Mode of Targeting to the Proteasome Determines GFP Fate

Eukaryotic Cells ◽

Polyubiquitin Chains ◽

Ubiquitin Proteasome ◽

Targeting Signal ◽

Green Fluorescent ◽

Ubiquitin Receptors ◽

Multiple Variants ◽

Domain Stability

ABSTRACTThe Ubiquitin-proteasome system (UPS) is the canonical pathway for protein degradation in eukaryotic cells. Green fluorescent protein (GFP) is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different stabilities. We previously found that the proteasome’s ability to unfold and degrade substrates is enhanced by polyubiquitin chains on the substrate, and that proteasomal ubiquitin receptors mediate this activation. Herein we investigate how the fate of GFP variants of differing stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, while the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates.

Mode of targeting to the proteasome determines GFP fate

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015235 ◽

2020 ◽

Vol 295 (47) ◽

pp. 15892-15901

Author(s):

Christopher Eric Bragança ◽

Daniel Adam Kraut

Keyword(s):

Protein Degradation ◽

Proteasomal Degradation ◽

Canonical Pathway ◽

Eukaryotic Cells ◽

Reporter Protein ◽

Ubiquitin Proteasome ◽

Targeting Signal ◽

Multiple Variants ◽

Domain Stability

The ubiquitin–proteasome system is the canonical pathway for protein degradation in eukaryotic cells. GFP is frequently used as a reporter in proteasomal degradation assays. However, there are multiple variants of GFP in use, and these variants have different intrinsic stabilities. Further, there are multiple means by which substrates are targeted to the proteasome, and these differences could also affect the proteasome's ability to unfold and degrade substrates. Herein we investigate how the fate of GFP variants of differing intrinsic stabilities is determined by the mode of targeting to the proteasome. We compared two targeting systems: linear Ub4 degrons and the UBL domain from yeast Rad23, both of which are commonly used in degradation experiments. Surprisingly, the UBL degron allows for degradation of the most stable sGFP-containing substrates, whereas the Ub4 degron does not. Destabilizing the GFP by circular permutation allows degradation with either targeting signal, indicating that domain stability and mode of targeting combine to determine substrate fate. Difficult-to-unfold substrates are released and re-engaged multiple times, with removal of the degradation initiation region providing an alternative clipping pathway that precludes unfolding and degradation; the UBL degron favors degradation of even difficult-to-unfold substrates, whereas the Ub4 degron favors clipping. Finally, we show that the ubiquitin receptor Rpn13 is primarily responsible for the enhanced ability of the proteasome to degrade stable UBL-tagged substrates. Our results indicate that the choice of targeting method and reporter protein are critical to the design of protein degradation experiments.

Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

10.1101/592378 ◽

2019 ◽

Author(s):

Mary D. Cundiff ◽

Christina M. Hurley ◽

Jeremy D. Wong ◽

Aarti Bashyal ◽

Jake Rosenberg ◽

...

Keyword(s):

Protein Degradation ◽

Motor Proteins ◽

Physical Models ◽

Protein Domain ◽

Eukaryotic Cells ◽

Multiple Receptors ◽

Ubiquitin Proteasome ◽

Ubiquitin Receptors ◽

Partial Redundancy

ABSTRACTThe ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

Fluorescent reporters for the ubiquitin–proteasome system

Essays in Biochemistry ◽

10.1042/bse0410113 ◽

2005 ◽

Vol 41 ◽

pp. 113-128 ◽

Cited By ~ 6

Author(s):

Florian A. Salomons ◽

Lisette G.G.C. Verhoef ◽

Nico P. Dantuma

Keyword(s):

High Efficiency ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Fluorescent Reporters ◽

