9 Ubiquitin-Proteasome Pathway of Intracellular Protein Degradation

Background: Proteasome inhibitors such as bortezomib, a chemotherapeutic used to treat multiple myeloma, induce thrombocytopenia within days of initiation. The mechanism for this thrombocytopenia has been tied to data revealing that proteasome activity is essential for platelet formation. The major pathway of selective protein degradation uses ubiquitin as a marker that targets proteins for proteolysis by the proteasome. This pathway is previously unexplored in megakaryocytes (MKs). Objectives: We aim to define the mechanism by which the ubiquitin-proteasome pathway affects MK maturation and platelet production. Results: Pharmacologic inhibition of proteasome activity blocks proplatelet formation in megakaryocytes. To further characterize how this degradation was occurring, we probed distinct ubiquitin pathways. Inhibition of the ubiquitin-activating enzyme E1 significantly inhibited proplatelet formation up to 73%. In addition, inhibition of the deubiquitinase proteins UCHL5 and USP14 significantly inhibited proplatelet formation up to 83%. These data suggest that an intact ubiquitin pathway is necessary for proplatelet formation. Proteomic and polysome analyses of MKs undergoing proplatelet formation revealed a subset of proteins decreased in proplatelet-producing megakaryocytes, consistent with data showing that protein degradation is necessary for proplatelet formation. Specifically, the centrosome stabilizing proteins Aurora kinase (Aurk) A/B, Tpx2, Cdk1, and Plk1 were decreased in proplatelet-producing MKs. Furthermore, inhibition of AurkA and Plk1, but not Cdk1, significantly inhibited proplatelet formation in vitro over 83%. Conclusions: We hypothesize that proplatelet formation is triggered by centrosome destabilization and disassembly, and that the ubiquitin-proteasome pathway plays a crucial role in this transformation. Specifically, regulation of the AurkA/Plk1/Tpx2 pathway may be key in centrosome integrity and initiation of proplatelet formation. Determination of the mechanism by which the ubiquitin-proteasome pathway regulates the centrosome and facilitates proplatelet formation will allow us to design better strategies to target and reverse thrombocytopenia.

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Intracellular Protein Degradation: From a Vague Idea, Through the Lysosome and the Ubiquitin—Proteasome System, and onto Human Diseases and Drug Targeting (Nobel Lecture)

ChemInform ◽

10.1002/chin.200549272 ◽

2005 ◽

Vol 36 (49) ◽

Author(s):

Aaron Ciechanover

Keyword(s):

Protein Degradation ◽

Drug Targeting ◽

Ubiquitin Proteasome System ◽

Human Diseases ◽

Intracellular Protein ◽

Nobel Lecture ◽

Intracellular Protein Degradation ◽

Ubiquitin Proteasome

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Intracellular Protein Degradation: From a Vague Idea, through the Lysosome and the Ubiquitin-Proteasome System, and onto Human Diseases and Drug Targeting (Nobel Lecture)

Angewandte Chemie International Edition ◽

10.1002/anie.200501428 ◽

2005 ◽

Vol 44 (37) ◽

pp. 5944-5967 ◽

Cited By ~ 113

Author(s):

Aaron Ciechanover

Keyword(s):

Protein Degradation ◽

Drug Targeting ◽

Ubiquitin Proteasome System ◽

Human Diseases ◽

Intracellular Protein ◽

Nobel Lecture ◽

Intracellular Protein Degradation ◽

Ubiquitin Proteasome

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Mechanisms of Cancer Cachexia

Physiological Reviews ◽

10.1152/physrev.00016.2008 ◽

2009 ◽

Vol 89 (2) ◽

pp. 381-410 ◽

Cited By ~ 672

Author(s):

Michael J. Tisdale

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Adipose Tissue ◽

Protein Degradation ◽

Initiation Factor ◽

Tissue Loss ◽

Α Subunit ◽

Ubiquitin Proteasome Pathway ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the α-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NFκB. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-α, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment.

