SCF Complex Research Articles

Arabidopsis AT2G02870 Loss of Function Mutants Lead to Enhanced Production of Hydroperoxide Lyase Pathway Genes and Products

F-box proteins containing variable C-terminal domains make an essential part of SKP1-Cullin-Ring box-F box (SCF) complex. SCF complex catalyzes the final step to link the ubiquitin tag with the target protein, destined for degradation, through F-box protein that confer overall substrate specificity to the complex. In this study, we analyzed the role of At2g02870, a Kelch containing F-box protein from Arabidopsis thaliana, by using reverse genetics strategy. At2g02870 loss of function mutant lines (at2g02870) were analyzed and compared with wild type plants for the expression of genes and products of hydroperoxide lyase (HPL) branch of oxylipin pathway. We found that the at2g02870 plants have enhanced expression of HPL pathway genes and produce more green leaf volatiles (GLV) than the wild type plants. Our results suggested that the gene is involved in the regulation of HPL pathway, possibly through the degradation of enzymes or/and the regulatory factors of the pathway.

The F-Box Domain-Dependent Activity of EMI1 Regulates PARPi Sensitivity in Triple-Negative Breast Cancers

The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.

Biallelic variants in FBXL3 cause intellectual disability, delayed motor development and short stature.

FBXL3 (F-Box and Leucine Rich Repeat Protein 3) encodes a protein that contains an F-box and several tandem leucine-rich repeats (LRR) domains. FBXL3 is part of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complex that binds and leads to phosphorylation-dependent degradation of the central clock protein cryptochromes (CRY1 and CRY2) by the proteasome and its absence causes circadian phenotypes in mice and behavioral problems. No FBXL3-related phenotypes have been described in humans. By a combination of exome sequencing and homozygosity mapping, we analyzed two consanguineous families with intellectual disability and identified homozygous loss-of-function (LoF) variants in FBXL3. In the first family, from Pakistan, an FBXL3 frameshift variant [NM_012158.2:c.885delT:p.(Leu295Phefs*25)] was the onlysegregating variant in five affected individuals in two family loops (LOD score: 3.12). In the second family, from Lebanon, we identified a nonsense variant [NM_012158.2:c.445C>T:p.(Arg149*)]. In a third patient from Italy, a likely deleterious non-synonymous variant [NM_012158.2:c.1072T>C:p.(Cys358Arg)] was identified in homozygosity. Protein 3D modeling predicted that the Cys358Arg change influences the binding with CRY2 by destabilizing the structure of the FBXL3, suggesting that this variant is also likely to be LoF. The eight affected individuals from the three families presented with a similar phenotype that included intellectual disability, developmental delay, short stature and mild facial dysmorphism, mainly large nose with a bulbous tip. The phenotypic similarity and the segregation analysis suggest that FBXL3 biallelic, LoF variants link this gene with syndromic autosomal recessive developmental delay/intellectual disability.

Proteasome lid bridges mitochondrial stress with Cdc53/Cullin1 NEDDylation status

Cycles of Cdc53/Cullin1 rubylation (a.k.a NEDDylation) protect ubiquitin-E3 SCF (Skp1-Cullin1-F-box protein) complexes from self-destruction and play an important role in mediating the ubiquitination of key protein substrates involved in cell cycle progression, development, and survival. Cul1 rubylation is balanced by the COP9 signalosome (CSN), a multi-subunit derubylase that shows 1:1 paralogy to the 26S proteasome lid. The turnover of SCF substrates and their relevance to various diseases is well studied, yet, the extent by which environmental perturbations influence Cul1 rubylation/derubylation cycles per se is still unclear. In this study, we show that the level of cellular oxidation serves as a molecular switch, determining Cullin1 rubylation/derubylation ratio. We describe a mutant of the proteasome lid subunit, Rpn11 that exhibits accumulated levels of Cullin1-Rub1 conjugates, a characteristic phenotype of csn mutants. By dissecting between distinct phenotypes of rpn11 mutants, proteasome and mitochondria dysfunction, we were able to recognize the high reactive oxygen species (ROS) production during the transition of cells into mitochondrial respiration, as a checkpoint of Cullin1 rubylation in a reversible manner. Thus, the study adds the rubylation cascade to the list of cellular pathways regulated by redox homeostasis.

