applying single-particle cryo-electron microscopy (cryo-EM) and image classification to samples in the presence of different nucleotides and nucleotide analogs, we were able to observe four distinct conformational states (s1 to s4). The resolution of the four conformers allowed for the construction of atomic models of the AAA+ ATPase module as it progresses through the functional cycle. In a hitherto unobserved state (s4), the gate controlling access to the CP is open. The structures described in this study allow us to put forward a model for the 26S functional cycle driven by ATP hydrolysis.

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

2016 ◽  
Vol 113 (28) ◽  
pp. 7816-7821 ◽  
Author(s):  
Andreas Schweitzer ◽  
Antje Aufderheide ◽  
Till Rudack ◽  
Florian Beck ◽  
Günter Pfeifer ◽  
...  
Keyword(s):  
Ubiquitin Proteasome System ◽  
Structural Features ◽  
26S Proteasome ◽  
The Other ◽  
Aaa Atpase ◽  
Loop Region ◽  
C Terminus ◽  
Proteasome Subunits ◽  
Atpase Subunits ◽  
Substrate Processing

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

scholarly journals Structural basis for dynamic regulation of the human 26S proteasome

2016 ◽  
Vol 113 (46) ◽  
pp. 12991-12996 ◽  
Author(s):  
Shuobing Chen ◽  
Jiayi Wu ◽  
Ying Lu ◽  
Yong-Bei Ma ◽  
Byung-Hoon Lee ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ground State ◽  
Atp Hydrolysis ◽  
26S Proteasome ◽  
Structural Basis ◽  
Core Particle ◽  
Dynamic Regulation ◽  
Aaa Atpase ◽  
The Core ◽  
Aaa Atpases

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near–atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

scholarly journals AAA+ ATPases in Protein Degradation: Structures, Functions and Mechanisms

Biomolecules ◽  
2020 ◽  
Vol 10 (4) ◽  
pp. 629 ◽  
Author(s):  
Shuwen Zhang ◽  
Youdong Mao
Keyword(s):  
Protein Degradation ◽  
Conformational Changes ◽  
Dynamic Models ◽  
Three Dimensional ◽  
26S Proteasome ◽  
Aaa Atpase ◽  
Substrate Protein ◽  
Function Relationship ◽  
Atp Binding And Hydrolysis ◽  
Aaa Atpases

Adenosine triphosphatases (ATPases) associated with a variety of cellular activities (AAA+), the hexameric ring-shaped motor complexes located in all ATP-driven proteolytic machines, are involved in many cellular processes. Powered by cycles of ATP binding and hydrolysis, conformational changes in AAA+ ATPases can generate mechanical work that unfolds a substrate protein inside the central axial channel of ATPase ring for degradation. Three-dimensional visualizations of several AAA+ ATPase complexes in the act of substrate processing for protein degradation have been resolved at the atomic level thanks to recent technical advances in cryogenic electron microscopy (cryo-EM). Here, we summarize the resulting advances in structural and biochemical studies of AAA+ proteases in the process of proteolysis reactions, with an emphasis on cryo-EM structural analyses of the 26S proteasome, Cdc48/p97 and FtsH-like mitochondrial proteases. These studies reveal three highly conserved patterns in the structure–function relationship of AAA+ ATPase hexamers that were observed in the human 26S proteasome, thus suggesting common dynamic models of mechanochemical coupling during force generation and substrate translocation.

scholarly journals Mechanistic insights into dynamic mutual regulation of USP14 and proteasome

2021 ◽  
Author(s):  
Shuwen Zhang ◽  
Shitao Zou ◽  
Deyao Yin ◽  
Daniel Finley ◽  
Zhaolong Wu ◽  
...  
Keyword(s):  
Atp Hydrolysis ◽  
26S Proteasome ◽  
State Transitions ◽  
Ubiquitin Chain ◽  
Time Resolved ◽  
Aaa Atpase ◽  
Cryo Electron Microscopy ◽  
Mutual Regulation ◽  
Conformational Landscape ◽  
Multiple Levels

Proteasomal degradation of ubiquitylated proteins is sophisticatedly regulated at multiple levels. A primary regulatory checkpoint is the removal of ubiquitin chains from substrates by the deubiquitylating enzyme USP14 that associates reversibly with the proteasome. How USP14 is activated and regulates the proteasome function remains unknown. Here we report cryo-electron microscopy (cryo-EM) structures of human USP14 in complex with the 26S proteasome in nine conformational states at 3.0-3.6 &Aring resolution, captured during polyubiquitylated protein degradation. Time-resolved cryo-EM analysis of the conformational continuum revealed two parallel pathways of proteasome state transitions induced by USP14 and captured transient conversion of substrate-engaged intermediates into substrate-inhibited intermediates. On the substrate-engaged pathway, USP14 activation allosterically reprograms conformational landscape of the AAA-ATPase motor and stimulates opening of the core particle gate, enabling observation of a near-complete cycle of asymmetric ATP hydrolysis around the ATPase ring during processive substrate unfolding. Dynamic USP14-ATPase interactions decouple the ATPase activity from RPN11-catalysed deubiquitylation and kinetically introduce three regulatory checkpoints on the proteasome, at the steps of ubiquitin recognition, substrate translocation initiation and ubiquitin chain recycling. These findings provide unprecedented insights into the complete functional cycles of USP14-regulated proteasome and of USP14 activation-deubiquitylation-disassembly and establish mechanistic foundations for USP14-targeted therapeutic discovery.

