Journal articles: 'SCF E3 ubiquitin ligase'

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Horn-Ghetko, Daniel, David T. Krist, J. Rajan Prabu, Kheewoong Baek, Monique P. C. Mulder, Maren Klügel, Daniel C. Scott, Huib Ovaa, Gary Kleiger, and Brenda A. Schulman. "Ubiquitin ligation to F-box protein targets by SCF–RBR E3–E3 super-assembly." Nature 590, no. 7847 (February 3, 2021): 671–76. http://dx.doi.org/10.1038/s41586-021-03197-9.

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AbstractE3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates1,2. However, rather than functioning individually, many neddylated cullin–RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family—which together account for nearly half of all ubiquitin ligases in humans—form E3–E3 super-assemblies3–7. Here, by studying CRLs in the SKP1–CUL1–F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3–E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3–E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3–E3 super-assembly may therefore underlie widespread ubiquitylation.

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Kawakami, T. "NEDD8 recruits E2-ubiquitin to SCF E3 ligase." EMBO Journal 20, no. 15 (August 1, 2001): 4003–12. http://dx.doi.org/10.1093/emboj/20.15.4003.

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Gorelik, Maryna, Stephen Orlicky, Maria A. Sartori, Xiaojing Tang, Edyta Marcon, Igor Kurinov, Jack F. Greenblatt, et al. "Inhibition of SCF ubiquitin ligases by engineered ubiquitin variants that target the Cul1 binding site on the Skp1–F-box interface." Proceedings of the National Academy of Sciences 113, no. 13 (March 14, 2016): 3527–32. http://dx.doi.org/10.1073/pnas.1519389113.

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Skp1–Cul1–F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.

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Sun, Yi. "Sag/Rbx2 E3 Ubiquitin Ligase: From Target Validation to Drug Discovery." Proceedings 22, no. 1 (November 14, 2019): 102. http://dx.doi.org/10.3390/proceedings2019022102.

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Risseeuw, Eddy P., Timothy E. Daskalchuk, Travis W. Banks, Enwu Liu, Julian Cotelesage, Hanjo Hellmann, Mark Estelle, David E. Somers, and William L. Crosby. "Protein interaction analysis of SCF ubiquitin E3 ligase subunits fromArabidopsis." Plant Journal 34, no. 6 (June 2003): 753–67. http://dx.doi.org/10.1046/j.1365-313x.2003.01768.x.

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Chang, Shu-Chun, Chin-Sheng Hung, Bo-Xiang Zhang, Tsung-Han Hsieh, Wayne Hsu, and Jeak Ling Ding. "A Novel Signature of CCNF-Associated E3 Ligases Collaborate and Counter Each Other in Breast Cancer." Cancers 13, no. 12 (June 8, 2021): 2873. http://dx.doi.org/10.3390/cancers13122873.

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Breast cancer (BRCA) malignancy causes major fatalities amongst women worldwide. SCF (Skp1-cullin-F-box proteins) E3 ubiquitin ligases are the most well-known members of the ubiquitination–proteasome system (UPS), which promotes cancer initiation and progression. Recently, we demonstrated that FBXL8, a novel F-box protein (SCFF-boxes) of SCF E3 ligase, accelerates BRCA advancement and metastasis. Since SCFF-boxes is a key component of E3 ligases, we hypothesized that other SCFF-boxes besides FBXL8 probably collaborate in regulating breast carcinogenesis. In this study, we retrospectively profiled the transcriptome of BRCA tissues and found a notable upregulation of four SCFF-box E3 ligases (FBXL8, FBXO43, FBXO15, and CCNF) in the carcinoma tissues. Similar to FBXL8, the knockdown of FBXO43 reduced cancer cell viability and proliferation, suggesting its pro-tumorigenic role. The overexpression of CCNF inhibited cancer cell progression, indicating its anti-tumorigenic role. Unexpectedly, CCNF protein was markedly downregulated in BRCA tissues, although its mRNA level was high. We showed that both E3 ligases, FBXL8 and FZR1, pulled down CCNF. Double knockdown of FBXL8 and FZR1 caused CCNF accumulation. On the other hand, CCNF itself pulled down a tumorigenic factor, RRM2, and CCNF overexpression reduced RRM2. Altogether, we propose a signature network of E3 ligases that collaboratively modulates CCNF anti-cancer activity. There is potential to target BRCA through modulation of the partnership axes of (i) CCNF-FBXL8, (ii) CCNF-FZR1, and (iii) CCNF-RRM2, particularly, via CCNF overexpression and activation and FBXL8/FZR1 suppression.

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Willems, A. R., T. Goh, L. Taylor, I. Chernushevich, A. Shevchenko, and M. Tyers. "SCF ubiquitin protein ligases and phosphorylation–dependent proteolysis." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 354, no. 1389 (September 29, 1999): 1533–50. http://dx.doi.org/10.1098/rstb.1999.0497.

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Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1–Cdc53–F–box protein (SCF) complexes. SCF complexes physically link substrate proteins to the E2 ubiquitin–conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome. SCF complexes contain a variable subunit called an F–box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53. Here, we review the substrates and pathways regulated by the yeast F–box proteins Cdc4, Grr1 and Met30. The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2. Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F–box proteins that appear to participate in SCF–like complexes. As many F–box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF–dependent proteolysis.

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Chen, Wei-Yi, Jui-Hsia Weng, Chen-Che Huang, and Bon-chu Chung. "Histone Deacetylase Inhibitors Reduce Steroidogenesis through SCF-Mediated Ubiquitination and Degradation of Steroidogenic Factor 1 (NR5A1)." Molecular and Cellular Biology 27, no. 20 (August 20, 2007): 7284–90. http://dx.doi.org/10.1128/mcb.00476-07.

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ABSTRACT Histone deacetylase (HDAC) inhibitors such as trichostatin A and valproic acid modulate transcription of many genes by inhibiting the activities of HDACs, resulting in the remodeling of chromatin. Yet this effect is not universal for all genes. Here we show that HDAC inhibitors suppressed the expression of steroidogenic gene CYP11A1 and decreased steroid secretion by increasing the ubiquitination and degradation of SF-1, a factor important for the transcription of all steroidogenic genes. This was accompanied by increased expression of Ube2D1 and SKP1A, an E2 ubiquitin conjugase and a subunit of the E3 ubiquitin ligase in the Skp1/Cul1/F-box protein (SCF) family, respectively. Reducing SKP1A expression with small interfering RNA resulted in recovery of SF-1 levels, demonstrating that the activity of SCF E3 ubiquitin ligase is required for the SF-1 degradation induced by HDAC inhibitors. Overexpression of exogenous SF-1 restored steroidogenic activities even in the presence of HDAC inhibitors. Thus, increased SF-1 degradation is the cause of the reduction in steroidogenesis caused by HDAC inhibitors. The increased SKP1A expression and SCF-mediated protein degradation could be the mechanism underlying the mode of action of HDAC inhibitors.

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Rogers, Gregory C., Nasser M. Rusan, David M. Roberts, Mark Peifer, and Stephen L. Rogers. "The SCFSlimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication." Journal of Cell Biology 184, no. 2 (January 26, 2009): 225–39. http://dx.doi.org/10.1083/jcb.200808049.

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Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCFSlimb as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCFSlimb target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

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Jia, L., and Y. Sun. "SCF E3 Ubiquitin Ligases as Anticancer Targets." Current Cancer Drug Targets 11, no. 3 (March 1, 2011): 347–56. http://dx.doi.org/10.2174/156800911794519734.

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Gearhart, Micah D., Connie M. Corcoran, Joseph A. Wamstad, and Vivian J. Bardwell. "Polycomb Group and SCF Ubiquitin Ligases Are Found in a Novel BCOR Complex That Is Recruited to BCL6 Targets." Molecular and Cellular Biology 26, no. 18 (September 15, 2006): 6880–89. http://dx.doi.org/10.1128/mcb.00630-06.