The critical role of AMPK in driving Akt activation under stress, tumorigenesis and drug resistance

PI3K/Akt signaling is activated in cancers and governs tumor initiation and progression, but how Akt is activated under diverse stresses is poorly understood. Here we identify AMPK as an essential regulator for Akt activation by various stresses. Surprisingly, AMPK is also activated by growth factor EGF through Ca2+/Calmodulin-dependent kinase and is essential for EGF-mediated Akt activation and biological functions. AMPK phosphorylates Skp2 at S256 and promotes the integrity and E3 ligase activity of Skp2 SCF complex leading to K63-linked ubiquitination and activation of Akt and subsequent oncogenic processes. Importantly, AMPK-mediated Skp2 S256 phosphorylation promotes breast cancer progression in mouse tumor models, correlates with Akt and AMPK activation in breast cancer patients, and predicts poor survival outcomes. Finally, targeting AMPK-mediated Skp2 S256 phosphorylation sensitizes cells to anti-EGF receptor targeted therapy. Our study sheds light on how stress and EGF induce Akt activation and new mechanisms for AMPK-mediated oncogenesis and drug resistance.

Bmi1 Regulates IκBα Degradation via Association with the SCF Complex.

Bmi1 is a polycomb group protein and regulator that stabilizes the ubiquitination complex PRC1 in the nucleus with no evidently direct link to the NF-κB pathway. In this study, we report a novel function of Bmi1: its regulation of IκBα ubiquitination in the cytoplasm. A deficiency of Bmi1 inhibited NF-κB-mediated gene expression in vitro and a NF-κB-mediated mouse model of arthritis in vivo. Mechanistic analysis showed that Bmi1 associated with the SCF ubiquitination complex via its N terminus and with phosphorylation by an IKKα/β-dependent pathway, leading to the ubiquitination of IκBα. These effects on NF-κB-related inflammation suggest Bmi1 in the SCF complex is a potential therapeutic target for various diseases and disorders, including autoimmune diseases.

Proteasome activity determines pupation timing through the degradation speed of timer molecule Blimp-1.

The transcriptional repressor Blimp-1 is a labile protein. This characteristic is key for determining pupation timing because the timing of the disappearance of Blimp-1 affects pupation timing by regulating the expression of its target βftz-f1. However, the molecular mechanisms that regulate the protein turnover of Blimp-1 are still unclear. Here, we demonstrate that Blimp-1 is regulated by the ubiquitin proteasome system. We show that Blimp-1 degradation is inhibited by proteasome inhibitor MG132. Pupation timing was delayed in mutants of 26S proteasome subunits as well as FBXO11, which recruits target proteins to the 26S proteasome as a component of the SCF ubiquitin ligase complex by slowing down the degradation speed of Blimp-1. Delay in pupation timing in the FBXO11 mutant was suppressed by the induction of βFTZ-F1. Furthermore, fat-body-specific knockdown of proteasomal activity was sufficient to induce a delay in pupation timing. These results suggest that Blimp-1 is degraded by the 26S proteasome and is recruited by FBXO11 in the fat body, which is important for determining pupation timing.

In vitro effects of FBXW7 mutation in serous endometrial cancer: Increased levels of potentially druggable proteins and sensitivity to SI-2 and dinaciclib.

