Determination of ubiquitin fitness landscapes under different chemical stresses in a classroom setting

Ubiquitin is essential for eukaryotic life and varies in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies indicate that ubiquitin is highly tolerant to single mutations. We hypothesized that this tolerance would be reduced by chemically induced physiologic perturbations. To test this hypothesis, a class of first year UCSF graduate students employed deep mutational scanning to determine the fitness landscape of all possible single residue mutations in the presence of five different small molecule perturbations. These perturbations uncover 'shared sensitized positions' localized to areas around the hydrophobic patch and the C-terminus. In addition, we identified perturbation specific effects such as a sensitization of His68 in HU and a tolerance to mutation at Lys63 in DTT. Our data show how chemical stresses can reduce buffering effects in the ubiquitin proteasome system. Finally, this study demonstrates the potential of lab-based interdisciplinary graduate curriculum.

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Determination of Ubiquitin Fitness Landscapes Under Different Chemical Stresses in a Classroom Setting

10.1101/025452 ◽

2015 ◽

Author(s):

David Mavor ◽

James Fraser ◽

Keyword(s):

Amino Acid ◽

Fitness Landscape ◽

Ubiquitin Proteasome System ◽

Single Amino Acid ◽

Graduate Curriculum ◽

Scanning Procedure ◽

C Terminus ◽

Rich Media ◽

Sequencing Studies ◽

Amino Acid Mutations

Ubiquitination is an essential post-translational regulatory process that can control protein stability, localization, and activity. Ubiquitin is essential for eukaryotic life and is highly conserved, varying in only 3 amino acid positions between yeast and humans. However, recent deep sequencing studies in S. cerevisiae indicate that ubiquitin is highly tolerant to single amino acid mutations. To resolve this paradox, we hypothesized that the set of tolerated substitutions would be reduced when the cultures are not grown in rich media conditions and that chemically induced physiologic perturbations might unmask constraints on the ubiquitin sequence. To test this hypothesis, a class of first year UCSF graduate students employed a deep mutational scanning procedure to determine the fitness landscape of a library of all possible single amino acid mutations of ubiquitin in the presence of one of five small molecule perturbations: MG132, Dithiothreitol (DTT), Hydroxyurea (HU), Caffeine, and DMSO. Our data reveal that the number of tolerated substitutions is greatly reduced by DTT, HU, or Caffeine, and that these perturbations uncover “shared sensitized positions” localized to areas around the hydrophobic patch and to the C-terminus. We also show perturbation specific effects including the sensitization of His68 in HU and tolerance to mutation at Lys63 in DTT. Taken together, our data suggest that chemical stress reduces buffering effects in the ubiquitin proteasome system, revealing previously hidden fitness defects. By expanding the set of chemical perturbations assayed, potentially by other classroom-based experiences, we will be able to further address the apparent dichotomy between the extreme sequence conservation and the experimentally observed mutational tolerance of ubiquitin. Finally, this study demonstrates the realized potential of a project lab-based interdisciplinary graduate curriculum.

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New strategies to inhibit KEAP1 and the Cul3-based E3 ubiquitin ligases

Biochemical Society Transactions ◽

10.1042/bst20130215 ◽

2014 ◽

Vol 42 (1) ◽

pp. 103-107 ◽

Cited By ~ 14

Author(s):

Peter Canning ◽

Alex N. Bullock

Keyword(s):