Cellular Processes ◽

The Status ◽

Ubiquitin Proteasome ◽

Green Fluorescent ◽

Specific Degradation

Regulated turnover of proteins in the cytosol and nucleus of eukaryotic cells is primarily performed by the ubiquitin–proteasome system (UPS). The UPS is involved in many essential cellular processes. Alterations in this proteolytic system are associated with a variety of human pathologies, such as neurodegenerative diseases, cancer, immunological disorders and inflammation. The precise role of the UPS in the pathophysiology of these diseases, however, remains poorly understood. Detection of UPS aberrations has been a major challenge because of the complexity of the system. Most studies focus on various aspects of the UPS, such as substrate recognition, ubiquitination, deubiquitination or proteasome activity, and do not provide a complete picture of the UPS as an integral system. To monitor the efficacy of the UPS, a number of reporter substrates have been developed based on fluorescent proteins, such as the green fluorescent protein and its spectral variants. These fluorescent UPS reporters contain specific degradation signals that target them with high efficiency and accuracy for proteasomal degradation. Several studies have shown that these reporters can probe the functionality of the UPS in cellular and animal models and provide us with important information on the status of the UPS under various conditions. Moreover, these reporters can aid the identification and development of novel anti-cancer and anti-inflammatory drugs based on UPS inhibition.

Ubiquitin receptors are required for substrate-mediated activation of the proteasome’s unfolding ability

Scientific Reports ◽

10.1038/s41598-019-50857-y ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 5

Author(s):

Mary D. Cundiff ◽

Christina M. Hurley ◽

Jeremy D. Wong ◽

Joseph A. Boscia ◽

Aarti Bashyal ◽

...

Keyword(s):

Protein Degradation ◽

Motor Proteins ◽

Physical Models ◽

Protein Domain ◽

Eukaryotic Cells ◽

Multiple Receptors ◽

Ubiquitin Proteasome ◽

Ubiquitin Receptors ◽

Partial Redundancy

Abstract The ubiquitin-proteasome system (UPS) is responsible for the bulk of protein degradation in eukaryotic cells, but the factors that cause different substrates to be unfolded and degraded to different extents are still poorly understood. We previously showed that polyubiquitinated substrates were degraded with greater processivity (with a higher tendency to be unfolded and degraded than released) than ubiquitin-independent substrates. Thus, even though ubiquitin chains are removed before unfolding and degradation occur, they affect the unfolding of a protein domain. How do ubiquitin chains activate the proteasome’s unfolding ability? We investigated the roles of the three intrinsic proteasomal ubiquitin receptors - Rpn1, Rpn10 and Rpn13 - in this activation. We find that these receptors are required for substrate-mediated activation of the proteasome’s unfolding ability. Rpn13 plays the largest role, but there is also partial redundancy between receptors. The architecture of substrate ubiquitination determines which receptors are needed for maximal unfolding ability, and, in some cases, simultaneous engagement of ubiquitin by multiple receptors may be required. Our results suggest physical models for how ubiquitin receptors communicate with the proteasomal motor proteins.

In situ dynamically monitoring the proteolytic function of the ubiquitin-proteasome system in cultured cardiac myocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.01233.2003 ◽

2004 ◽

Vol 287 (3) ◽

pp. H1417-H1425 ◽

Cited By ~ 46

Author(s):

Xin Dong ◽

Jinbao Liu ◽

Hanqiao Zheng ◽

Joseph W. Glasford ◽

Wei Huang ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cardiac Myocytes ◽