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ABSTRACT The corepressor BCOR potentiates transcriptional repression by the proto-oncoprotein BCL6 and suppresses the transcriptional activity of a common mixed-lineage leukemia fusion partner, AF9. Mutations in human BCOR cause male lethal, X-linked oculofaciocardiodental syndrome. We identified a BCOR complex containing Polycomb group (PcG) and Skp-Cullin-F-box subcomplexes. The PcG proteins include RING1, RYBP, NSPC1, a Posterior Sex Combs homolog, and RNF2, an E3 ligase for the mono-ubiquitylation of H2A. BCOR complex components and mono-ubiquitylated H2A localize to BCL6 targets, indicating that the BCOR complex employs PcG proteins to expand the repertoire of enzymatic activities that can be recruited by BCL6. This also suggests that BCL6 can target PcG proteins to DNA. In addition, the BCOR complex contains components of a second ubiquitin E3 ligase, namely, SKP1 and FBXL10 (JHDM1B). We show that BCOR coimmunoprecipitates isoforms of FBXL10 which contain a JmjC domain that recently has been determined to have histone H3K36 demethylase activity. The recruitment of two distinct classes of E3 ubiquitin ligases and a histone demethylase by BCOR suggests that BCOR uses a unique combination of epigenetic modifications to direct gene silencing.

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Arquint, Christian, Fabien Cubizolles, Agathe Morand, Alexander Schmidt, and Erich A. Nigg. "The SKP1-Cullin-F-box E3 ligase βTrCP and CDK2 cooperate to control STIL abundance and centriole number." Open Biology 8, no. 2 (February 2018): 170253. http://dx.doi.org/10.1098/rsob.170253.

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Deregulation of centriole duplication has been implicated in cancer and primary microcephaly. Accordingly, it is important to understand how key centriole duplication factors are regulated. E3 ubiquitin ligases have been implicated in controlling the levels of several duplication factors, including PLK4, STIL and SAS-6, but the precise mechanisms ensuring centriole homeostasis remain to be fully understood. Here, we have combined proteomics approaches with the use of MLN4924, a generic inhibitor of SCF E3 ubiquitin ligases, to monitor changes in the cellular abundance of centriole duplication factors. We identified human STIL as a novel substrate of SCF-βTrCP. The binding of βTrCP depends on a DSG motif within STIL, and serine 395 within this motif is phosphorylated in vivo . SCF-βTrCP-mediated degradation of STIL occurs throughout interphase and mutations in the DSG motif causes massive centrosome amplification, attesting to the physiological importance of the pathway. We also uncover a connection between this new pathway and CDK2, whose role in centriole biogenesis remains poorly understood. We show that CDK2 activity protects STIL against SCF-βTrCP-mediated degradation, indicating that CDK2 and SCF-βTrCP cooperate via STIL to control centriole biogenesis.

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Vadhvani, Mayur, Nicola Schwedhelm-Domeyer, Chaitali Mukherjee, and Judith Stegmüller. "The Centrosomal E3 Ubiquitin Ligase FBXO31-SCF Regulates Neuronal Morphogenesis and Migration." PLoS ONE 8, no. 2 (February 28, 2013): e57530. http://dx.doi.org/10.1371/journal.pone.0057530.

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Kwun, Hyun Jin, Yuan Chang, and Patrick S. Moore. "Protein-mediated viral latency is a novel mechanism for Merkel cell polyomavirus persistence." Proceedings of the National Academy of Sciences 114, no. 20 (May 1, 2017): E4040—E4047. http://dx.doi.org/10.1073/pnas.1703879114.

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Viral latency, in which a virus genome does not replicate independently of the host cell genome and produces no infectious particles, is required for long-term virus persistence. There is no known latency mechanism for chronic small DNA virus infections. Merkel cell polyomavirus (MCV) causes an aggressive skin cancer after prolonged infection and requires an active large T (LT) phosphoprotein helicase to replicate. We show that evolutionarily conserved MCV LT phosphorylation sites are constitutively recognized by cellular Fbw7, βTrCP, and Skp2 Skp-F-box-cullin (SCF) E3 ubiquitin ligases, which degrade and suppress steady-state LT protein levels. Knockdown of each of these E3 ligases enhances LT stability and promotes MCV genome replication. Mutations at two of these phosphoreceptor sites [serine (S)220 and S239] in the full viral genome increase LT levels and promote MCV virion production and transmission, which can be neutralized with anti-capsid antibody. Virus activation is not mediated by viral gene transactivation, given that these mutations do not increase late gene transcription in the absence of genome replication. Mechanistic target of rapamycin inhibition by either nutrient starvation or use of an active site inhibitor reduces Skp2 levels and stabilizes LT, leading to enhanced MCV replication and transmission. MCV can sense stresses in its intracellular environment, such as nutrient loss, through SCF E3 ligase activities, and responds by initiating active viral transmission. Protein-mediated viral latency through cellular SCF E3 ligase targeting of viral replication proteins is a unique form of latency that may promote chronic viral persistence for some small DNA and RNA viruses.

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Kim, Tai Young, Priscila F. Siesser, Kent L. Rossman, Dennis Goldfarb, Kathryn Mackinnon, Feng Yan, XianHua Yi, et al. "Substrate Trapping Proteomics Reveals Targets of the βTrCP2/FBXW11 Ubiquitin Ligase." Molecular and Cellular Biology 35, no. 1 (October 20, 2014): 167–81. http://dx.doi.org/10.1128/mcb.00857-14.

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Defining the full complement of substrates for each ubiquitin ligase remains an important challenge. Improvements in mass spectrometry instrumentation and computation and in protein biochemistry methods have resulted in several new methods for ubiquitin ligase substrate identification. Here we used the parallel adapter capture (PAC) proteomics approach to study βTrCP2/FBXW11, a substrate adaptor for the SKP1–CUL1–F-box (SCF) E3 ubiquitin ligase complex. The processivity of the ubiquitylation reaction necessitates transient physical interactions between FBXW11 and its substrates, thus making biochemical purification of FBXW11-bound substrates difficult. Using the PAC-based approach, we inhibited the proteasome to “trap” ubiquitylated substrates on the SCFFBXW11E3 complex. Comparative mass spectrometry analysis of immunopurified FBXW11 protein complexes before and after proteasome inhibition revealed 21 known and 23 putatively novel substrates. In focused studies, we found that SCFFBXW11bound, polyubiquitylated, and destabilized RAPGEF2, a guanine nucleotide exchange factor that activates the small GTPase RAP1. High RAPGEF2 protein levels promoted cell-cell fusion and, consequently, multinucleation. Surprisingly, this occurred independently of the guanine nucleotide exchange factor (GEF) catalytic activity and of the presence of RAP1. Our data establish new functions for RAPGEF2 that may contribute to aneuploidy in cancer. More broadly, this report supports the continued use of substrate trapping proteomics to comprehensively define targets for E3 ubiquitin ligases. All proteomic data are available via ProteomeXchange with identifier PXD001062.

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Lassot, Irina, Emmanuel Ségéral, Clarisse Berlioz-Torrent, Herve Durand, Lionel Groussin, Tsonwin Hai, Richard Benarous, and Florence Margottin-Goguet. "ATF4 Degradation Relies on a Phosphorylation-Dependent Interaction with the SCFβTrCPUbiquitin Ligase." Molecular and Cellular Biology 21, no. 6 (March 15, 2001): 2192–202. http://dx.doi.org/10.1128/mcb.21.6.2192-2202.2001.

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ABSTRACT The ubiquitin-proteasome pathway regulates gene expression through protein degradation. Here we show that the F-box protein βTrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for IκBα and β-catenin degradation, is colocalized in the nucleus with ATF4, a member of the ATF-CREB bZIP family of transcription factors, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation and on ATF4 serine residue 219 present in the context of DSGXXXS, which is similar but not identical to the motif found in other substrates of βTrCP. ATF4 ubiquitination in HeLa cells is enhanced in the presence of βTrCP. The F-box-deleted βTrCP protein behaves as a negative transdominant mutant that inhibits ATF4 ubiquitination and degradation and, subsequently, enhances its activity in cyclic AMP-mediated transcription. ATF4 represents a novel substrate for the SCFβTrCP complex, which is the first mammalian E3 ubiquitin ligase identified so far for the control of the degradation of a bZIP transcription factor.