Drug Targets ◽

Ubiquitin Ligases ◽

Ubiquitin Proteasome System ◽

E3 Ubiquitin Ligases ◽

E3 Ligases ◽

Btb Domain ◽

C Terminus ◽

Ubiquitin Proteasome ◽

New Gene ◽

And Function

E3 ubiquitin ligases that direct substrate proteins to the ubiquitin–proteasome system are promising, though largely unexplored drug targets both because of their function and their remarkable specificity. CRLs [Cullin–RING (really interesting new gene) ligases] are the largest group of E3 ligases and function as modular multisubunit complexes constructed around a Cullin-family scaffold protein. The Cul3-based CRLs uniquely assemble with BTB (broad complex/tramtrack/bric-à-brac) proteins that also homodimerize and perform the role of both the Cullin adapter and the substrate-recognition component of the E3. The most prominent member is the BTB–BACK (BTB and C-terminal Kelch)–Kelch protein KEAP1 (Kelch-like ECH-associated protein 1), a master regulator of the oxidative stress response and a potential drug target for common conditions such as diabetes, Alzheimer's disease and Parkinson's disease. Structural characterization of BTB–Cul3 complexes has revealed a number of critical assembly mechanisms, including the binding of an N-terminal Cullin extension to a bihelical ‘3-box’ at the C-terminus of the BTB domain. Improved understanding of the structure of these complexes should contribute significantly to the effort to develop novel therapeutics targeted to CRL3-regulated pathways.

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HSP70-binding motifs function as protein quality control degrons

10.1101/2021.12.22.473789 ◽

2021 ◽

Author(s):

Amanda B Abildgaard ◽

Søren D Petersen ◽

Fia B Larsen ◽

Caroline Kampmeyer ◽

Kristoffer E Johansson ◽

...

Keyword(s):

Quality Control ◽

Protein Quality ◽

Protein Quality Control ◽

Ubiquitin Proteasome System ◽

Misfolded Proteins ◽

Binding Motif ◽

Binding Motifs ◽

C Terminus ◽

Ubiquitin Proteasome ◽

Chaperone Binding

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for degradation through the ubiquitin-proteasome system (UPS). To uncover how PQC degrons function, we performed a screen in Saccharomyces cerevisiae by fusing a library of flexible tetrapeptides to the C-terminus of the Ura3-HA-GFP reporter. The identified degrons exhibited high sequence variation but with marked hydrophobicity. Notably, the best scoring degrons constitute predicted Hsp70-binding motifs. When directly tested, a canonical Hsp70 binding motif (RLLL) functioned as a dose-dependent PQC degron that was targeted by Hsp70, Hsp110, Fes1, several Hsp40 J-domain co-chaperones and the PQC E3 ligase Ubr1. Our results suggest that multiple PQC degrons overlap with chaperone-binding sites and that PQC-linked degradation achieves specificity via chaperone binding. Thus, the PQC system has evolved to exploit the intrinsic capacity of chaperones to recognize misfolded proteins, thereby placing them at the nexus of protein folding and degradation.

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Tying up loose ends: the N-degron and C-degron pathways of protein degradation

Biochemical Society Transactions ◽

10.1042/bst20191094 ◽

2020 ◽

Vol 48 (4) ◽

pp. 1557-1567 ◽

Cited By ~ 1

Author(s):

Richard T. Timms ◽

Itay Koren

Keyword(s):

Protein Degradation ◽

Protein Interactions ◽

Ubiquitin Proteasome System ◽

Protein Protein Interactions ◽

Cellular Functions ◽

Functional Roles ◽

C Terminus ◽

Ubiquitin Proteasome ◽

Cellular Machinery ◽

Substrate Proteins

Selective protein degradation by the ubiquitin-proteasome system (UPS) is thought to be governed primarily by the recognition of specific motifs — degrons — present in substrate proteins. The ends of proteins — the N- and C-termini – have unique properties, and an important subset of protein–protein interactions involve the recognition of free termini. The first degrons to be discovered were located at the extreme N-terminus of proteins, a finding which initiated the study of the N-degron (formerly N-end rule) pathways, but only in the last few years has it emerged that a diverse set of C-degron pathways target analogous degron motifs located at the extreme C-terminus of proteins. In this minireview we summarise the N-degron and C-degron pathways currently known to operate in human cells, focussing primarily on those that have been discovered in recent years. In each case we describe the cellular machinery responsible for terminal degron recognition, and then consider some of the functional roles of terminal degron pathways. Altogether, a broad spectrum of E3 ubiquitin ligases mediate the recognition of a diverse array of terminal degron motifs; these degradative pathways have the potential to influence a wide variety of cellular functions.