Serous endometrial cancers (ECs) are clinically aggressive tumors that frequently harbor somatic mutations in FBXW7 (F-box and WD repeat domain-containing 7). The FBXW7 tumor suppressor is part of a SCF (complex of SKP1, Cullin 1, F-box protein) ubiquitin ligase complex which controls the degradation of numerous substrates that, if not properly regulated, can contribute to the initiation or progression of tumorigenesis. Despite reports that up to 30% of serous ECs include somatic mutations in FBXW7, the molecular effects of mutated FBXW7 in ECs have not been determined. Here, we used transient transfection and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) editing in serous EC cell lines to interrogate the molecular effects of six recurrent FBXW7 mutations. We show that FBXW7 mutations lead to increased Cyclin E1, steroid receptor coactivator 3 (SRC-3), c-MYC, Rictor, glycogen synthase kinase 3 (GSK3), P70S6 kinase, and protein kinase B (AKT) phosphorylated protein levels in serous EC cells. Furthermore, we demonstrate that CRISPR-edited FBXW7-mutant ARK1 serous EC cells exhibit increased sensitivity to SI-2 (a SRC inhibitor) and dinaciclib (a cyclin dependent kinase (CDK) inhibitor) compared to parental ARK1 cells. Collectively, our findings reveal biochemical effects of FBXW7 mutations in the context of EC and provide in vitro evidence of sensitivity to targeted inhibitors.

Abstract 1913: Investigation of targeting SKP2 to improve the treatment response in glioma

Abstract Glioma is the most frequent tumor in central nervous system (CNS), constituting about 30% of CNS tumors and 80% of malignant brain tumors. The current standard strategy for glioma treatment is surgery plus adjuvant chemotherapy and concurrent radiotherapy. However, the prognosis of glioma is still poor. It has been proved that the existence of glioma stem cells and the timely repair of DNA damages are the main causes of treatment failure. Skp2 (S-phase kinase associated protein-2), a member of F-box proteins, forms the Skp2 SCF complex with Skp1, Cullin-1, and Rbx1, serves as one of the important E3-ligases in the process of ubiquitination, and is responsible for substrate recognition. Skp2 is overexpressed in a variety of human cancers and promotes cancer progression by inducing p27 degradation. Importantly, Skp2 deficiency profoundly restricts cancer progression in multiple genetic mouse tumor models. Our group has demonstrated that Skp2 is involved in the regulation of pool size and self-renewal ability of hematopoietic stem cells, as well as breast cancer stem cells and nasopharyngeal carcinoma stem cell-like cells. However, the exact role of Skp2 in the regulation of glioma stem cells and DNA damage repair in glioma has not been elucidated yet. We proposed that targeting Skp2 could promote radio- and chemotherapy sensitivity through suppressing function of glioma stem cells. We first detected the expression of SKP2 in glioma tumor tissues and found that SKP2 was highly expressed in glioma and related to the prognosis of glioma patients. We also adopted gene knockdown methods and inhibitor pretreatment with small molecules in order to suppress the level of Skp2, and then checked the sphere formation and self-renewal ability of glioma stem cell upon radio/chemo treatments. In this way we evaluated the role of Skp2 in repressing the glioma cell proliferation and subsequently triggering apoptosis. Our project will clarify the novel mechanisms about how Skp2 is involved in the glioma radio- and chemotherapy resistance, which will provide a potential target to improve the radio/chemo therapeutic effect through targeting one single target. Citation Format: Jing Wang, Hong-Kai Su, Zhong-Ping Chen, Yue Qu, Hai-Ping Cai. Investigation of targeting SKP2 to improve the treatment response in glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1913.

Arabidopsis SKP1-like protein13 (ASK13) positively regulates seed germination and seedling growth under abiotic stress.

SKP1 (S-phase kinase-associated protein1) proteins are key members of the SCF (SKP-cullin-F-box protein) E3 ligase complexes that ubiquitinate target proteins and play diverse roles in plant biology. However, in comparison with other members of the SCF complex, knowledge of SKP1-like proteins is very limited in plants. In the present work, we report that Arabidopsis SKP1-like protein13 (ASK13) is differentially regulated in different organs during seed development and germination and is up-regulated in response to abiotic stress. Yeast two-hybrid library screening and subsequent assessment of in vivo interactions through bimolecular fluorescence complementation analysis revealed that ASK13 not only interacts with F-box proteins but also with other proteins that are not components of SCF complexes. Biochemical analysis demonstrated that ASK13 not only exists as a monomer but also as a homo-oligomer or heteromer with other ASK proteins. Functional analysis using ASK13 overexpression and knockdown lines showed that ASK13 positively influences seed germination and seedling growth, particularly under abiotic stress. Taken together, our data strongly suggest that apart from participation to form SCF complexes, ASK13 interacts with several other proteins and is implicated in different cellular processes distinct from protein degradation.