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Suber, Tomeka, Jianxin Wei, Anastasia M. Jacko, Ina Nikolli, Yutong Zhao, Jing Zhao, and Rama K. Mallampalli. "SCFFBXO17 E3 ligase modulates inflammation by regulating proteasomal degradation of glycogen synthase kinase-3β in lung epithelia." Journal of Biological Chemistry 292, no. 18 (March 15, 2017): 7452–61. http://dx.doi.org/10.1074/jbc.m116.771667.

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Glycogen synthase kinase-3β (GSK3β) has diverse biological roles including effects on cellular differentiation, migration, and inflammation. GSK3β phosphorylates proteins to generate phosphodegrons necessary for recognition by Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligases leading to subsequent proteasomal degradation of these substrates. However, little is known regarding how GSK3β protein stability itself is regulated and how its stability may influence inflammation. Here we show that GSK3β is degraded by the ubiquitin-proteasome pathway in murine lung epithelial cells through lysine 183 as an acceptor site for K48 polyubiquitination. We have identified FBXO17 as an F-box protein subunit that recognizes and mediates GSK3β polyubiquitination. Both endogenous and ectopically expressed FBXO17 associate with GSK3β, and its overexpression leads to decreased protein levels of GSK3β. Silencing FBXO17 gene expression increased the half-life of GSK3β in cells. Furthermore, overexpression of FBXO17 inhibits agonist-induced release of keratinocyte-derived cytokine (KC) and interleukin-6 (IL-6) production by cells. Thus, the SCFFBXO17 E3 ubiquitin ligase complex negatively regulates inflammation by targeting GSK3β in lung epithelia.

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Wang, Quan, Vanessa Crnković, Christian Preisinger, and Judith Stegmüller. "The parkinsonism-associated protein FBXO7 cooperates with the BAG6 complex in proteasome function and controls the subcellular localization of the complex." Biochemical Journal 478, no. 12 (June 18, 2021): 2179–99. http://dx.doi.org/10.1042/bcj20201000.

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The regulation of proteasome activity is essential to cellular homeostasis and defects have been implicated in various disorders including Parkinson disease. The F-box protein FBXO7 has been implicated in early-onset parkinsonism and has previously been shown to have a regulatory role in proteasome activity and assembly. Here, we report the association of the E3 ubiquitin ligase FBXO7-SCF (SKP1, cullin-1, F-box protein) with the BAG6 complex, consisting of the subunits BAG6, GET4 and UBL4A. We identify the subunit GET4 as a direct interactor of FBXO7 and we show that the subunits GET4 and UBL4A are required for proper proteasome activity. Our findings demonstrate reduced binding of FBXO7 variants to GET4 and that FBXO7 variants bring about reduced proteasome activity. In addition, we find that GET4 is a non-proteolytic substrate of FBXO7, that binding of GET4 to BAG6 is enhanced in the presence of active FBXO7-SCF and that the cytoplasmic localization of the BAG6 complex is dependent on the E3 ubiquitin ligase activity. Taken together, our study shows that the parkinsonism-associated FBXO7 cooperates with the BAG6 complex in proteasome function and determines the subcellular localization of this complex.

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Williams, Clara, Patricia Fernández-Calvo, Maite Colinas, Laurens Pauwels, and Alain Goossens. "Jasmonate and auxin perception: how plants keep F-boxes in check." Journal of Experimental Botany 70, no. 13 (June 6, 2019): 3401–14. http://dx.doi.org/10.1093/jxb/erz272.

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Abstract Phytohormones regulate the plasticity of plant growth and development, and responses to biotic and abiotic stresses. Many hormone signal transduction cascades involve ubiquitination and subsequent degradation of proteins by the 26S proteasome. The conjugation of ubiquitin to a substrate is facilitated by the E1 activating, E2 conjugating, and the substrate-specifying E3 ligating enzymes. The most prevalent type of E3 ligase in plants is the Cullin–RING ligase (CRL)-type, with F-box proteins (FBPs) as the substrate recognition component. The activity of these SKP–Cullin–F-box (SCF) complexes needs to be tightly regulated in time and place. Here, we review the regulation of SCF function in plants on multiple levels, with a focus on the auxin and jasmonate SCF-type receptor complexes. We discuss in particular the relevance of protein–protein interactions and post-translational modifications as mechanisms to keep SCF functioning under control. Additionally, we highlight the unique property of SCFTIR1/AFB and SCFCOI1 to recognize substrates by forming co-receptor complexes. Finally, we explore how engineered selective agonists can be used to study and uncouple the outcomes of the complex auxin and jasmonate signaling networks that are governed by these FBPs.

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Ren, Yi, Qingzhu Hua, Jiayan Pan, Zhike Zhang, Jietang Zhao, Xinhua He, Yonghua Qin, and Guibing Hu. "SKP1-like protein, CrSKP1-e, interacts with pollen-specific F-box proteins and assembles into SCF-type E3 complex in ‘Wuzishatangju’ (Citrus reticulata Blanco) pollen." PeerJ 8 (December 22, 2020): e10578. http://dx.doi.org/10.7717/peerj.10578.

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S-ribonuclease (S-RNase)-based self-incompatibility (SI) mechanisms have been extensively studied in Solanaceae, Rosaceae and Plantaginaceae. S-RNase-based SI is controlled by two closely related genes, S-RNase and S-locus F-box (SLF), located at a polymorphic S-locus. In the SI system, the SCF-type (SKP1-CUL1-F-box-RBX1) complex functions as an E3 ubiquitin ligase complex for ubiquitination of non-self S-RNase. Pummelo (Citrus grandis) and several mandarin cultivars are suggested to utilize an S-RNase-based SI system. However, the molecular mechanism of the non-S-factors involved in the SI reaction is not straightforward in Citrus. To investigate the SCF-type E3 complex responsible for the SI reaction in mandarin, SLF, SKP1-like and CUL1 candidates potentially involved in the SI reaction of ‘Wuzishatangju’ (Citrus reticulata Blanco) were identified based on the genome-wide identification and expression analyses. Sixteen pollen-specific F-box genes (CrFBX1-CrFBX16), one pollen-specific SKP1-like gene (CrSKP1-e) and two CUL1 genes (CrCUL1A and CrCUL1B) were identified and cloned from ‘Wuzishatangju’. Yeast two-hybrid (Y2H) and in vitro binding assays showed that five CrFBX proteins could bind to CrSKP1-e, which is an ortholog of SSK1 (SLF-interacting-SKP1-like), a non-S-factor responsible for the SI reaction. Luciferase complementation imaging (LCI) and in vitro binding assays also showed that CrSKP1-e interacts with the N-terminal region of both CrCUL1A and CrCUL1B. These results indicate that CrSKP1-e may serve as a functional member of the SCF-type E3 ubiquitin ligase complex in ‘Wuzishatangju’.

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Lee, Albert, Stephanie L. Rayner, Alana De Luca, Serene S. L. Gwee, Marco Morsch, Vinod Sundaramoorthy, Hamideh Shahheydari, et al. "Casein kinase II phosphorylation of cyclin F at serine 621 regulates the Lys48-ubiquitylation E3 ligase activity of the SCF (cyclin F) complex." Open Biology 7, no. 10 (October 2017): 170058. http://dx.doi.org/10.1098/rsob.170058.

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Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3–5 years of diagnosis. Recently, we identified disease-linked mutations in the CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) (Williams KL et al . 2016 Nat. Commun. 7 , 11253. ( doi:10.1038/ncomms11253 )). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST (proline, glutamic acid, serine and threonine) sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF (cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF (cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.