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The intracellular mechanism of Berberine-induced inhibition of CYP3A4 activity

Current Pharmaceutical Design ◽

10.2174/1381612827666210715155809 ◽

2021 ◽

Vol 27 ◽

Author(s):

Pan-Feng Feng ◽

Long-Xun Zhu ◽

Jing Jie ◽

Peng-Xiang Yang ◽

Xia Chen

Keyword(s):

Constitutive Androstane Receptor ◽

Ubiquitin Proteasome System ◽

Isoquinoline Alkaloid ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Cyp3a4 Gene ◽

Underlying Mechanisms ◽

Novel Drug

Background: Berberine (BBR) is an isoquinoline alkaloid extracted from Chinese medicine, exerting various pharmacological effects. BBR is partially metabolized by cytochrome 3A4 (CYP3A4) in vivo. Some reports indicated that BBR could inhibit the activity of CYP3A4. However, the underlying mechanisms are not entirely understood. CYP3A4 is transcriptionally regulated by two nuclear receptors: nuclear transcription X receptor (PXR) and constitutive androstane receptor (CAR). It degraded via the ubiquitin-proteasome system. Hence, we tried to explore the mechanisms of CYP3A4 inhibition on both transcriptive and protein levels. Methods: Western Blot, RT-PCR, and Co-immunoprecipitation were used to perform the experiments. Results: Our results showed that BBR inhibited the transcription of CYP3A4 gene by downregulating PXR. In addition, BBR accelerated the degradation of CYP3A4 protein via the polyubiquitination pathway. Conclusion: These findings may lead to the determination of novel drug-drug interactions with BBR and contribute to the future clinical application of BBR.

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Application of SPRi Biosensors for Determination of 20S Proteasome and UCH-L1 Levels in the Serum and Urine of Transitional Bladder Cancer Patients

Applied Sciences ◽

10.3390/app11177835 ◽

2021 ◽

Vol 11 (17) ◽

pp. 7835

Author(s):

Anna Sankiewicz ◽

Tomasz Guszcz ◽

Ewa Gorodkiewicz

Keyword(s):

Bladder Cancer ◽

Cancer Progression ◽

Transitional Cell ◽

Ubiquitin Proteasome System ◽

Clinicopathological Parameters ◽

Control Group ◽

20S Proteasome ◽

Ubiquitin Proteasome ◽

Serum And Urine

The ubiquitin–proteasome system (UPS) participates in the degradation of proteins which play an important role in regulating the cell cycle, apoptosis, and angiogenesis, as well as in the immune system. These processes are important in carcinogenesis. Transitional cell carcinoma (TCC) is one of the predominant types of bladder cancer. The relationship between the ubiquitin–proteasome system and cancer progression has become a topic of increasing interest among researchers. In this work, we propose an application of surface plasmon resonance imaging (SPRi)-based biosensors for the detection of 20S proteasome and ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) in the blood serum and urine of patients with TCC. The aim of the study was to determine 20S proteasome and UCH-L1 concentrations and to correlate the results with clinicopathological parameters. The group of subjects consisted of 82 patients with confirmed TCC, in addition to a control group of 27 healthy volunteers. It was found that 20S proteasome and UCH-L1 concentrations were significantly elevated in both the serum and urine of TCC patients, compared with the healthy subjects. There was a correlation between 20S proteasome concentrations in serum and urine, as well as between serum proteasome and UCH-L1 concentration. The SPRi biosensor sensitive to 20S proteasome using PSI inhibitor as the receptor, and the SPRi biosensor sensitive to the UCH-L1 protein using the protein-specific antibody as the receptor is suitable for the determination of 20S proteasome and UCH-L1 in body fluids and can serve as useful tools in the investigation of cancer biomarkers.

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The ubiquitin–proteasome system and skeletal muscle wasting

Essays in Biochemistry ◽

10.1042/bse0410173 ◽

2005 ◽

Vol 41 ◽

pp. 173-186 ◽

Cited By ~ 110

Author(s):

Didier Attaix ◽

Sophie Ventadour ◽

Audrey Codran ◽

Daniel Béchet ◽

Daniel Taillandier ◽

...

Keyword(s):

Skeletal Muscle ◽

Muscle Wasting ◽

Proteolytic Enzymes ◽

Cathepsin L ◽

Ubiquitin Proteasome System ◽

Complete Breakdown ◽

Skeletal Muscle Proteins ◽

Ubiquitin Proteasome ◽

Tripeptidyl Peptidase Ii ◽

F Box Protein

The ubiquitin–proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin–protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients.

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