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The effect of denervation on protein synthesis and degradation in adult rat diaphragm muscle

Journal of Applied Physiology ◽

10.1152/japplphysiol.91247.2008 ◽

2009 ◽

Vol 107 (2) ◽

pp. 438-444 ◽

Cited By ~ 41

Author(s):

Heather M. Argadine ◽

Nathan J. Hellyer ◽

Carlos B. Mantilla ◽

Wen-Zhi Zhan ◽

Gary C. Sieck

Keyword(s):

Protein Synthesis ◽

Protein Degradation ◽

Caspase 3 ◽

Diaphragm Muscle ◽

Rat Diaphragm ◽

Ubiquitin Proteasome Pathway ◽

Protein Ubiquitination ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Net Protein

Previous studies showed that unilateral denervation (DNV) of the rat diaphragm muscle (DIAm) results in loss of myosin heavy chain protein by 1 day after DNV. We hypothesize that DNV decreases net protein balance as a result of activation of the ubiquitin-proteasome pathway. In DIAm strips, protein synthesis was measured by incorporation of 3H-Tyr, and protein degradation was measured by Tyr release at 1, 3, 5, 7, and 14 days after DNV. Total protein ubiquitination, caspase-3 expression/activity, and actin fragmentation were analyzed by Western analysis. We found that, at 3 days after DNV, protein synthesis increased by 77% relative to sham controls. Protein synthesis remained elevated at 5 (85%), 7 (53%), and 14 days (123%) after DNV. At 5 days after DNV, protein degradation increased by 43% relative to sham controls and remained elevated at 7 (49%) and 14 days (74%) after DNV. Thus, by 5 days after DNV, net protein balance decreased by 43% compared with sham controls and was decreased compared with sham at 7 (49%) and 14 days (72%) after DNV. Protein ubiquitination increased at 5 days after DNV and remained elevated. DNV had no effect on caspase-3 activity or actin fragmentation, suggesting that the ubiquitin-proteasome pathway rather than caspase-3 activation is important in the DIAm response to DNV. Early loss of contractile proteins, such as myosin heavy chain, is likely the result of selective protein degradation rather than generalized protein breakdown. Future studies should evaluate this selective effect of DNV.

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Involvement of the Ubiquitin-Proteasome System in the Formation of Experimental Postsurgical Peritoneal Adhesions

Mediators of Inflammation ◽

10.1155/2012/194723 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 5

Author(s):

Clara Di Filippo ◽

Pasquale Petronella ◽

Fulvio Freda ◽

Marco Scorzelli ◽

Marco Ferretti ◽

...

Keyword(s):

Protein Degradation ◽

Ubiquitin Proteasome System ◽

20S Proteasome ◽

Intracellular Protein ◽

Peritoneal Adhesions ◽

Proteasome Subunit ◽

Peritoneal Tissue ◽

Intracellular Protein Degradation ◽

Ubiquitin Proteasome

We investigated the Ubiquitin-Proteasome System (UPS), major nonlysosomal intracellular protein degradation system, in the genesis of experimental postsurgical peritoneal adhesions. We assayed the levels of UPS within the adhered tissue along with the development of peritoneal adhesions and used the specific UPS inhibitor bortezomib in order to assess the effect of the UPS blockade on the peritoneal adhesions. We found a number of severe postsurgical peritoneal adhesions at day 5 after surgery increasing until day 10. In the adhered tissue an increased values of ubiquitin and the 20S proteasome subunit, NFkB, IL-6, TNF-αand decreased values of IkB-beta were found. In contrast, bortezomib-treated rats showed a decreased number of peritoneal adhesions, decreased values of ubiquitin and the 20S proteasome, NFkB, IL-6, TNF-α, and increased levels of IkB-beta in the adhered peritoneal tissue. The UPS system, therefore, is primarily involved in the formation of post-surgical peritoneal adhesions in rats.

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A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment

Biomolecules ◽

10.3390/biom11101512 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1512

Author(s):

Gang Chen ◽

Yu Kong ◽

You Li ◽

Ailing Huang ◽

Chunyu Wang ◽

...

Keyword(s):

Protein Degradation ◽

Fluorescent Protein ◽

Cultured Cells ◽

Dependent Manner ◽

Intracellular Protein ◽

Full Size ◽

Intracellular Protein Degradation ◽

Potential Applications ◽

Ubiquitin Proteasome ◽

Green Fluorescent

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin–proteasome and autophagy–lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

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Combination of Artesunate and WNT974 Induces KRAS Protein Degradation by Upregulating E3 Ligase ANACP2 and β-TrCP in the Ubiquitin–Proteasome Pathway

10.21203/rs.3.rs-744967/v2 ◽

2021 ◽

Author(s):

RUIHONG GONG ◽

Minting Chen ◽

Chunhua Huang Huang ◽

Hoi Leong Xavier Wong ◽

Hiu Yee Kwan ◽

...