SCF\u03b2-TRCP E3 ubiquitin ligase targets the tumor suppressor ZNRF3 for ubiquitination and degradation

Wnt signaling has emerged as a major regulator of tissue development by governing the self-renewal and maintenance of stem cells in most tissue types. As a key upstream regulator of the Wnt pathway, the transmembrane E3 ligase ZNRF3 has recently been established to play a role in negative regulation of Wnt signaling by targeting Frizzled (FZD) receptor for ubiquitination and degradation. However, the upstream regulation of ZNRF3, in particular the turnover of ZNRF3, is still unclear. Here we report that ZNRF3 is accumulated in the presence of proteasome inhibitor treatment independent of its E3-ubiquitin ligase activity. Furthermore, the Cullin 1-specific SCF complex containing β-TRCP has been identified to directly interact with and ubiquitinate ZNRF3 thereby regulating its protein stability. Similar with the degradation of β-catenin by β-TRCP, ZNRF3 is ubiquitinated by β-TRCP in both CKI-phosphorylation- and degron-dependent manners. Thus, our findings not only identify a novel substrate for β-TRCP oncogenic regulation, but also highlight the dual regulation of Wnt signaling by β-TRCP in a context-dependent manner where β-TRCP negatively regulates Wnt signaling by targeting β-catenin, and positively regulates Wnt signaling by targeting ZNRF3.

FBW7 suppresses cell proliferation and G2/M cell cycle transition via promoting γ-catenin K63-linked ubiquitylation

FBW7 is an E3 ubiquitin ligase and frequently mutated in various types of cancer. As a component of SCF ubiquitin ligase complex, FBW7 usually targets the substrates via K11 or K48-linked ubiquitylation and subsequent degradation of target proteins. Nevertheless, the role of FBW7 in mediating non-degradable ubiquitin signaling remains unknown in human cancers. In this study, we identified γ-catenin as a new binding protein of FBW7 by TAP-MS (tandem affinity purification-mass spectrum). Knockdown of FBW7 did not affect the stability of γ-catenin, but significantly reduced the K63-linked ubiquitin of γ-catenin, resulting in decreased expression of γ-catenin downstream gene 14-3-3σ. Rescue experiment revealed that γ-catenin promoted the expression of 14-3-3σ in a K63-linked ubiquitin signaling dependent manner. Furthermore, we showed that FBW7 cooperated with γ-catenin to inhibit G2/M cell cycle transition and cell proliferation. Taken together, our study uncovered a novel mechanism that FBW7 associated with γ-catenin and promoted its K63-linked ubiquitylation, providing new insights in understanding the role of FBW7 in inhibiting G2/M cell cycle transition and tumor cell proliferation.

The function of replication and SCF complex during Drosophila wing development.

Chromosomal DNA replication machinery functions in the growing cells and organs in multicellular organisms. We previously demonstrated that its knockdown in several tissues of Drosophila led to a rough eye phenotype, the loss of bristles in the eye and female sterile. In this paper, I investigated in detail the wing phenotype using RNAi flies, and observed that the knockdown not only of Mcm10 but also of some other prereplicative complex components including Cdt1, Polα-primase, RPA, Psf2 (partner of SLD five 2; a subunit of GINS (Go, Ichi, Nii, and San; five, one, two, and three in Japanese) and Rfc3 (replication factor C 3; a subunit of RFC complex) demonstrated wing phenotypes, using Gal4-driver flies. Surprisingly, some SCF complex components, which control cell cycle progression via protein degradation, also showed the wing phenotype. These results showed that the DNA replication machinery contributes to wing development independently of growth, probably through defects in DNA replication and protein degradation at specific places and times.