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Wu, Kenneth, Serge Y. Fuchs, Angus Chen, Peilin Tan, Carlos Gomez, Ze'ev Ronai, and Zhen-Qiang Pan. "The SCFHOS/β-TRCP-ROC1 E3 Ubiquitin Ligase Utilizes Two Distinct Domains within CUL1 for Substrate Targeting and Ubiquitin Ligation." Molecular and Cellular Biology 20, no. 4 (February 15, 2000): 1382–93. http://dx.doi.org/10.1128/mcb.20.4.1382-1393.2000.

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ABSTRACT We describe a purified ubiquitination system capable of rapidly catalyzing the covalent linkage of polyubiquitin chains onto a model substrate, phosphorylated IκBα. The initial ubiquitin transfer and subsequent polymerization steps of this reaction require the coordinated action of Cdc34 and the SCFHOS/β-TRCP-ROC1 E3 ligase complex, comprised of four subunits (Skp1, cullin 1 [CUL1], HOS/β-TRCP, and ROC1). Deletion analysis reveals that the N terminus of CUL1 is both necessary and sufficient for binding Skp1 but is devoid of ROC1-binding activity and, hence, is inactive in catalyzing ubiquitin ligation. Consistent with this, introduction of the N-terminal CUL1 polypeptide into cells blocks the tumor necrosis factor alpha-induced and SCF-mediated degradation of IκB by forming catalytically inactive complexes lacking ROC1. In contrast, the C terminus of CUL1 alone interacts with ROC1 through a region containing the cullin consensus domain, to form a complex fully active in supporting ubiquitin polymerization. These results suggest the mode of action of SCF-ROC1, where CUL1 serves as a dual-function molecule that recruits an F-box protein for substrate targeting through Skp1 at its N terminus, while the C terminus of CUL1 binds ROC1 to assemble a core ubiquitin ligase.

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Morel, Marion, Krushangi N. Shah, and Weiwen Long. "The F-box protein FBXL16 up-regulates the stability of C-MYC oncoprotein by antagonizing the activity of the F-box protein FBW7." Journal of Biological Chemistry 295, no. 23 (April 28, 2020): 7970–80. http://dx.doi.org/10.1074/jbc.ra120.012658.

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F-box proteins, such as F-box/WD repeat-containing protein 7 (FBW7), are essential components of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases. They bind to S-phase kinase-associated protein 1 (SKP1) through the F-box motif and deliver their protein substrate to the E3 ligase complex for ubiquitination and subsequent degradation. F-box and leucine-rich repeat protein 16 (FBXL16) is a poorly studied F-box protein. Because it does not interact with the scaffold protein cullin 1 (CUL1), we hypothesized that FBXL16 might not form a functional SCF-E3 ligase complex. In the present study, we found that FBXL16 up-regulates the levels of proteins targeted by SCF-E3 ligases, such as C-MYC, β-catenin, and steroid receptor coactivator 3 (SRC-3). Focusing on C-MYC, a well-known oncoprotein overexpressed in most human cancers, we show that FBXL16 stabilizes C-MYC by antagonizing FBW7-mediated C-MYC ubiquitination and degradation. Further, we found that, although FBXL16 does not interact with CUL1, it interacts with SKP1 via its N-terminal F-box domain and with its substrate C-MYC via its C-terminal leucine-rich repeats (LRRs) domain. We found that both the F-box domain and the LRR domain are important for FBXL16-mediated C-MYC stabilization. In line with its role in up-regulating the levels of the C-MYC and SRC-3 oncoproteins, FBXL16 promoted cancer cell growth and migration and colony formation in soft agar. Our findings reveal that FBXL16 is an F-box protein that antagonizes the activity of another F-box protein, FBW7, and thereby increases C-MYC stability, resulting in increased cancer cell growth and invasiveness.

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Kitagawa, Kyoko, and Masatoshi Kitagawa. "The SCF-type E3 Ubiquitin Ligases as Cancer Targets." Current Cancer Drug Targets 16, no. 2 (January 5, 2016): 119–29. http://dx.doi.org/10.2174/1568009616666151112122231.

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Merzetti, Eric M., Lindsay A. Dolomount, and Brian E. Staveley. "The FBXO7 homologue nutcracker and binding partner PI31 in Drosophila melanogaster models of Parkinson’s disease." Genome 60, no. 1 (January 2017): 46–54. http://dx.doi.org/10.1139/gen-2016-0087.

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Parkinsonian-pyramidal syndrome (PPS) is an early onset form of Parkinson’s disease (PD) that shows degeneration of the extrapyramidal region of the brain to result in a severe form of PD. The toxic protein build-up has been implicated in the onset of PPS. Protein removal is mediated by an intracellular proteasome complex: an E3 ubiquitin ligase, the targeting component, is essential for function. FBXO7 encodes the F-box component of the SCF E3 ubiquitin ligase linked to familial forms of PPS. The Drosophila melanogaster homologue nutcracker (ntc) and a binding partner, PI31, have been shown to be active in proteasome function. We show that altered expression of either ntc or PI31 in dopaminergic neurons leads to a decrease in longevity and locomotor ability, phenotypes both associated with models of PD. Furthermore, expression of ntc-RNAi in an established α-synuclein-dependent model of PD rescues the phenotypes of diminished longevity and locomotor control.

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Bassermann, Florian C., Silvia Muench, Christine von Klitzing, Stephan W. Morris, Christian Peschel, and Justus Duyster. "Inactivation of NIPA Results in Premature Mitotic Entry and Subsequent Mitotic Catastrophe." Blood 106, no. 11 (November 16, 2005): 1364. http://dx.doi.org/10.1182/blood.v106.11.1364.1364.

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Abstract Ubiquitin-mediated destruction of regulatory proteins marks a vital means of controlling cell cycle progresssion. The E3 ubiquitin-ligases are prominent in this process as they determine specificity of the ubiquitination process and thus regulate proteasomal degradation of target proteins. Recently, a class of E3 ligases referred to as SCF complexes has been identified. The substrate binding specifity within this class of E3 ligases is mediated by a class of molecules termed F-box proteins. We previously reported the cloning of NIPA (nuclear interaction partner of ALK) in complex with constitutively-active oncogenic fusions of ALK, which contribute to the development of certain lymphomas and sarcomas. Subsequently we characterized NIPA as a human F-box protein that defines an oscillating ubiquitin ligase (SCF-NIPA) which targets nuclear cyclin B1 in interphase. We have now determined the consequence of inactivating NIPA with regard to cell cycle regulation using an RNAi approach. Kinetic analysis of cell cycle phase transition times revealed a premature onset of Cdk1/cyclin B1 kinase activity and early mitotic entry in cells treated with NIPA siRNA. Cyclin B1 was shown to accumulate within the nucleus in these cells correlative to a reduced ubiquitination activity of the SCF-NIPA complex. Subsequent to premature mitotic entry, NIPA inactivated cells arrested in prometaphase and mitotic catastrophe was observed thereafter. We searched for relevant proteins involved in this process and found Survivin, a member of the inhibitor of apoptosis proteins (IAP) to be strongly downregulated in NIPA siRNA treated cells. Survivin has been shown to function as a mitotic checkpoint molecule that induces mitotic catastrophe subsequent to aberant mitosis when inactivated or downregulated. While the precise functional relationship between Survivin and NIPA is still under investigation, the current data distinguishes NIPA as a central molecule in timing mitotic entry. Given this function, NIPA directly influences the fidelity of DNA replication and segregation. Interference with NIPA function may therefore be an oncogenic principle that favours genomic instability in tumor cells.

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Pavlides, Savvas C., Kuang-Tzu Huang, Dylan A. Reid, Lily Wu, Stephanie V. Blank, Khushbakhat Mittal, Lankai Guo, et al. "Inhibitors of SCF-Skp2/Cks1 E3 Ligase Block Estrogen-Induced Growth Stimulation and Degradation of Nuclear p27kip1: Therapeutic Potential for Endometrial Cancer." Endocrinology 154, no. 11 (November 1, 2013): 4030–45. http://dx.doi.org/10.1210/en.2013-1757.

