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Targeted protein degradation with Proteolysis Targeting Chimeras (PROTACs) is a powerful therapeutic modality for eliminating disease-causing proteins through targeted ubiquitination and proteasome-mediated degradation. Most PROTACs have exploited substrate receptors of Cullin-RING E3 ubiquitin ligases such as cereblon and VHL. Whether core, shared, and essential components of the Cullin-RING E3 ubiquitin ligase complex can be used for PROTAC applications remains less explored. Here, we discovered a cysteine-reactive covalent recruiter EN884 against the SKP1 adapter protein of the SKP1-CUL1-F-box containing SCF complex. We further showed that this recruiter can be used in PROTAC applications to degrade neo-substrate proteins such as BRD4 and the androgen receptor in a SKP1- and proteasome-dependent manner. Our studies demonstrate that core and essential adapter proteins within the Cullin-RING E3 ubiquitin ligase complex can be exploited for targeted protein degradation applications and that covalent chemoproteomic strategies can enable recruiter discovery against these targets., Competing Interests: Competing Financial Interests Statement OWH was an employee of Bristol Myers Squibb when this study was initiated, but is now an employee of Lyterian Therapeutics. IEW was an employee of Bristol Myers Squibb when this study was initiated but is now a co-founder and the CEO of Lyterian Therapeutics. IEW is on the Scientific Advisory Boards of PAIVBio and Firefly Biologics. DKN is a co-founder, shareholder, and scientific advisory board member for Frontier Medicines and Vicinitas Therapeutics. DKN is a member of the board of directors for Vicinitas Therapeutics. DKN is also on the scientific advisory board of The Mark Foundation for Cancer Research, Photys Therapeutics, and Apertor Pharmaceuticals. DKN is also an Investment Advisory Partner for a16z Bio, an Advisory Board member for Droia Ventures, and an iPartner for The Column Group.

Through catalyzing the ubiquitination of key regulatory proteins, cullin-RING ubiquitin ligases (CRLs) play essential biological roles and their activities are controlled by multiple mechanisms including neddylation, the conjugation of NEDD8 to cullins. Upon neddylation, a CRL, such as the CUL1-based CRL1, undergoes conformational changes that accelerate substrate ubiquitination. Given the structural diversity across subfamilies of CRLs and their substrates, to what extent neddylation modulates the activity of individual CRLs remains to be evaluated. Here, through reconstituting the CRL2 ubiquitination reaction in vitro, we showed that neddylation promotes CRL2VHL -dependent degradation of both full-length HIF1α and the degron peptide of HIF1α, resulting in more than 10-fold increase in the rate of substrate ubiquitination. Consistently, pevonedistat (also known as MLN4924), an inhibitor of neddylation, inhibits the degradation of HIF1α in RCC4 cells stably expressing VHL in cycloheximide chase assays. However, such inhibitory effect of pevonedistat on HIF1α degradation was not observed in HEK293 cells, which was further found to be due to CRL2VHL -independent degradation that was active in HEK293 but not RCC4 cells. After truncating HIF1α to its Carboxy-terminal Oxygen-Dependent Degradation (CODD) domain, we showed that pevonedistat inhibited the degradation of CODD and increased its half-life by six-fold in HEK293 cells. Our results demonstrate that neddylation plays a significant role in activating CRL2, and the cellular activity of CRL2VHL is better reflected by the degradation of CODD than that of HIF1α, especially under conditions where CRL2-independent degradation of HIF1α is active. This article is protected by copyright. All rights reserved.

Thirty years of research have revealed the fundamental role of the ubiquitin-proteasome system in diverse aspects of cellular regulation in eukaryotes. The ubiquitin-protein ligases or E3s are central to the ubiquitin-proteasome system since they determine the specificity of ubiquitylation. The cullin-RING ligases (CRLs) constitute one large class of E3s that can be subdivided based on the cullin isoform and the substrate adapter. SCF complexes, composed of CUL1 and the SKP1/F-box protein substrate adapter, are perhaps the best characterized in plants. More recently, accumulating evidence has demonstrated the essential roles of CRL3 E3s, consisting of a CUL3 protein and a BTB/POZ substrate adaptor. In this Review, we describe the variety of CRL3s functioning in plants and the wide range of processes that they regulate. Furthermore, we illustrate how different classes of E3s may cooperate to regulate specific pathways or processes.