Identification of Targetable Surfaces of Cks1 for Development of New Cancer Therapeutics

Cyclin-dependent protein kinase regulatory subunit 1 (Cks1) is involved in cell cycle progressionthrough interactions with cyclin-dependent kinases (CDK) and ubiquitination of cyclin-dependentkinase inhibitors (CKI). Dysfunction of CDK dependent associations can affect the entrance of acell into mitosis, particularly the G1-S phase transition. Abnormal assistance from Cks1 with themultiprotein complex SCF (Skp2) in the ubiquitination of CDKN1B (p27Kip1) can disrupt the mitoticregulatory protein levels escalating to cancer development. In this study, we use computationalmethods to investigate interactions in three different complexes sharing Cks1 in order to create atargeted pharmaceutical solution that will assist in regulation. Our analysis is performed using thecrystal structure of Cks1 in three complexes to include involvement between one ubiquitin ligaseand two CDKs. The assessment is based on the intermolecular electrostatic interactions, such ashydrogen bonds and charge-charge interactions. We observe that charge and hydrogen bonding playsa significant role in the stability between Cks1 and adjacent proteins in each complex. Due to largebinding interfaces and varied distribution of charge across the main contact regions, we decided thata condensed pocket on Cks1 that interacts with phosphorylated p27Kip1 should be selected for furtherin-depth examination.

Glycosylation Promotes the Random Coil to Helix Transition in a Region of a Protist Skp1 Associated with F-Box Binding.

Cullin-ring-ligases mediate protein polyubiquitination, a signal for degradation in the 26S proteasome. The CRL1 class consists of Skp1/cullin-1/F-box protein/Rbx1 (SCF) complexes that cyclically associate with ubiquitin-E2 to build the polyubiquitin chain. Within the SCF complex, the 162-amino acid DdSkp1 from Dictyostelium bridges cullin-1 with an F-box protein (FBP), the specificity factor for substrate selection. The hydroxylation-dependent glycosylation of Pro143 of DdSkp1 by a pentasaccharide forms the basis of a novel O2-sensing mechanism in the social amoeba Dictyostelium and other protists. Previous evidence indicated that glycosylation promotes increased α-helical content correlating with enhanced interaction with three F-box proteins. To localize these differences, we used nuclear magnetic resonance (NMR) methods to compare nonglycosylated DdSkp1 and a glycoform with a single GlcNAc sugar (Gn-DdSkp1). We report NMR assignments of backbone 1HN, 15N, 13Cα, and 13CO nuclei as well as side-chain 13Cβ and methyl 13C/1H nuclei of Ile(δ1), Leu, and Val in both unmodified DdSkp1 and Gn-DdSkp1. The random coil index and 15N{1H} HNOE indicate that the C-terminal region, which forms a helix-loop-helix motif centered on Pro143 at the crystallographically defined binding interface with F-box domains, remains dynamic in both DdSkp1 and Gn-DdSkp1. Chemical shifts indicate that the variation of conformation in Gn-DdSkp1, relative to DdSkp1, is limited to this region and characterized by increased helical fold. Extension of the glycan chain results in further changes, also limited to this region. Thus, glycosylation may control F-box protein interactions via a local effect on DdSkp1 conformation, by a mechanism that may be general to many unicellular eukaryotes.

Characterization and comparative expression analysis of CUL1 genes in rice.

Cullin-RING E3 ubiquitin ligase (CRL) complex is known as the largest family of E3 ligases. The most widely characterized CRL, SCF complex (CRL1), utilizes CUL1 as a scaffold protein to assemble the complex components. To better understand CRL1-mediated cellular processes in rice, three CUL1 genes (OsCUL1s) were isolated in Oryza sativa. Although all OsCUL1 proteins exhibited high levels of amino acid similarities with each other, OsCUL1-3 had a somewhat distinct structure from OsCUL1-1 and OsCUL1-2. Basal expression levels of OsCUL1-3 were much lower than those of OsCUL1-1 and OsCUL1-2 in all selected samples, showing that OsCUL1-1 and OsCUL1-2 play predominant roles relative to OsCUL1-3 in rice. OsCUL1-1 and OsCUL1-2 genes were commonly upregulated in dry seeds and by ABA and salt/drought stresses, implying their involvement in ABA-mediated processes. These genes also showed similar expression patterns in response to various hormones and abiotic stresses, alluding to their functional redundancy. Expression of the OsCUL1-3 gene was also induced in dry seeds and by ABA-related salt and drought stresses, implying their participation in ABA responses. However, its expression pattern in response to hormones and abiotic stresses was somehow different from those of the OsCUL1-1 and OsCUL1-2 genes. Taken together, these findings suggest that the biological role and function of OsCUL1-3 may be distinct from those of OsCUL1-1 and OsCUL1-2. The results of expression analysis of OsCUL1 genes in this study will serve as a useful platform to better understand overlapping and distinct roles of OsCUL1 proteins and CRL1-mediated cellular processes in rice plants.