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In many human cancers, the tumor suppressor, p27kip1 (p27), a cyclin-dependent kinase inhibitor critical to cell cycle arrest, undergoes perpetual ubiquitin-mediated proteasomal degradation by the E3 ligase complex SCF-Skp2/Cks1 and/or cytoplasmic mislocalization. Lack of nuclear p27 causes aberrant cell cycle progression, and cytoplasmic p27 mediates cell migration/metastasis. We previously showed that mitogenic 17-β-estradiol (E2) induces degradation of p27 by the E3 ligase Skp1-Cullin1-F-Box- S phase kinase-associated protein2/cyclin dependent kinase regulatory subunit 1 in primary endometrial epithelial cells and endometrial carcinoma (ECA) cell lines, suggesting a pathogenic mechanism for type I ECA, an E2-induced cancer. The current studies show that treatment of endometrial carcinoma cells-1 (ECC-1) with small molecule inhibitors of Skp2/Cks1 E3 ligase activity (Skp2E3LIs) stabilizes p27 in the nucleus, decreases p27 in the cytoplasm, and prevents E2-induced proliferation and degradation of p27 in endometrial carcinoma cells-1 and primary ECA cells. Furthermore, Skp2E3LIs increase p27 half-life by 6 hours, inhibit cell proliferation (IC50, 14.3μM), block retinoblastoma protein (pRB) phosphorylation, induce G1 phase block, and are not cytotoxic. Similarly, using super resolution fluorescence localization microscopy and quantification, Skp2E3LIs increase p27 protein in the nucleus by 1.8-fold. In vivo, injection of Skp2E3LIs significantly increases nuclear p27 and reduces proliferation of endometrial epithelial cells by 42%–62% in ovariectomized E2-primed mice. Skp2E3LIs are specific inhibitors of proteolytic degradation that pharmacologically target the binding interaction between the E3 ligase, SCF-Skp2/Cks1, and p27 to stabilize nuclear p27 and prevent cell cycle progression. These targeted inhibitors have the potential to be an important therapeutic advance over general proteasome inhibitors for cancers characterized by SCF-Skp2/Cks1-mediated destruction of nuclear p27.

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Kainulainen, Markus, Simone Lau, Charles E. Samuel, Veit Hornung, and Friedemann Weber. "NSs Virulence Factor of Rift Valley Fever Virus Engages the F-Box Proteins FBXW11 and β-TRCP1 To Degrade the Antiviral Protein Kinase PKR." Journal of Virology 90, no. 13 (April 27, 2016): 6140–47. http://dx.doi.org/10.1128/jvi.00016-16.

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ABSTRACTRift Valley fever virus (RVFV, familyBunyaviridae, genusPhlebovirus) is a relevant pathogen of both humans and livestock in Africa. The nonstructural protein NSs is a major virulence factor known to suppress the type I interferon (IFN) response by inhibiting host cell transcription and by proteasomal degradation of a major antiviral IFN effector, the translation-inhibiting protein kinase PKR. Here, we identified components of the modular SCF (Skp1, Cul1, F-box protein)-type E3 ubiquitin ligases as mediators of PKR destruction by NSs. Small interfering RNAs (siRNAs) against the conserved SCF subunit Skp1 protected PKR from NSs-mediated degradation. Consequently, RVFV replication was severely reduced in Skp1-depleted cells when PKR was present. SCF complexes have a variable F-box protein subunit that determines substrate specificity for ubiquitination. We performed an siRNA screen for all (about 70) human F-box proteins and found FBXW11 to be involved in PKR degradation. The partial stabilization of PKR by FBXW11 depletion upregulated PKR autophosphorylation and phosphorylation of the PKR substrate eIF2α and caused a shutoff of host cell protein synthesis in RVFV-infected cells. To maximally protect PKR from the action of NSs, knockdown of structurally and functionally related FBXW1 (also known as β-TRCP1), in addition to FBXW11 deletion, was necessary. Consequently, NSs was found to interact with both FBXW11 and β-TRCP1. Thus, NSs eliminates the antiviral kinase PKR by recruitment of SCF-type E3 ubiquitin ligases containing FBXW11 and β-TRCP1 as substrate recognition subunits. This antagonism of PKR by NSs is essential for efficient RVFV replication in mammalian cells.IMPORTANCERift Valley fever virus is a pathogen of humans and animals that has the potential to spread from Africa and the Arabian Peninsula to other regions. A major virulence mechanism is the proteasomal degradation of the antiviral kinase PKR by the viral protein NSs. Here, we demonstrate that NSs requires E3 ubiquitin ligase complexes of the SCF (Skp1, Cul1, F-box protein) type to destroy PKR. SCF-type complexes can engage variant ubiquitination substrate recognition subunits, and we found the F-box proteins FBXW11 and β-TRCP1 to be relevant for the action of NSs against PKR. Thus, we identified the host cell factors that are critically needed by Rift Valley fever virus to uphold its replication against the potent antiviral kinase PKR.

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Thompson, Laura L., Kailee A. Rutherford, Chloe C. Lepage, and Kirk J. McManus. "The SCF Complex Is Essential to Maintain Genome and Chromosome Stability." International Journal of Molecular Sciences 22, no. 16 (August 9, 2021): 8544. http://dx.doi.org/10.3390/ijms22168544.

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The SKP1, CUL1, F-box protein (SCF) complex encompasses a group of 69 SCF E3 ubiquitin ligase complexes that primarily modify protein substrates with poly-ubiquitin chains to target them for proteasomal degradation. These SCF complexes are distinguishable by variable F-box proteins, which determine substrate specificity. Although the function(s) of each individual SCF complex remain largely unknown, those that have been characterized regulate a wide array of cellular processes, including gene transcription and the cell cycle. In this regard, the SCF complex regulates transcription factors that modulate cell signaling and ensures timely degradation of primary cell cycle regulators for accurate replication and segregation of genetic material. SCF complex members are aberrantly expressed in a myriad of cancer types, with altered expression or function of the invariable core SCF components expected to have a greater impact on cancer pathogenesis than that of the F-box proteins. Accordingly, this review describes the normal roles that various SCF complexes have in maintaining genome stability before discussing the impact that aberrant SCF complex expression and/or function have on cancer pathogenesis. Further characterization of the SCF complex functions is essential to identify and develop therapeutic approaches to exploit aberrant SCF complex expression and function.

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Oh, Kwang-Jin, Anna Kalinina, Jing Wang, Keiko Nakayama, Keiichi I. Nakayama, and Srilata Bagchi. "The Papillomavirus E7 Oncoprotein Is Ubiquitinated by UbcH7 and Cullin 1- and Skp2-Containing E3 Ligase." Journal of Virology 78, no. 10 (May 15, 2004): 5338–46. http://dx.doi.org/10.1128/jvi.78.10.5338-5346.2004.

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ABSTRACT Recurrent infections with high-risk human papillomaviruses (HPVs) are associated with human cervical cancers. All HPV-associated cancer tissues express the viral oncoproteins E6 and E7, which stimulate cell growth. The expression of E7 is crucial for both the initiation and the maintenance of HPV-associated cancer. Recent studies showed that the level of E7 in cancer cells is regulated by ubiquitin-dependent proteolysis through the 26S proteasome. In this study, we characterized the enzymes involved in the ubiquitin-dependent proteolysis of E7. We show that UbcH7, an E2 ubiquitin-conjugating enzyme, is specifically involved in the ubiquitination of E7. Furthermore, we show that E7 interacts with the SCF (Skp-Cullin-F box) ubiquitin ligase complex containing Cullin 1 (Cul1) and Skp2 and can be ubiquitinated by the Cul1-containing ubiquitin ligase in vitro. Coimmunoprecipitation analyses revealed that E7 interacts with Skp2 and Cul1 in vivo. Finally, the half-life of E7 was found to be significantly longer in Skp2−/− mouse embryo fibroblasts (MEFs) than in wild-type MEFs. Taken together, these results suggest that the Cul1- and Skp2-containing ubiquitin ligase plays a role in the ubiquitination and proteolysis of E7. In HPV type 16-containing cervical carcinoma cell line Caski, E7 localizes to both the cytoplasm and the nucleus. Brief treatment of Caski cells with MG132 (a proteasome inhibitor) causes the accumulation of E7 in discrete nuclear bodies. These nuclear bodies are detergent insoluble and contain polyubiquitinated E7. We suggest that E7 relocates to specific nuclear bodies for proteolysis in HPV-containing epithelial cells.