Fusarium oxysporum causes significant economic losses in many crop plants by causing root rot, necrosis, and wilting symptoms. Homology and molecular dynamics studies are promising tools for the detection in F. oxysporum of the systemic resistance compound, salicylic acid, for control of the SKP1-CUL1-F-box protein complex. The structure of SKP1-CUL1-F-box subunit Skp1 from F. oxysporum is produced by Modeler 9v7 for the conduct of docking studies. The Skp1 structure is based on the yeast Cdc4/Skp1 (PDB ID: 3MKS A) crystal structure collected by the Protein data bank. Applying molecular dynamic model simulation methods to the final predicted structure and further evaluated by 3D and PROCHECK test programmers, the final model is verified to be accurate. Applying GOLD 3.0.1, SCF Complex Skp1 is used to prevent stress-tolerant operation. The SKP1-CUL1-F-box model is predicted to be stabilized and tested as a stable docking structure. The predicted model of the SCF structure has been stabilized and confirmed to be a reliable structure for docking studies. The results indicated that GLN8, LYS9, VAL10, TRP11, GLU48, ASN49 in SCF complex are important determinant residues in binding as they have strong hydrogen bonding with salicylic acid, which showed best docking results with SKP1-CUL1-F-box complex subunit Skp1 with docking score 25.25KJ/mol. Insilco studies have been used to determine the mode of action of salicylic acid for Fusarium control. Salicylic acid hinders the SKP1-CUL1-F-box complex, which is important in protein-like interactions through hydrogen bodings. Results from docking studies have shown that the best energy for SKP1-CUL1-F-box was salicylic acid.Communicated by Ramaswamy H. Sarma.

The SKP1, CUL1, F-box protein (SCF) complex represents a family of 69 E3 ubiquitin ligases that poly-ubiquitinate protein substrates marking them for proteolytic degradation via the 26S proteasome. Established SCF complex targets include transcription factors, oncoproteins and tumor suppressors that modulate cell cycle activity and mitotic fidelity. Accordingly, genetic and epigenetic alterations involving SCF complex member genes are expected to adversely impact target regulation and contribute to disease etiology. To gain novel insight into cancer pathogenesis, we determined the prevalence of genetic and epigenetic alterations in six prototypic SCF complex member genes (SKP1, CUL1, RBX1, SKP2, FBXW7 and FBXO5) from patient datasets extracted from The Cancer Genome Atlas (TCGA). Collectively, ~45% of observed SCF complex member mutations are predicted to impact complex structure and/or function in 10 solid tumor types. In addition, the distribution of encoded alterations suggest SCF complex members may exhibit either tumor suppressor or oncogenic mutational profiles in a cancer type dependent manner. Further bioinformatic analyses reveal the potential functional implications of encoded alterations arising from missense mutations by examining predicted deleterious mutations with available crystal structures. The SCF complex also exhibits frequent copy number alterations in a variety of cancer types that generally correspond with mRNA expression levels. Finally, we note that SCF complex member genes are differentially methylated across cancer types, which may effectively phenocopy gene copy number alterations. Collectively, these data show that SCF complex member genes are frequently altered at the genetic and epigenetic levels in many cancer types, which will adversely impact the normal targeting and timely destruction of protein substrates, which may contribute to the development and progression of an extensive array of cancer types.