Keyword(s):

Mouse Model ◽

Synergistic Effect ◽

Protein Degradation ◽

Combination Treatment ◽

Ubiquitin Proteasome Pathway ◽

Protein Levels ◽

Ubiquitin Proteasome ◽

Proteasome Pathway ◽

Kras Protein ◽

Gsk 3Β

Abstract BackgroundKRAS mutation is one of the dominant gene mutations in colorectal cancer (CRC). Up to present, targeting KRAS for CRC treatment remains a clinical challenge. WNT974 (LGK974) is a porcupine inhibitor that interferes Wnt signaling pathway. Artesunate (ART) is a water-soluble semi-synthetic derivative of artemisinin.MethodsThe synergistic effect of ART and WNT974 combination in reducing CRC cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RT-PCR was utilized for the mRNA levels of KRAS, CUL7, ANAPC2, UBE2M, RNF123, SYVN1, or β-TrCP. Western blot assay was utilized for the protein levels of KRAS, ANAPC2, β-TrCP, or GSK-3β. Xenograft mouse model assay was performed for the anti-CRC effect of combination of ART and WNT974 in vivo. IHC assay was utilized for the levels of KRAS, β-TrCP, or GSK-3β in tumor tissues. Results Our study shows that the combination of WNT974 and ART exhibits synergistic effect in reducing CRC growth. The combination treatment significantly reduces KRAS protein level and activity in CRC cells. Interestingly, the combination treatment increases E3 ligases ANAPC2 expression. Our data show that overexpression of ANAPC2 significantly reduces KRAS protein levels, which is reversed by MG132. Knockdown of ANAPC2 in CRC abolishes the combination treatment-reduce KRAS expression. Besides, the treatment also increases the expressions of GSK-3β and E3 ligase β-TrCP that is known to degrade GSK-3β-phosphorylated KRAS protein. Knockdown of β-TrCP- and inhibition of GSK-3β abolish the combination treatment-induce KRAS ubiquitination and reduction in expression.ConclusionsOur data clearly show that the combination treatment significantly enhances KRAS protein degradation via the ubiquitination ubiquitin–proteasome pathway, which is also demonstrated in xenograft mouse model. The study provides strong scientific evidence for the development of the combination of WNT974 and ART as KRAS-targeting therapeutics for CRC treatment.

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PROTEASOME INHIBITION IN MULTIPLE MYELOMA: Therapeutic Implication

The Annual Review of Pharmacology and Toxicology ◽

10.1146/annurev.pharmtox.45.120403.100037 ◽

2005 ◽

Vol 45 (1) ◽

pp. 465-476 ◽

Cited By ~ 98

Author(s):

Dharminder Chauhan ◽

Teru Hideshima ◽

Kenneth C. Anderson

Keyword(s):

Multiple Myeloma ◽

Proteasome Inhibitors ◽

Proteasome Inhibition ◽

Protein Translation ◽

26S Proteasome ◽

Intracellular Protein ◽

Cycle Growth ◽

Intracellular Protein Degradation ◽

Ubiquitin Proteasome ◽

Proteasome Pathway

Normal cellular functioning requires processing of proteins regulating cell cycle, growth, and apoptosis. The ubiquitin-proteasome pathway (UBP) modulates intracellular protein degradation. Specifically, the 26S proteasome is a multienzyme protease that degrades misfolded or redundant proteins; conversely, blockade of the proteasomal degradation pathways results in accumulation of unwanted proteins and cell death. Because cancer cells are more highly proliferative than normal cells, their rate of protein translation and degradation is also higher. This notion led to the development of proteasome inhibitors as therapeutics in cancer. The FDA recently approved the first proteasome inhibitor bortezomib (Velcade™), formerly known as PS-341, for the treatment of newly diagnosed and relapsed/refractory multiple myeloma (MM). Ongoing studies are examining other novel proteasome inhibitors, in addition to bortezomib, for the treatment of MM and other cancers.

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