Fluorescent Protein ◽

Proteasome Inhibition ◽

Great Promise ◽

Dynamic Changes ◽

Ubiquitin Proteasome ◽

The ubiquitin-proteasome system (UPS) is responsible for turnover of most cellular proteins in eukaryotes. Protein degradation by the UPS serves quality control and regulatory functions. Proteasome inhibition showed great promise in effectively treating cancer and restenosis. UPS dysfunction in cardiac hypertrophy and failure has recently been suspected but remains to be investigated. A system capable of monitoring dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ would no doubt benefit significantly efforts to decipher the pathogenic significance of UPS dysfunction in the heart and to evaluate the effect of proteasome inhibition on cardiac myocytes. We successfully established such a system in cultured cardiac myocytes by delivering and expressing a modified green fluorescence protein (GFPu) gene using recombinant adenoviruses. GFPu contains a ubiquitination signal sequence fused to the COOH terminus. Fluorescence microscopy and Western blots revealed that protein abundance of modified green fluorescent protein (GFPu), but not wild-type green fluorescent protein, in cultured cardiac myocytes was incrementally increased when function of the proteasomes was inhibited in various degrees by specific inhibitors. The increase in GFPu protein levels and fluorescence intensity is paralleled by a decrease in the in vitro peptidase activity of the proteasomes. Our results demonstrate that GFPu can be used as a surrogate marker to monitor dynamic changes in proteolytic function of the UPS in cardiac myocytes in situ. Application of this novel system reveals that moderate levels of H2O2, a reactive oxygen species generator, impair proteolytic function of the UPS in cultured cardiac myocytes.

Dysregulation of the Ubiquitin Proteasome System in Human Malignancies: A Window for Therapeutic Intervention

Cancers ◽

10.3390/cancers13071513 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1513

Author(s):

Chee Wai Fhu ◽

Azhar Ali

Keyword(s):

Protein Quality ◽

26S Proteasome ◽

Eukaryotic Cells ◽

Lysosomal Degradation ◽

Polyubiquitin Chains ◽

Protein Levels ◽

Cellular Processes ◽

Ubiquitin Proteasome

The ubiquitin proteasome system (UPS) governs the non-lysosomal degradation of oxidized, damaged, or misfolded proteins in eukaryotic cells. This process is tightly regulated through the activation and transfer of polyubiquitin chains to target proteins which are then recognized and degraded by the 26S proteasome complex. The role of UPS is crucial in regulating protein levels through degradation to maintain fundamental cellular processes such as growth, division, signal transduction, and stress response. Dysregulation of the UPS, resulting in loss of ability to maintain protein quality through proteolysis, is closely related to the development of various malignancies and tumorigenesis. Here, we provide a comprehensive general overview on the regulation and roles of UPS and discuss functional links of dysregulated UPS in human malignancies. Inhibitors developed against components of the UPS, which include U.S. Food and Drug Administration FDA-approved and those currently undergoing clinical trials, are also presented in this review.

Motif trap: a rapid method to clone motifs that can target proteins to defined subcellular localisations

Journal of Cell Science ◽

10.1242/jcs.112.23.4207 ◽

1999 ◽

Vol 112 (23) ◽

pp. 4207-4211

Author(s):

L.A. Bejarano ◽

C. Gonzalez

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Eukaryotic Cells ◽

Subcellular Localisation ◽

Dna Fragments ◽

Rt Pcr ◽

Rapid Procedure ◽

Target Proteins ◽

Protein Motifs ◽

We have developed a rapid procedure termed Motif Trap (MT) to identify protein motifs that are able to target proteins to a distinct subcellular localisation in eukaryotic cells. By expressing random DNA fragments fused to green fluorescent protein (GFP), individual cells with the GFP localisation of interest are readily isolated allowing for the expressed DNA fragments to be cloned by RT-PCR. These can then be used to identify the corresponding full-length cDNAs. Using MT, we have identified patterns of GFP localisation which correspond to every major organelle and compartment. We have shown that MT is useful to identify new sequences that determine subcellular localisation as well as known targeting motifs.

Accurate and live peroxisome biogenesis evaluation achieved by lentiviral expression of a green fluorescent protein fused to a peroxisome targeting signal 1

Histochemistry and Cell Biology ◽

10.1007/s00418-020-01855-z ◽

2020 ◽

Vol 153 (5) ◽

pp. 295-306 ◽

Cited By ~ 2

Author(s):

Tanguy Demaret ◽

Guillaume E. Courtoy ◽

Joachim Ravau ◽

Patrick Van Der Smissen ◽

Mustapha Najimi ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Peroxisome Biogenesis ◽

Targeting Signal ◽

Efficient APC/C substrate degradation in cells undergoing mitotic exit depends on K11 ubiquitin linkages