Cancer is one of the world's major causes of mortality, and it plays a most important role in the world's declining life expectancy. F-box and WD-40 domain protein 7 (FBXW7), a typical participant of the F-box family of proteins, has been considered as an antitumor protein and one of the maximum deregulated ubiquitin-proteasome system proteins in uterine carcinosarcoma, endometrial clear cell carcinoma and cervical carcinoma with the greatest prevalence of alterations. FBXW7 variants with known clinical significance, as well as nsSNP’s in the F-Box and WD40 domains, were evaluated using functionality prediction web resources. Upon analysing the seventy-three deleterious nsSNP’s impact on protein stability and function, we identified that forty-one nsSNP’s of WD40 domain and three of F-Box domain imply decreased stability of the FBXW7 structure. Next to TP53 and PTEN, FBXW7 was reported with the highest percentage of arginine substitution among mutations related to cancer. The current research concentrated on two arginine residue locations (Arg465, Arg505) within the WD40-repeat domain, which is vital for substrate binding. Computational analysis revealed that significant deviation in stability and structural configuration of mutants R505L, R465H, R465P, R505G, R505C, R465C R505S and R505L structures. Protein–protein interaction network of FBXW7 populated with promising hub proteins NOTCH1, c-Myc, CCNE1, STYX, KLG5, SREB1, NFKB2, SKP1, CUL1, thus alteration in the FBXW7 leads to aberration in their signalling pathways as well as their substrate binding ability makes this protein as attractive target for personalized therapeutic intervention.

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.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein‐specific receptor for substrate in the SKP1/CUL1/F‐box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin‐mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar‐binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin‐mediated xenophagy against GAS. Thus, SCF(FBXO2) recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

The Hedgehog (Hh) signaling pathway is crucial for the development of vertebrate and invertebrate animals alike. Hh ligand binds its receptor Patched (Ptc), allowing the activation of the obligate signal transducer Smoothened (Smo). The levels and localizations of both Ptc and Smo are regulated by ubiquitination, and Smo is under additional regulation by phosphorylation and SUMOylation. Downstream of Smo, the Ci/Gli family of transcription factors regulates the transcriptional responses to Hh. Phosphorylation, ubiquitination and SUMOylation are important for the stability and localization of Ci/Gli proteins and Hh signaling output. Finally, Suppressor of Fused directly regulates Ci/Gli proteins and itself is under proteolytic regulation that is critical for normal Hh signaling.

Chemical control of cullin neddylation is attracting increased attention based largely on the successes of the NEDD8-activating enzyme (E1) inhibitor Pevonedistat. Recently reported chemical probes enable selective and time-dependent inhibition of downstream members of the neddylation tri-enzymatic cascade including the co-E3, DCN1. In this work, we report the optimization of a novel class of small molecule inhibitors of the DCN1-UBE2M interaction. Rational X-ray co-structure enabled optimization afforded a 25-fold improvement in potency relative to the initial screening hit. The potency gains are largely attributed to additional hydrophobic interactions mimicking the N-terminal acetyl group that drives binding of UBE2M to DCN1. The compounds inhibit the protein-protein interaction, block NEDD8 transfer in biochemical assays, engage DCN1 in cells, and selectively reduce the steady-state neddylation of Cul1 and Cul3 in two squamous carcinoma cell lines harboring DCN1 amplification.

The Cullin-RING E3 ubiquitin ligase (CRL) complex is the most common E3 ligase, and the SCF complex (CRL1) has the most diverse functions. Cullin1(CUL1) is a scaffolding protein for assembly of the complex. SCF has been shown to participate in the non-biological stress response pathways. In this study, a classic CUL1 protein was identified in Solanum tuberosum, StCUL1. A full-length cDNA of the StCUL1 gene was obtained from ED13 (a potato variety) by Ralstonia solanacearum inoculation using the RACE method. Sequence analysis indicated that the gene comprised 2662 bp, with an open reading frame of 2229 bp encoding 743 amino acids. The expression levels of the StCUL1 gene in potato treated with R. solanacearum and exogenous hormones (such as salicylic acid, jasmonic acid methyl ester and abscisic acid) at different time points were determined by real-time PCR. The results indicated that StCUL1 was induced not only by pathogenic bacteria, but also by exogenous hormones, with sustained high expression. However, there were some differences in the modes of expression. Tissue localization analysis indicated that its expression was tissue specific, and it was mainly in the phloem of the vascular system of stems and leaves.