Recent Patents on Micro-EDM Milling

2020 ◽  
Vol 13 (3) ◽  
pp. 219-229
Author(s):  
Baocheng Xie ◽  
Jianguo Liu ◽  
Yongqiu Chen
Keyword(s):  
Electrical Discharge ◽  
Electrical Discharge Machining ◽  
Three Dimensional ◽  
Processing Method ◽  
Electrode Wear ◽  
Micro Edm ◽  
Processing Efficiency ◽  
Processing Methods ◽  
Machining Quality ◽  
Micro Electrical Discharge Machining

Background: Micro-Electrical Discharge Machining (EDM) milling is widely used in the processing of complex cavities and micro-three-dimensional structures, which is a more effective processing method for micro-precision parts. Thus, more attention has been paid on the micro-EDM milling. Objective : To meet the increasing requirement of machining quality and machining efficiency of micro- EDM milling, the processing devices and processing methods of micro-EDM milling are being improved continuously. Methods: This paper reviews various current representative patents related to the processing devices and processing methods of micro-EDM milling. Results: Through summarizing a large number of patents about processing devices and processing methods of micro-EDM milling, the main problems of current development, such as the strategy of electrode wear compensation and the development trends of processing devices and processing methods of micro-EDM milling are discussed. Conclusion: The optimization of processing devices and processing methods of micro-EDM milling are conducive to solving the problems of processing efficiency and quality. More relevant patents will be invented in the future.

scholarly journals 3D Reconstruction of Cultural Heritage Sites as an Educational Approach. The Sanctuary of Delphi

2021 ◽  
Vol 11 (8) ◽  
pp. 3635
Author(s):  
Ioannis Liritzis ◽  
Pantelis Volonakis ◽  
Spyros Vosinakis
Keyword(s):  
University Students ◽  
3D Reconstruction ◽  
Cultural Heritage ◽  
New Technology ◽  
Three Dimensional ◽  
Computer Gaming ◽  
Educational Approach ◽  
Heritage Sites ◽  
3D Environment ◽  
Almost All

In the field of cultural heritage, three-dimensional (3D) reconstruction of monuments is a usual activity for many professionals. The aim in this paper focuses on the new technology educational application combining science, history, and archaeology. Being involved in almost all stages of implementation steps and assessing the level of participation, university students use tools of computer gaming platform and participate in ways of planning the virtual environment which improves their education through e-Learning. The virtual 3D environment is made with different imaging methods (helium-filled balloon, Structure for motion, 3D repository models) and a developmental plan has been designed for use in many future applications. Digital tools were used with 3D reconstructed buildings from the museum archive to Unity 3D for the design. The pilot study of Information Technology work has been employed to introduce cultural heritage and archaeology to university syllabuses. It included students with a questionnaire which has been evaluated accordingly. As a result, the university students were inspired to immerse themselves into the virtual lab, aiming to increasing the level of interaction. The results show a satisfactory learning outcome by an easy to use and real 3D environment, a step forward to fill in needs of contemporary online sustainable learning demands.

scholarly journals Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals

2021 ◽  
Vol 11 (2) ◽  
pp. 94
Author(s):  
Masaki Kumondai ◽  
Akio Ito ◽  
Evelyn Marie Gutiérrez Rico ◽  
Eiji Hishinuma ◽  
Akiko Ueda ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Three Dimensional ◽  
Warfarin Dose ◽  
Catalytic Efficiency ◽  
Therapeutic Window ◽  
Wild Type ◽  
Drug Metabolizing Enzyme ◽  
Novel Variants ◽  
Metabolizing Enzyme ◽  
Almost All

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype–phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

The Cryo-EM Effect: Structural Biology of Neurodegenerative Disease Proteostasis Factors

2021 ◽  
Author(s):  
Benjamin C Creekmore ◽  
Yi-Wei Chang ◽  
Edward B Lee
Keyword(s):  
Neurodegenerative Diseases ◽  
Protein Aggregation ◽  
Neurodegenerative Disease ◽  
Electron Tomography ◽  
Ubiquitin Proteasome System ◽  
26S Proteasome ◽  
X Ray Crystallography ◽  
New Information ◽  
A Cell ◽  
Regulate Protein

Abstract Neurodegenerative diseases are characterized by the accumulation of misfolded proteins. This protein aggregation suggests that abnormal proteostasis contributes to aging-related neurodegeneration. A better fundamental understanding of proteins that regulate proteostasis may provide insight into the pathophysiology of neurodegenerative disease and may perhaps reveal novel therapeutic opportunities. The 26S proteasome is the key effector of the ubiquitin-proteasome system responsible for degrading polyubiquitinated proteins. However, additional factors, such as valosin-containing protein (VCP/p97/Cdc48) and C9orf72, play a role in regulation and trafficking of substrates through the normal proteostasis systems of a cell. Nonhuman AAA+ ATPases, such as the disaggregase Hsp104, also provide insights into the biochemical processes that regulate protein aggregation. X-ray crystallography and cryo-electron microscopy (cryo-EM) structures not bound to substrate have provided meaningful information about the 26S proteasome, VCP, and Hsp104. However, recent cryo-EM structures bound to substrate have provided new information about the function and mechanism of these proteostasis factors. Cryo-EM and cryo-electron tomography data combined with biochemical data have also increased the understanding of C9orf72 and its role in maintaining proteostasis. These structural insights provide a foundation for understanding proteostasis mechanisms with near-atomic resolution upon which insights can be gleaned regarding the pathophysiology of neurodegenerative diseases.

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