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Sahasrabuddhe, Anagh A., Xiaofei Chen, Fuzon Chung, Thirunavukkarasu Velusamy, Megan S. Lim, and Kojo S. J. Elenitoba-Johnson. "SCF-β-TrCP E3 Ubiquitin Ligase-Mediated Proteolysis Of EZH2 Regulates H3K27 Trimethylation Activity In Germinal Center-Derived B-Cell Lymphomas." Blood 122, no. 21 (November 15, 2013): 244. http://dx.doi.org/10.1182/blood.v122.21.244.244.

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Abstract EZH2 is a critical enzymatic subunit of the PRC2 which trimethylates histone H3 (H3K27) to mediate gene repression. While overexpression of EZH2 and its increased H3K27 methylation have generally been associated with hematologic malignancies and solid tumors, recurrent gain of function somatic mutations in EZH2 have been recently reported in germinal center B-cell (GCB) derived lymphomas. These mutations, which replace a single tyrosine in the SET domain of the EZH2 protein (Y641), occur in 7.2% of FLs (follicular lymphoma) and 21.7% of GCB DLBCL (diffuse large B-cell lymphoma) and are absent from ABC DLBCLs. Consequently, B-cell lymphoma cell lines and lymphoma primary samples harboring heterozygous EZH2Y641mutations exhibit increased levels of histone H3 Lys27 trimethylation (H3K27me3) and promote a lymphoproliferative phenotype with expansion of the GC B cell compartment. Although extensive studies have been conducted to understand the role of polycomb group of proteins in cancer pathogenesis, few studies have been aimed at understanding the post-translational regulation of these proteins. Skp1/Cullin1/F- box protein (SCF) ubiquitin ligase complexes are the largest family of multicomponent ring E3 ubiquitin ligases that control the degradation of many important regulatory proteins and have been implicated in the regulation of epigenetic regulators. However, the mechanism of EZH2 degradation and its implication in PRC2 mediated methyl transferase activity in a cancer-specific context is not fully understood. Cullin-ring ubiquitin ligases (CRLs) form the largest known class of multicomponent E3 ubiqutin ligase family in eukaryotes and target a wide array of substrates involved in several biological processes. To investigate a CRL-mediated basis for post-translational EZH2 regulation, we screened a panel of cullin family E3 ligases for interaction with EZH2 and discovered that EZH2 was immunoprecipitated by cullin1 exclusively among all other cullins (cul2,3,4a,4b,5 and 7). We engineered and expressed a cullin1 dominant negative protein which resulted in EZH2 accumulation in a dose dependent manner. Using co-immunoprecipitation approach, we screened 8 different F box proteins and observed that SCF E3 ubiquitin ligase β-TrCP (FBXW1) uniquely and specifically interacted with EZH2. Further, RNAi-mediated silencing of β-TrCP resulted in EZH2 stabilization with attendant increase in H3K27 trimethylation activity. Importantly, the Y641 mutants recurrently identified and relevant in lymphoma pathogenesis were unable to bind β-TrCP. Further, cycloheximide chase experiments showed that Y641 mutants endogenously expressed in lymphoma-derived cells exhibit increased EZH2 stability as well as enhanced H3K27 trimethylation activity. Our studies demonstrate that β-TrCP plays an important role in controlling H3K27 trimethylation activity via an unusual recognition mechanism targeting EZH2 for proteasomal degradation. Our findings delineate a novel β-TrCP/EZH2 axis requiring an intact Y641 residue for EZH2 regulation by β-TrCP. Oncogenic mutations in Y641 in GCB-cell derived lymphomas confer increased stability to EZH2 and render it resistant to β-TrCP-mediated degradation. This newly identified mechanism might help in design potential novel therapeutic approaches for clinical management of cancer potentially driven by aberrant accumulation/function of EZH2. Disclosures: No relevant conflicts of interest to declare.

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Inui, Naoki, Satoshi Sakai, and Masatoshi Kitagawa. "Molecular Pathogenesis of Pulmonary Fibrosis, with Focus on Pathways Related to TGF-β and the Ubiquitin-Proteasome Pathway." International Journal of Molecular Sciences 22, no. 11 (June 5, 2021): 6107. http://dx.doi.org/10.3390/ijms22116107.

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Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. During the past decade, novel pathogenic mechanisms of IPF have been elucidated that have shifted the concept of IPF from an inflammatory-driven to an epithelial-driven disease. Dysregulated repair responses induced by recurrent epithelial cell damage and excessive extracellular matrix accumulation result in pulmonary fibrosis. Although there is currently no curative therapy for IPF, two medications, pirfenidone and nintedanib, have been introduced based on understanding the pathogenesis of the disease. In this review, we discuss advances in understanding IPF pathogenesis, highlighting epithelial–mesenchymal transition (EMT), the ubiquitin-proteasome system, and endothelial cells. TGF-β is a central regulator involved in EMT and pulmonary fibrosis. HECT-, RING finger-, and U-box-type E3 ubiquitin ligases regulate TGF-β-Smad pathway-mediated EMT via the ubiquitin-proteasome pathway. p27 degradation mediated by the SCF-type E3 ligase, Skp2, contributes to the progression of pulmonary fibrosis by promotion of either mesenchymal fibroblast proliferation, EMT, or both. In addition to fibroblasts as key effector cells in myofibroblast differentiation and extracellular matrix deposition, endothelial cells also play a role in the processes of IPF. Endothelial cells can transform into myofibroblasts; therefore, endothelial–mesenchymal transition can be another source of myofibroblasts.

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Zheng, Jianyu, Xiaoming Yang, Jennifer M. Harrell, Sophia Ryzhikov, Eun-Hee Shim, Karin Lykke-Andersen, Ning Wei, Hong Sun, Ryuji Kobayashi, and Hui Zhang. "CAND1 Binds to Unneddylated CUL1 and Regulates the Formation of SCF Ubiquitin E3 Ligase Complex." Molecular Cell 10, no. 6 (December 2002): 1519–26. http://dx.doi.org/10.1016/s1097-2765(02)00784-0.

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Jia, Lijun, and Yi Sun. "RBX1/ROC1-SCF E3 ubiquitin ligase is required for mouse embryogenesis and cancer cell survival." Cell Division 4, no. 1 (2009): 16. http://dx.doi.org/10.1186/1747-1028-4-16.

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Chen, B. B., T. A. Coon, J. R. Glasser, and R. K. Mallampalli. "Calmodulin Antagonizes a Calcium-Activated SCF Ubiquitin E3 Ligase Subunit, FBXL2, To Regulate Surfactant Homeostasis." Molecular and Cellular Biology 31, no. 9 (February 22, 2011): 1905–20. http://dx.doi.org/10.1128/mcb.00723-10.

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Choudhury, Rajarshi, Thomas Bonacci, Xianxi Wang, Andrew Truong, Anthony Arceci, Yanqiong Zhang, Christine A. Mills, Jennifer L. Kernan, Pengda Liu, and Michael J. Emanuele. "The E3 Ubiquitin Ligase SCF(Cyclin F) Transmits AKT Signaling to the Cell-Cycle Machinery." Cell Reports 20, no. 13 (September 2017): 3212–22. http://dx.doi.org/10.1016/j.celrep.2017.08.099.

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Barford, David. "Structure, function and mechanism of the anaphase promoting complex (APC/C)." Quarterly Reviews of Biophysics 44, no. 2 (November 22, 2010): 153–90. http://dx.doi.org/10.1017/s0033583510000259.