The AURKA gene, encoding Aurora kinase-A (Aurora-A), is frequently amplified and overexpressed across multiple cancer types correlating with poor prognosis. Although the AURKA gene is frequently amplified in human cancers, underlying mechanism(s) for Aurora-A protein stability through different phases of cell cycle are not well elucidated. Inhibiting the kinase activity and promoting protein degradation are two well-validated conceptual strategies for targeting protein kinases in cancers. Here, we demonstrate that Eukaryotic Elongation Factor 1 Alpha 2 (eEF1A2) facilitates PTEN-GSK3β mediated Aurora-A protein degradation through the SCF complex (SKP1-Cul1-FBXW7) during the S/G2 phase of proliferating cells. In contrast, this mechanism is inactivated in cancer cells accompanying PTEN-GSK3β pathway deficiency. Mechanistically, eEF1A2 interacts with Aurora-A, GSK3β, FBXW7 and Cul1-E3 ligase, as the SCF complex, to facilitate Aurora-A polyubiquitination for 26S proteasomal degradation. eEF1A2 promotes PTEN phosphorylation at T366 and stability, inactivates AKT and activates GSK3β which in turn phosphorylates Aurora-A at S283, S284 and S342. The phosphorylation of Aurora-A at S342 is detected during S/G2 phase of cell mitosis in parallel with eEF1A2-SCF complex formation with active form of GSK3β and neddylated Cul1. Conversely, genetic ablation of EEF1A2 and PTEN, activation of AKT, inhibition of GSK3β, expression of Aurora-A phosphodeficient-mutant attenuates the Aurora-A protein degradation which is corroborated in Aurora-A overexpressing mouse mammary carcinomas and human breast carcinomas. This study identifies a novel mechanism of Aurora-A protein degradation mediated eEF1A2-PTEN-GSK3β pathway and provides a framework for the discovery of Aurora-A therapeutic targets in breast cancer that harbors deficiency of PTEN tumor suppressor pathway. Citation Format: Treekitkarnmongkol W, Solis LM, Kai K, Thompson AM, Tian W, Wistuba II, Sasai K, Jltsumori Y, Sahin AA, Hawke DH, Lee JM, Qin L, Bawa-Khalfe T, Rad R, Wong KK, Abbott CM, Katayama H, Sen S. eEF1A2 facilitates PTEN-GSK3β mediated Aurora-A protein degradation during S-G2 phase inactivated in PTEN-deficient breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P1-05-05.

BackgroundLate-onset preeclampsia (PE) is preeclampsia occurring after 34 weeks of gestational age or later. Cullin 1 (CUL1), a proangiogenic protein, is expressed in the placenta, where an imbalance between proangiogenic and antiangiogenic proteins during gestation can cause disturbance of trophoblast invasion. This defect results in vascular ischemia that may produce preeclampsia. The objective of this study was to determine the correlation between CUL1 as proangiogenic factor and late-onset preeclampsia. MethodsThis study was of analytical observational cross-sectional design and involved 44 preeclampsia patients with ³34 weeks of gestational age (late-onset PE). The CUL1 level in the subjects’ sera, taken before they gave birth, and in homogenates of their placenta, obtained per vaginam or by cesarean section, were examined by the ELISA technique. Statistical analysis was performed with the Spearman correlation test with significant p value of

Activation of the NACHT, leucine-rich repeat, and pyrin domains-containing protein 3 (collectively known as NLRP3) inflammasome plays a key role in host immune response, which is the first line of defense against cellular stresses and pathogen infections. However, excessive inflammasome activation damages host cells, and therefore it must be precisely controlled. Here, we discover that Cullin1 (CUL1), a key component of the Skp1-Cullin1-F-box E3 ligase, plays a critical role in controlling the NLRP3 inflammasome. CUL1 represses inflammasome assembly in cultured cells, suppresses NLRP3 function in human monocytic cell line macrophages, and attenuates inflammatory responses in mouse model. Detailed studies demonstrate that CUL1 interacts with NLRP3 and promotes NLRP3 ubiquitination, but not protein degradation, to repress the NLRP3 inflammasome activation. Moreover, upon inflammatory stimuli, including ATP and nigericin treatments, CUL1 disassociates from NLRP3 to release the repression of the NLRP3 inflammasome. Thus, this study reveals a distinct and unique mechanism underlying the control of systematic activation of the NLRP3 inflammasome.-Wan, P., Zhang, Q., Liu, W., Jia, Y., Ai, S., Wang, T., Wang, W., Pan, P., Yang, G., Xiang, Q., Huang, S., Yang, Q., Zhang, W., Liu, F., Tan, Q., Zhang, W., Wu, K., Liu, Y., Wu, J. Cullin1 binds and promotes NLRP3 ubiquitination to repress systematic inflammasome activation.

