Harnessing the Ubiquitination Machinery to Target the Degradation of Specific Cellular Proteins

Zhou, Pengbo; Bogacki, Robert; McReynolds, Lisa J.; Howley, Peter M.

doi:10.1016/s1097-2765(00)00074-5

Cited by 151 publications

(115 citation statements)

References 20 publications

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“…As ␤-catenin is also required for the normal growth of the cell, targeted cells can not survive when the ␤-catenin gene is completely silenced with current knockout technologies (18). Targeting proteins to SCF ubiquitination machinery promises an alternative gene-silencing tool (19)(20)(21). In this study, we demonstrate that SCF provides a powerful means of eradicating pathogenic ␤-catenin in CRC.…”

mentioning

confidence: 74%

“…These features have prompted several earlier studies to explore SCF ubiquitin-conjugation machinery as a possible protein knockout tool (19)(20)(21). One study demonstrated SCF-mediated degradation of retinoblastoma protein (20). A very recent study showed that an artificially designed ''Protacs'' effectively eradicated a targeted methionine aminopeptidase 2 protein (21).…”

Section: Discussionmentioning

confidence: 99%

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Eradication of pathogenic β-catenin by Skp1/Cullin/F box ubiquitination machinery

Ishikawa

Kojima

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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The use of Skp1͞Cull 1͞F box (SCF) ubiquitin-conjugation machinery as a potential knockout tool offers a means of eradicating diseasecausing proteins. Here a chimeric F box protein (CFP) was engineered to achieve selective eradication of pathogenic ␤-catenin in colorectal cancer. We show that CFP specifically searches for and subsequently links the abnormal ␤-catenin to the cellular SCF ubiquitination complex. Introduction of the CFP to colorectal cancer cells induced targeted ubiquitination and proteolytic degradation of nuclear and cytoplasmic free ␤-catenin while preserving its normal cellular adhesion counterpart. Elimination of pathogenic ␤-catenin suppressed constitutive Wingless͞Wnt signaling and inhibited in vitro and in vivo tumor cell growth. This study demonstrates a practical utility of a SCF-based knockout system as a tool in targeting an abnormal protein that affects growth and transformation.

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mentioning

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Eradication of pathogenic β-catenin by Skp1/Cullin/F box ubiquitination machinery

Ishikawa

Kojima

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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“…Results obtained with the ODC͞AZ system and other previously reported approaches for inducing proteasome-dependent degradation of specific proteins (2,3,(5)(6)(7)(8) should be interpreted with understanding that the particular protein ligand chosen may have multiple cellular targets, including unknown or unanticipated protein targets in addition to known interacting proteins intended for targeted degradation. Thus, phenotypes created by these targeted protein-degradation methods could potentially reflect the loss of expression of several interacting proteins.…”

Section: Discussionmentioning

confidence: 97%

“…1). Accordingly, expression systems have been described in which whole proteins or fragments constituting functional proteininteraction domains or peptide ligands are expressed as chimeric fusion proteins together with modified versions of ubiquitin (2,3), ubiquitin ligases (e.g., HECT-domain-containing proteins) (4), or adapter proteins that associate with ubiquitin ligase complexes (e.g., F-box proteins of Skp1͞cullin͞F-box protein complexes) (5)(6)(7)(8), with the intention of inducing polyubiquitination of cellular proteins that bind these chimeric proteins, thus creating holes in pathways that reveal cellular phenotypes.…”