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AbstractThe complex molecular events responsible for coordinating chromosome replication and segregation with cell division and growth are collectively known as the cell cycle. Progression through the cell cycle is orchestrated by the interplay between controlled protein synthesis and degradation and protein phosphorylation. Protein degradation is primarily regulated through the ubiquitin proteasome system, mediated by two related E3 protein ubiquitin ligases, the Skp1 cullin F-box (SCF) and the anaphase promoting complex (also known as the cyclosome) (APC/C). The APC/C is a multi-subunit cullin-RING E3 ubiquitin ligase that regulates progression through the mitotic phase of the cell cycle and controls entry into S phase by catalysing the ubiquitylation of cyclins and other cell cycle regulatory proteins. Selection of APC/C targets is controlled through recognition of short destruction motifs, predominantly the D-box and KEN-box. APC/C-mediated coordination of cell cycle progression is achieved through the temporal regulation of APC/C activity and substrate specificity, exerted through a combination of co-activator subunits, reversible phosphorylation and inhibitory proteins and complexes. The aim of this article is to discuss the APC/C from a structural and mechanistic perspective. Although an atomic structure of the APC/C is still lacking, a combination of genetic, biochemical, electron microscopy studies of intact APC/C and crystallographic analysis of individual subunits, together with analogies to evolutionarily related E3 ligases of the RING family, has provided deep insights into the molecular mechanisms of catalysis and substrate recognition, and structural organisation of the APC/C.

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Chen, Wei, Sheng Xiong, Jin Li, Xiuying Li, Yuan Liu, Chunbin Zou, and Rama K. Mallampalli. "The Ubiquitin E3 Ligase SCF-FBXO24 Recognizes Deacetylated Nucleoside Diphosphate Kinase A To Enhance Its Degradation." Molecular and Cellular Biology 35, no. 6 (January 12, 2015): 1001–13. http://dx.doi.org/10.1128/mcb.01185-14.

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The Skp-Cul-F box (SCF) ubiquitin E3 ligase machinery recognizes predominantly phosphodegrons or, less commonly, an (I/L)Q molecular signature within substrates to facilitate their recruitment in mediating protein ubiquitination and degradation. Here, we examined the molecular signals that determine the turnover of the multifunctional enzyme nucleoside diphosphate kinase A (NDPK-A) that controls cell proliferation. NDPK-A protein exhibits a half-life of ∼6 h in HeLa cells and is targeted for ubiquitylation through actions of the F-box protein FBXO24. SCF-FBXO24 polyubiquitinates NDPK-A at K85, and two NH 2 -terminal residues, L55 and K56, were identified as important molecular sites for FBXO24 interaction. Importantly, K56 acetylation impairs its interaction with FBXO24, and replacing K56 with Q56, an acetylation mimic, reduces NDPK-A FBXO24 binding capacity. The acetyltransferase GCN5 catalyzes K56 acetylation within NDPK-A, thereby stabilizing NDPK-A, whereas GCN5 depletion in cells accelerates NDPK-A degradation. Cellular expression of an NDPK-A acetylation mimic or FBXO24 silencing increases NDPK-A life span which, in turn, impairs cell migration and wound healing. We propose that lysine acetylation when presented in the appropriate context may be recognized by some F-box proteins as a unique inhibitory molecular signal for their recruitment to restrict substrate degradation.

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Nelson, David E., and Heike Laman. "A Competitive Binding Mechanism between Skp1 and Exportin 1 (CRM1) Controls the Localization of a Subset of F-box Proteins." Journal of Biological Chemistry 286, no. 22 (March 4, 2011): 19804–15. http://dx.doi.org/10.1074/jbc.m111.220079.

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SCF-type E3 ubiquitin ligases are crucial regulators of cell cycle progression. As the F-box protein is the substrate-specifying subunit of this family of ligases, their availability dictates the timing and the location of the ubiquitination of substrates. We report here our investigation into the regulation of the localization of F-box proteins, in particular Fbxo7, whose mislocalization is associated with human disease. We identified a motif in Fbxo7 that we have characterized as a functional leucine-rich nuclear export sequence (NES), and which allowed binding to the nuclear export protein, exportin 1 (CRM1). Unusually, the NES was embedded within the F-box domain, which is bound by Skp1 and enables the F-box protein to form part of an E3 ubiquitin ligase. The NES of Fbxo7 controlled its localization and was conserved in Fbxo7 homologues in other species. Skp1 binding prevented Fbxo7 from contacting CRM1. We propose that this competitive binding allowed Fbxo7 to accumulate within the nucleus starting at the G1/S transition. More than ten other F-box proteins also contain an NES at the same location in their F-box domains, indicating that this competitive binding mechanism may contribute to the regulation of a sixth of the known F-box proteins.

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Sadowski, Martin, Randy Suryadinata, Xianning Lai, Jörg Heierhorst, and Boris Sarcevic. "Molecular Basis for Lysine Specificity in the Yeast Ubiquitin-Conjugating Enzyme Cdc34." Molecular and Cellular Biology 30, no. 10 (March 1, 2010): 2316–29. http://dx.doi.org/10.1128/mcb.01094-09.

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ABSTRACT Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

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Blanchette, Paola, Chi Ying Cheng, Qin Yan, Gary Ketner, David A. Ornelles, Thomas Dobner, Ronald C. Conaway, Joan Weliky Conaway, and Philip E. Branton. "Both BC-Box Motifs of Adenovirus Protein E4orf6 Are Required To Efficiently Assemble an E3 Ligase Complex That Degrades p53." Molecular and Cellular Biology 24, no. 21 (November 1, 2004): 9619–29. http://dx.doi.org/10.1128/mcb.24.21.9619-9629.2004.

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ABSTRACT Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway. We have shown previously that p53 degradation relies on interactions of E4orf6 with the cellular proteins Cul5, Rbx1, and elongins B and C to form an E3 ligase similar to the SCF and VBC complexes. Here we show that, like other elongin BC-interacting proteins, including elongin A, von Hippel-Lindau protein, and Muf1, the interaction of E4orf6 is mediated by the BC-box motif; however, E4orf6 uniquely utilizes two BC-box motifs for degradation of p53 and another target, Mre11. In addition, our data suggest that the interaction of E1B55K with E4orf6 depends on the ability of E4orf6 to form the E3 ligase complex and that such complex formation may be required for all E4orf6-E1B55K functions.

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Dove, Katja K., Hilary A. Kemp, Kristin R. Di Bona, Katherine H. Reiter, Luke J. Milburn, David Camacho, David S. Fay, Dana L. Miller, and Rachel E. Klevit. "Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development." Proceedings of the National Academy of Sciences 114, no. 32 (July 24, 2017): E6576—E6584. http://dx.doi.org/10.1073/pnas.1705060114.

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Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans. These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

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Bochis, Ovidiu Vasile, Bogdan Fetica, Catalin Vlad, Patriciu Achimas-Cadariu, and Alexandru Irimie. "The importance of ubiquitin E3 ligases, SCF and APC/C, in human cancers." Medicine and Pharmacy Reports 88, no. 1 (January 28, 2015): 9–14. http://dx.doi.org/10.15386/cjmed-377.

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A normal evolution of the cell-cycle phases consists of multiple consecutive events, which makes it a highly complex process. Its preservation is regulated by Cyclin-Cdks (cyclin-dependent kinases) interactions and protein degradation, which is often controlled by the ubiquitin-mediated proteolysis.The goal of this review is to emphasize the most important features of the regulation of the cell-cycle involved in cancerogenesis, by presenting the involvement of E3 ubiquitin ligases SCF (Skp1-Cul1-F-box protein) and APC/C (Anaphase-promoting complex/cyclosome) in human malignancies. Also, we discuss the importance of the ubiquitin proteasome pathway blockade in cancer treatment. We know that a better understanding of the regulatory biology of the cell cycle can lead to the development of new target therapies for cancer.

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Liu, Tong-Bao, Yina Wang, Sabriya Stukes, Qing Chen, Arturo Casadevall, and Chaoyang Xue. "The F-Box Protein Fbp1 Regulates Sexual Reproduction and Virulence in Cryptococcus neoformans." Eukaryotic Cell 10, no. 6 (April 8, 2011): 791–802. http://dx.doi.org/10.1128/ec.00004-11.