Maintaining stable tryptophan levels is required to control neuronal and immune activity. We report that tryptophan homeostasis is largely controlled by the stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme responsible for tryptophan catabolism. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding non-catalytic exosites, resulting in rapid catabolism of tryptophan. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers, and unmasks a 4-amino-acid degron that triggers TDO polyubiquitination by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. The non-metabolizable analog alpha-methyl-tryptophan stabilizes tetrameric TDO, and thereby stably reduces tryptophanemia. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia, as required for both neurological and immune homeostasis.

Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a major gene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ub proteins are covalently linked onto a large array of UPS substrates with the help of three enzymes (E1 activating, E2 conjugating, and E3 ligating enzymes) and direct them for turnover in the 26S proteasome complex. The S-phase Kinase-associated Protein 1 (Skp1), CUL1, F-box (FBX) protein (SCF) complexes have been identified as the largest E3 ligase group in plants due to the dramatic number expansion of the FBX genes in plant genomes. Since it is the FBX proteins that recognize and determine the specificity of SCF substrates, much effort has been done to characterize their genomic, physiological, and biochemical roles in the past two decades of functional genomic studies. However, the sheer size and high sequence diversity of the FBX gene family demands new approaches to uncover unknown functions. In this work, we first identified 82 known FBX members that have been functionally characterized up to date in Arabidopsis thaliana. Through comparing the genomic structure, evolutionary selection, expression patterns, domain compositions, and functional activities between known and unknown FBX gene members, we developed a neural network machine learning approach to predict whether an unknown FBX member is likely functionally active in Arabidopsis, thereby facilitating its future functional characterization.

PurposeEmerging evidence has shown that SKP1-cullin-1-F-box-protein (SCF) E3 ligases contribute to the pathogenesis of different cancers by mediating the ubiquitination and degradation of tumor suppressors. However, the functions of SCF E3 ligases in the pathogenesis of colorectal cancer (CRC) remain obscure. Materials and MethodsThe cancerous and adjacent noncancerous tissues from CRC patients were collected, and protein levels were analyzed. Lentiviral short hairpin RNA (shRNA) and plasmid transfection were used to knock down and overexpress gene expression in CRC cell lines. Immunoprecipitation (IP), mass spectrometry, and co-IP analyses were used to determine protein interactions and the assembly of the SCF complex. Cell proliferation, migration, and tumor xenograft assays were performed to examine the effects of SCF members on CRC cell growth in vitro and in vivo.ResultsHypoxia activated the docking of hypoxia-inducible factor 1α (HIF1α) onto the CUL1 promoter and induced CUL1 expression in CRC cells. CUL1 coupled with RBX1, SKP1, and FBXL1 to assemble the SCFFBXL1 complex in CRC biopsies and cells. The SCFFBXL1 E3 ligase specifically ubiquitinated and degraded the MEN1 tumor suppressor. Knockdown of HIF1α or SCFFBXL1 members, or blockage of SCFFBXL1 by two inhibitors (DT204 and SZLP1-41) caused the accumulation of MEN1 protein and led to a significant decrease in cell proliferation and migration in vitro and tumor growth in vivo.ConclusionThe SCFFBXL1 E3 ligase is required for the ubiquitination of MEN1, and disruption of this complex may represent a new therapeutic strategy for the treatment of CRC.