Molecular Biology of the Cell ◽

10.1091/mbc.e15-02-0102 ◽

2015 ◽

Vol 26 (24) ◽

pp. 4325-4332 ◽

Cited By ~ 33

Author(s):

Mingwei Min ◽

Tycho E. T. Mevissen ◽

Maria De Luca ◽

David Komander ◽

Catherine Lindon

Keyword(s):

Cell Cycle Progression ◽

Degradation Kinetics ◽

Mitotic Exit ◽

Anaphase Promoting Complex ◽

Polyubiquitin Chains ◽

Substrate Degradation ◽

Ubiquitin Proteasome ◽

In Cells

The ubiquitin proteasome system (UPS) directs programmed destruction of key cellular regulators via posttranslational modification of its targets with polyubiquitin chains. These commonly contain Lys-48 (K48)–directed ubiquitin linkages, but chains containing atypical Lys-11 (K11) linkages also target substrates to the proteasome—for example, to regulate cell cycle progression. The ubiquitin ligase called the anaphase-promoting complex/cyclosome (APC/C) controls mitotic exit. In higher eukaryotes, the APC/C works with the E2 enzyme UBE2S to assemble K11 linkages in cells released from mitotic arrest, and these are proposed to constitute an improved proteolytic signal during exit from mitosis. We tested this idea by correlating quantitative measures of in vivo K11-specific ubiquitination of individual substrates, including Aurora kinases, with their degradation kinetics tracked at the single-cell level. All anaphase substrates tested by this methodology are stabilized by depletion of K11 linkages via UBE2S knockdown, even if the same substrates are significantly modified with K48-linked polyubiquitin. Specific examination of substrates depending on the APC/C coactivator Cdh1 for their degradation revealed Cdh1-dependent enrichment of K11 chains on these substrates, whereas other ubiquitin linkages on the same substrates added during mitotic exit were Cdh1-independent. Therefore we show that K11 linkages provide the APC/C with a means to regulate the rate of substrate degradation in a coactivator-specified manner.

An Unusual ERAD-Like Complex Is Targeted to the Apicoplast of Plasmodium falciparum

Eukaryotic Cell ◽

10.1128/ec.00083-09 ◽

2009 ◽

Vol 8 (8) ◽

pp. 1134-1145 ◽

Cited By ~ 112

Author(s):

Simone Spork ◽

Jan A. Hiss ◽

Katharina Mandel ◽

Maik Sommer ◽

Taco W. A. Kooij ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Molecular Mechanisms ◽

Protein Transport ◽

Fluorescent Protein ◽

Imaging Techniques ◽

Encoded Proteins ◽

Red Algal ◽

Endosymbiotic Event ◽

Targeting Signal ◽

ABSTRACT Many apicomplexan parasites, including Plasmodium falciparum, harbor a so-called apicoplast, a complex plastid of red algal origin which was gained by a secondary endosymbiotic event. The exact molecular mechanisms directing the transport of nuclear-encoded proteins to the apicoplast of P. falciparum are not well understood. Recently, in silico analyses revealed a second copy of proteins homologous to components of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) system in organisms with secondary plastids, including the malaria parasite P. falciparum. These proteins are predicted to be endowed with an apicoplast targeting signal and are suggested to play a role in the transport of nuclear-encoded proteins to the apicoplast. Here, we have studied components of this ERAD-derived putative preprotein translocon complex in malaria parasites. Using transfection technology coupled with fluorescence imaging techniques we can demonstrate that the N terminus of several ERAD-derived components targets green fluorescent protein to the apicoplast. Furthermore, we confirm that full-length PfsDer1-1 and PfsUba1 (homologues of yeast ERAD components) localize to the apicoplast, where PfsDer1-1 tightly associates with membranes. Conversely, PfhDer1-1 (a host-specific copy of the Der1-1 protein) localizes to the ER. Our data suggest that ERAD components have been “rewired” to provide a conduit for protein transport to the apicoplast. Our results are discussed in relation to the nature of the apicoplast protein transport machinery.