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ABSTRACTCryptococcus neoformansis the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Althoughfbp1mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, asfbp1mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating betweenfbp1mutants, even though normal dikaryotic hyphae were observed during mating.In vitroassays of stress responses revealed thatfbp1mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in bothSaccharomyces cerevisiaeandC. neoformansvia its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence inC. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.

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Schaefer, Kristina N., Mira I. Pronobis, Clara E. Williams, Shiping Zhang, Lauren Bauer, Dennis Goldfarb, Feng Yan, M. Ben Major, and Mark Peifer. "Wnt regulation: exploring Axin-Disheveled interactions and defining mechanisms by which the SCF E3 ubiquitin ligase is recruited to the destruction complex." Molecular Biology of the Cell 31, no. 10 (May 1, 2020): 992–1014. http://dx.doi.org/10.1091/mbc.e19-11-0647.

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Wnt signaling plays key roles in embryonic development and adult stem cell homeostasis and is altered in human cancer. We explore β-catenin transfer from the destruction complex to the E3 ligase, and test models suggesting Dishevelled and APC2 compete for association with Axin.

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Shen, Mingzhe, Chae Jin Lim, Junghoon Park, Jeong Eun Kim, Dongwon Baek, Jaesung Nam, Sang Yeol Lee, et al. "HOS15 is a transcriptional corepressor of NPR1-mediated gene activation of plant immunity." Proceedings of the National Academy of Sciences 117, no. 48 (November 16, 2020): 30805–15. http://dx.doi.org/10.1073/pnas.2016049117.

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Transcriptional regulation is a complex and pivotal process in living cells. HOS15 is a transcriptional corepressor. Although transcriptional repressors generally have been associated with inactive genes, increasing evidence indicates that, through poorly understood mechanisms, transcriptional corepressors also associate with actively transcribed genes. Here, we show that HOS15 is the substrate receptor for an SCF/CUL1 E3 ubiquitin ligase complex (SCFHOS15) that negatively regulates plant immunity by destabilizing transcriptional activation complexes containing NPR1 and associated transcriptional activators. In unchallenged conditions, HOS15 continuously eliminates NPR1 to prevent inappropriate defense gene expression. Upon defense activation, HOS15 preferentially associates with phosphorylated NPR1 to stimulate rapid degradation of transcriptionally active NPR1 and thus limit the extent of defense gene expression. Our findings indicate that HOS15-mediated ubiquitination and elimination of NPR1 produce effects contrary to those of CUL3-containing ubiquitin ligase that coactivate defense gene expression. Thus, HOS15 plays a key role in the dynamic regulation of pre- and postactivation host defense.

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Gil-Bernabé, Ana M., Francisco Romero, M. Cristina Limón-Mortés, and María Tortolero. "Protein Phosphatase 2A Stabilizes Human Securin, Whose Phosphorylated Forms Are Degraded via the SCF Ubiquitin Ligase." Molecular and Cellular Biology 26, no. 11 (June 1, 2006): 4017–27. http://dx.doi.org/10.1128/mcb.01904-05.

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ABSTRACT Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.

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McGinnis, Karen M., Stephen G. Thomas, Jonathan D. Soule, Lucia C. Strader, Janice M. Zale, Tai-ping Sun, and Camille M. Steber. "The Arabidopsis SLEEPY1 Gene Encodes a Putative F-Box Subunit of an SCF E3 Ubiquitin Ligase." Plant Cell 15, no. 5 (April 10, 2003): 1120–30. http://dx.doi.org/10.1105/tpc.010827.

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Mfarej, Michael G., and Robert V. Skibbens. "DNA damage induces Yap5-dependent transcription of ECO1/CTF7 in Saccharomyces cerevisiae." PLOS ONE 15, no. 12 (December 29, 2020): e0242968. http://dx.doi.org/10.1371/journal.pone.0242968.

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Yeast Eco1 (ESCO2 in humans) acetyltransferase converts chromatin-bound cohesins to a DNA tethering state, thereby establishing sister chromatid cohesion. Eco1 establishes cohesion during DNA replication, after which Eco1 is targeted for degradation by SCF E3 ubiquitin ligase. SCF E3 ligase, and sequential phosphorylations that promote Eco1 ubiquitination and degradation, remain active throughout the M phase. In this way, Eco1 protein levels are high during S phase, but remain low throughout the remaining cell cycle. In response to DNA damage during M phase, however, Eco1 activity increases—providing for a new wave of cohesion establishment (termed Damage-Induced Cohesion, or DIC) which is critical for efficient DNA repair. To date, little evidence exists as to the mechanism through which Eco1 activity increases during M phase in response to DNA damage. Possibilities include that either the kinases or E3 ligase, that target Eco1 for degradation, are inhibited in response to DNA damage. Our results reveal instead that the degradation machinery remains fully active during M phase, despite the presence of DNA damage. In testing alternate models through which Eco1 activity increases in response to DNA damage, the results reveal that DNA damage induces new transcription of ECO1 and at a rate that exceeds the rate of Eco1 turnover, providing for rapid accumulation of Eco1 protein. We further show that DNA damage induction of ECO1 transcription is in part regulated by Yap5—a stress-induced transcription factor. Given the role for mutated ESCO2 (homolog of ECO1) in human birth defects, this study highlights the complex nature through which mutation of ESCO2, and defects in ESCO2 regulation, may promote developmental abnormalities and contribute to various diseases including cancer.

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Illert, Anna L., Florian Bassermann, Christine von Klitzing, Petra Seipel, Stephan W. Morris, Christian Peschel, and Justus Duyster. "Multisite Phosphorylation of NIPA at G2/M." Blood 110, no. 11 (November 16, 2007): 3348. http://dx.doi.org/10.1182/blood.v110.11.3348.3348.

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Abstract The regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome, with the F-Box subunit of the SCF specifically recruiting a given substrate to the SCF core. We previously reported the cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, which contributes to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein (FBP) that defines an oscillating ubiquitin E3 ligase. The SCFNIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1. Here, we identify the region of NIPA that mediates binding to its substrate cyclin B1. In addition to the recently described serine residue 354, we specify 2 new residues, Ser-359 and Ser-395, implicated in the phosphorylation process at G2M within this region. Moreover, we found cyclin B1/Cdk1 to phosphorylate NIPA at Ser-395 in mitosis. Mutation of both Ser-359 and Ser-395 impaired effective inactivation of the SCFNIPA complex, resulting in reduced levels of mitotic cyclin B1. Furthermore, we aimed to identify the kinases involved in the initial phosphorylation of Ser-345. Therefore, we tested a panel of different kinases active at the G2M transition such as GSK3?, Casein kinase 2, PLK-1 and Erk1. Effective in vitro phosphorylation of NIPA could only be demonstrated with Erk-1. Moreover, we demonstrate an interaction of Erk-1 and NIPA at G2M but not in interphase cells. Binding of Erk-1 and NIPA led to phosphorylation at Ser-354 in vivo and could be blocked by the MEK-1/MEK-2 inhibitor PD98059. Together these data suggest a process of sequential phosphorylation where NIPA is initially phosphorylated by Erk-1 leading to the dissociation of NIPA from the SCF core complex. Once Ser-354 is phosphorylated, cyclin B1/CDK1 amplifies phosphorylation of NIPA, thus contributing to the regulation of its own abundance in early mitosis. In ALK positive lymphomas enhanced phosphorylation of NIPA at Ser 354 can be observed. We demonstrate that NPM-ALK leads to the activation of Erk-1, thereby phosphorylating and inactivating the SCFNIPA E3 ligase. Inactivation of SCFNIPA may have an important impact on the cell cycle turnover of lymphoma cells and thus for the pathogenesis of NPM-ALK induced lymphomas.

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Journal articles on the topic 'SCF E3 ubiquitin ligase'

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