The ubiquitin-proteasome system is essential for cell cycle progression. Cyclin F is a cell cycle-regulated substrate adapter F-box protein for the Skp1, CUL1, and F-box protein (SCF) family of E3 ubiquitin ligases. Despite its importance in cell cycle progression, identifying cyclin F-bound SCF complex (SCFCyclin F) substrates has remained challenging. Since cyclin F overexpression rescues a yeast mutant in the cdc4 gene, we considered the possibility that other genes that genetically modify cdc4 mutant lethality could also encode cyclin F substrates. We identified the mitochondrial and cytosolic deacylating enzyme sirtuin 5 (SIRT5) as a novel cyclin F substrate. SIRT5 has been implicated in metabolic processes, but its connection to the cell cycle is not known. We show that cyclin F interacts with and controls the ubiquitination, abundance, and stability of SIRT5. We show SIRT5 knockout results in a diminished G1 population and a subsequent increase in both S and G2/M. Global proteomic analyses reveal cyclin-dependent kinase (CDK) signaling changes congruent with the cell cycle changes in SIRT5 knockout cells. Together, these data demonstrate that SIRT5 is regulated by cyclin F and suggest a connection between SIRT5, cell cycle regulation, and metabolism.

Amino acids blood levels are remarkably constant, in spite of fasting periods and large variations in diet supply. Systemic homeostasis is particularly important for tryptophan, given its key role in many cellular processes, including immunity and neuronal activity. In humans, tryptophanemia is maintained around 60µM. Here, we showed that tryptophan homeostasis is largely controlled by the post-translational stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme catalyzing the first step of tryptophan catabolism along the kynurenine pathway. TDO, located at the strategic crossroad of the portal and systemic circulation, degrades more than 90% of food-derived tryptophan. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding non-catalytic exosites, promoting rapid blood tryptophan catabolism. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers, and unmasks a degron. This triggers the polyubiquitination of TDO by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. Artificial stabilization of tetrameric TDO with the non-metabolizable analog alpha-methyl-tryptophan in vivo stably reduces blood tryptophan levels. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia, as required for both neurological and immune homeostasis. La concentration plasmatique des acides aminés est remarquablement constante en dépit des variations de nos apports alimentaires. L’homéostasie systémique des acides aminés est particulièrement importante pour le tryptophane, en raison de son rôle clef dans de nombreux processus biologiques, incluant l’immunité et l’activité neuronale. Chez l’homme, la tryptophanémie est en permanence maintenue à 60µM. Nos découvertes révèlent que l’homéostasie du tryptophane est majoritairement contrôlée par la stabilité post-traductionnelle de la tryptophane 2,3-dioxygenase (TDO), l’enzyme hépatique catalysant la première réaction du catabolisme du tryptophane dans la voie métabolique de la kynurénine. Localisée au carrefour de l’artère hépatique et de la veine porte, la protéine TDO occupe une position stratégique pour filtrer le sang et dégrade plus de 90% du tryptophane d’origine alimentaire. Lorsque la tryptophanémie augmente, le tryptophane active la protéine TDO en stabilisant sa conformation tétramérique au moyen de quatre exosites non catalytiques, promouvant ainsi le catabolisme du tryptophane plasmatique. Une fois la tryptophanémie faible, le tryptophane libère les exosites et provoque la déstabilisation du tétramère en dimères et en monomères inactifs, libérant ainsi un motif de dégradation ou dégron reconnu par des ubiquitines ligases SKP1-CUL1-F-box. Elles déclenchent alors la polyubiquitination de TDO, laquelle conduit à la dégradation de l’enzyme par le protéasome et à l’arrêt du catabolisme du tryptophane. In vivo, la stabilisation de la protéine TDO hépatique avec un analogue non métabolisable, l’alpha-méthyl-tryptophane, réduit de manière permanente la concentration plasmatique du tryptophane. Nos résultats décrivent un mécanisme d’adaptation rapide du catabolisme du tryptophane permettant une dégradation de l’acide aminé en excès et prévenant son catabolisme en dessous de sa teneur physiologique. Cette boucle de rétrocontrôle assure une régulation fine de la tryptophanémie dont le maintien est nécessaire à l’homéostasie neurologique et immunitaire. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

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’.

Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence-based techniques in ubiquitination studies remain underexplored at least partly because of challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, FRET diubiquitination, to track substrate ubiquitination by fluorescence. This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The diubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET diubiquitination relies on Ub–substrate fusion, which can be implemented using either one of the two validated strategies. Method 1 is the use of recombinant substrate–Ub fusion, applicable to all substrate peptides that can bind to E3. Method 2 is a chemoenzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based diubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.

The identification and functional characterization of natural variants in plants are essential for understanding phenotypic adaptation. Here we identify a molecular variation in At2g47310 that contributes to the natural variation in flowering time in Arabidopsis thaliana accessions. This gene, which we term SISTER of FCA (SSF), functions in an antagonistic manner to its close homolog FCA. Genome-wide association analysis screens two major haplotypes of SSF associated with the natural variation in FLC expression, and a single polymorphism, SSF-N414D, is identified as a main contributor. The SSF414N protein variant interacts more strongly with CUL1, a component of the E3 ubiquitination complex, than the SSF414D form, mediating differences in SSF protein degradation and FLC expression. FCA and SSF appear to have arisen through gene duplication after dicot-monocot divergence, with the SSF-N414D polymorphism emerging relatively recently within A. thaliana. This work provides a good example for deciphering the functional importance of natural polymorphisms in different organisms., Natural variation represents valuable source for gene discovery. Here, the authors show that a homolog of Flowering Control Locus A (FCA) functions in an antagonistic manner to FCA in regulating Arabidopsis flowering time through interacting with CUL1-E3 and modulating FLC expression.

Transcription is regulated through a dynamic interplay of DNA-associated proteins, and the composition of gene-regulatory complexes is subject to continuous adjustments. Protein alterations include post-translational modifications and elimination of individual polypeptides. Spatially and temporally controlled protein removal is, therefore, essential for gene regulation and accounts for the short half-life of many transcription factors. The ubiquitin–proteasome system is responsible for site- and target-specific ubiquitination and protein degradation. Specificity of ubiquitination is conferred by ubiquitin ligases. Cullin-RING complexes, the largest family of ligases, require multi-unit assembly around one of seven cullin proteins. To investigate the direct role of cullins in ubiquitination of DNA-bound proteins and in gene regulation, we analyzed their subcellular locations and DNA-affinities. We found CUL4A and CUL7 to be largely excluded from the nucleus, whereas CUL4B was primarily nuclear. CUL1,2,3, and 5 showed mixed cytosolic and nuclear expression. When analyzing chromatin affinity of individual cullins, we discovered that CUL1 preferentially associated with active promoter sequences and co-localized with 23% of all DNA-associated protein degradation sites. CUL1 co-distributed with c-MYC and specifically repressed nuclear-encoded mitochondrial and splicing-associated genes. These studies underscore the relevance of spatial control in chromatin-associated protein ubiquitination and define a novel role for CUL1 in gene repression.

F-box proteins, as substrates for S phase kinase-associated protein 1 (SKP1)-cullin 1 (CUL1)-F-box protein (SCF) ubiquitin ligase complexes, mediate the degradation of a large number of regulatory proteins involved in cancer processes. In this study, we found that F-box only protein 2 (FBXO2) was up-regulated in 21 endometrial carcinoma (EC) samples compared with five normal endometrium samples based on our Fudan cohort RNA-sequencing. The increased FBXO2 expression was associated with tumor stage, tumor grade, and histologic tumor type, and poor prognosis based on The Cancer Genome Atlas (TCGA) database. FBXO2 knockdown inhibited EC cell proliferation, and FBXO2 overexpression promoted the parental cell phenotype in vivo and in vitro. Fibrillin1 (FBN1) was also identified as a substrate for FBXO2 using a ubiquitination-proteome approach. In addition, promotion of EC proliferation by FBXO2 was regulated by specific proteins of the cell cycle (CDK4, CyclinD1, CyclinD2, and CyclinA1) and the autophagy signaling pathway (ATG4A and ATG4D) based on RNA sequencing (RNA-seq). We concluded that FBXO2 acts as an E3 ligase that targets FBN1 for ubiquitin-dependent degradation, so as to promote EC proliferation by regulating the cell cycle and the autophagy signaling pathway. Targeting FBXO2 may represent a potential therapeutic target for EC.