mentioning

confidence: 99%

Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway

Matsuzawa

Cuddy

Fukushima

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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With the euchromatic portion of several mammalian genomes now sequenced, emphasis has turned to ascertaining the functions of gene products. A method for targeting destruction of selected proteins in mammalian cells is described, based on the ubiquitinindependent mechanism by which ornithine decarboxylase (ODC) is degraded by the 26S proteasome in collaboration with antizyme (AZ). We show that expressing whole proteins, protein domains, or peptide ligands fused to the N terminus of ODC promotes proteasome-dependent degradation of these chimeric fusion proteins and their interacting cellular target proteins. Moreover, the degradation of the interacting (targeted) protein depends on coexpression of AZ in about half of cases, providing an inducible switch for triggering the degradation process. By using 12 pairs of interacting proteins for testing, direct comparisons with several alternative strategies for achieving targeted protein destruction based on the concept of induced ubiquitination revealed advantages of the ODC͞AZ system, which does not require posttranslational attachment of ubiquitin to target proteins. As proof of concept, the ODC͞AZ system was used to ablate expression of specific endogenous proteins (e.g., TRAF6; Rb), and was shown to create the expected lesions in cellular pathways that require these proteins. Altogether, these findings reveal a strategy for achieving targeted destruction of cellular proteins, thus providing an additional tool for revealing the cellular phenotypes of gene products.antizyme ͉ ornithine decarboxylase ͉ proteasome ͉ protein degradation F unctions are known for fewer than half the genes identified in mammalian genomes to date, indicating a need for gene functionalization technologies that can reveal the phenotypes of genes and their encoded proteins in various cellular contexts. Methods targeting gene-expression pathways at the level of mRNA, such as antisense oligonucleotides and small interfering RNA, offer exquisite specificity for gene silencing through Watson-Crick-type hybridization of synthetic or vector-derived nucleotide sequences to target mRNAs. These gene-silencing methods, however, sometimes prove ineffective, either because the targeted mRNAs encode long-lived stable proteins or because of functional redundancy among members of multigene families. These deficiencies have prompted attempts to inducibly target degradation of the protein products of genes by tapping into components of the eukaryotic ubiquitination machinery, which induces proteasome-dependent degradation of ubiquitin-modified proteins (reviewed in ref. 1). Accordingly, expression systems have been described in which whole proteins or fragments constituting functional proteininteraction domains or peptide ligands are expressed as chimeric fusion proteins together with modified versions of ubiquitin (2, 3), ubiquitin ligases (e.g., HECT-domain-containing proteins) (4), or adapter proteins that associate with ubiquitin ligase complexes (e.g., F-box proteins of Skp1͞cullin͞F-box protein complexes...

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“…In most of these cases, intrabodies were used as means to alter the intracellular trafficking and localization of their targets, for example, by trapping oncogene products (e.g., epidermal growth factor receptor and ErbB2) that were destined for cell surface expression in the endoplasmic reticulum or isolating transcription factors (e.g., Cyclin E) in the cytoplasm. Alternatively, intrabodies have been used to block protein-protein or protein-nucleic acid interactions (51), to directly promote cell death by inducing apoptosis-related proteolysis cascade (52 -54), or, albeit indirectly, to facilitate selective degradation of target proteins by recruitment to the ubiquitin-proteasome pathway (55). In this study, by targeting the enzymatic activity of an intracellular kinase (i.e., Etk) with an intrabody, we showed that we could modulate the role of a kinase plays in cell transformation in an extremely specific manner.…”

Section: Discussionmentioning

confidence: 99%

Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation

Paz¹,

Brennan²,

Iacolina³

et al. 2005

Molecular Cancer Therapeutics

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Etk, the 70-kDa member of the Tec family of nonreceptor protein tyrosine kinases, is expressed in a variety of hematopoietic, epithelial, and endothelial cells and was shown to be involved in several cellular processes, including proliferation, differentiation, and motility. In this study, we describe a novel approach using a human singledomain antibody phage display library for the generation of intrabodies directed against Etk. These single-domain antibodies bind specifically to recombinant Etk and efficiently block its kinase activity. When expressed in transformed cells, these antibodies associated tightly with Etk, leading to significant blockade of Etk enzymatic activity and inhibition of clonogenic cell growth in soft agar. Our results indicate that Etk may play a role in Src-induced cellular transformation and thus may represent a good target for cancer intervention. Furthermore, our single-domain antibody-based intrabody system proves to be an excellent tool for future intracellular targeting of other signaling molecules. [Mol Cancer Ther 2005;4(11):1801 -9]

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Harnessing the Ubiquitination Machinery to Target the Degradation of Specific Cellular Proteins

Cited by 151 publications

References 20 publications

Eradication of pathogenic β-catenin by Skp1/Cullin/F box ubiquitination machinery

Eradication of pathogenic β-catenin by Skp1/Cullin/F box ubiquitination machinery

Method for targeting protein destruction by using a ubiquitin-independent, proteasome-mediated degradation pathway

Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation

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