Sterile alpha motif (SAM) and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) acts as a restriction factor for several RNA and DNA viruses by limiting the intracellular pool of deoxynucleoside triphosphates. Here, we investigated the regulation of SAMHD1 expression during human cytomegalovirus (HCMV) infection. SAMHD1 knockdown using shRNA increased the activity of the viral UL99 late gene promoter in human fibroblasts by 7- to 9-fold, confirming its anti-HCMV activity. We also found that the level of SAMHD1 was initially increased by HCMV infection but decreased partly at the protein level at late stages of infection. SAMHD1 loss was not observed with UV-inactivated virus and required viral DNA replication. This reduction of SAMHD1 was effectively blocked by MLN4924, an inhibitor of the Cullin-RING-E3 ligase (CRL) complexes, but not by bafilomycin A1, an inhibitor of vacuolar-type H+-ATPase. Indirect immunofluorescence assays further supported the CRL-mediated SAMHD1 loss at late stages of virus infection. Knockdown of CUL2 and to a lesser extent CUL1 using siRNA stabilized SAMHD1 in normal fibroblasts and inhibited SAMHD1 loss during virus infection. Altogether, our results demonstrate that SAMHD1 inhibits the growth of HCMV, but HCMV causes degradation of SAMHD1 at late stages of viral infection through the CRL complexes.

Funder: Wildy Fellowship Department of Pathology, Funder: Addenbrooke's Charitable Trust, Cambridge University Hospitals; doi: http://dx.doi.org/10.13039/501100002927, Funder: The Mark Foundation, In cancer, many genes are mutated by genome rearrangement, but our understanding of the functional consequences of this remains rudimentary. Here we report the F-box protein encoded by FBXL17 is disrupted in the region of the gene that encodes its substrate-binding leucine rich repeat (LRR) domain. Truncating Fbxl17 LRRs impaired its association with the other SCF holoenzyme subunits Skp1, Cul1 and Rbx1, and decreased ubiquitination activity. Loss of the LRRs also differentially affected Fbxl17 binding to its targets. Thus, genomic rearrangements in FBXL17 are likely to disrupt SCFFbxl17-regulated networks in cancer cells. To investigate the functional effect of these rearrangements, we performed a yeast two-hybrid screen to identify Fbxl17-interacting proteins. Among the 37 binding partners Uap1, an enzyme involved in O-GlcNAcylation of proteins was identified most frequently. We demonstrate that Fbxl17 binds to UAP1 directly and inhibits its phosphorylation, which we propose regulates UAP1 activity. Knockdown of Fbxl17 expression elevated O-GlcNAcylation in breast cancer cells, arguing for a functional role for Fbxl17 in this metabolic pathway.

Background High-grade serous ovarian cancer (HGSOC) is the most common and lethal ovarian cancer histotype. Chromosome instability (CIN, an increased rate of chromosome gains and losses) is believed to play a fundamental role in the development and evolution of HGSOC. Importantly, overexpression of Cyclin E1 protein induces CIN, and genomic amplification of CCNE1 contributes to HGSOC pathogenesis in ~20% of patients. Cyclin E1 levels are normally regulated in a cell cycle-dependent manner by the SCF (SKP1–CUL1–FBOX) complex, an E3 ubiquitin ligase that includes the proteins SKP1 and CUL1. Conceptually, diminished SKP1 or CUL1 expression is predicted to underlie increases in Cyclin E1 levels and induce CIN. Methods This study employs fallopian tube secretory epithelial cell models to evaluate the impact diminished SKP1 or CUL1 expression has on Cyclin E1 and CIN in both short-term (siRNA) and long-term (CRISPR/Cas9) studies. Results Single-cell quantitative imaging microscopy approaches revealed changes in CIN-associated phenotypes and chromosome numbers and increased Cyclin E1 in response to diminished SKP1 or CUL1 expression. Conclusions These data identify SKP1 and CUL1 as novel CIN genes in HGSOC precursor cells that may drive early aetiological events contributing to HGSOC development.