Ubiquitin conjugating enzyme E2 L3 promoted tumor growth of NSCLC through accelerating p27kip1 ubiquitination and degradation.

The molecular pathogenesis of human lung cancer has not been completely clarified. Here, we reported that UBE2L3, a member of the ubiquitin-conjugating enzymes (E2s), were overexpressed in non-small-cell lung cancer (NSCLC) tissues compared with the non-tumor tissues. High expression of UBE2L3 was correlated with advanced tumor stage and adverse outcomes. Knockdown of UBE2L3 inhibited NSCLC cell growth while ectopic expression of UBE2L3 promoted NSCLC cell growth in a cell cycle dependent manner. The results of subcutaneous tumor xenograft studies revealed that knockdown of UBE2L3 attenuated the in vivo tumor growth. Mechanistically, we observed that UBE2L3 could interact with F-box protein Skp2, a member of the SCF (Skp2) ubiquitin ligase complex, and thus promoted the ubiquitination and proteasomal degradation of p27kip1. Furthermore, NSCLC cases with high level of UBE2L3 and low level of p27kip1 had worst prognosis, suggesting that combination of UBE2L3 and p27kip1 is a more powerful prognostic marker for NSCLC patients. Taken together, the current study presented a novel marker for predicting prognosis and a potential therapeutic target for NSCLC patients.

Ubiquitination of exposed glycoproteins by SCFFBXO27 directs damaged lysosomes for autophagy

Ubiquitination functions as a signal to recruit autophagic machinery to damaged organelles and induce their clearance. Here, we report the characterization of FBXO27, a glycoprotein-specific F-box protein that is part of the SCF (SKP1/CUL1/F-box protein) ubiquitin ligase complex, and demonstrate that SCFFBXO27 ubiquitinates glycoproteins in damaged lysosomes to regulate autophagic machinery recruitment. Unlike F-box proteins in other SCF complexes, FBXO27 is subject to N-myristoylation, which localizes it to membranes, allowing it to accumulate rapidly around damaged lysosomes. We also screened for proteins that are ubiquitinated upon lysosomal damage, and identified two SNARE proteins, VAMP3 and VAMP7, and five lysosomal proteins, LAMP1, LAMP2, GNS, PSAP, and TMEM192. Ubiquitination of all glycoproteins identified in this screen increased upon FBXO27 overexpression. We found that the lysosomal protein LAMP2, which is ubiquitinated preferentially on lysosomal damage, enhances autophagic machinery recruitment to damaged lysosomes. Thus, we propose that SCFFBXO27 ubiquitinates glycoproteins exposed upon lysosomal damage to induce lysophagy.

The ubiquitin-conjugating enzyme CDC34 is essential for cytokinesis in contrast to putative subunits of a SCF complex in Trypanosoma brucei.

The ubiquitin-proteasome system is a post-translational regulatory pathway for controlling protein stability and activity that underlies many fundamental cellular processes, including cell cycle progression. Target proteins are tagged with ubiquitin molecules through the action of an enzymatic cascade composed of E1 ubiquitin activating enzymes, E2 ubiquitin conjugating enzymes, and E3 ubiquitin ligases. One of the E3 ligases known to be responsible for the ubiquitination of cell cycle regulators in eukaryotes is the SKP1-CUL1-F-box complex (SCFC). In this work, we identified and studied the function of homologue proteins of the SCFC in the life cycle of Trypanosoma brucei, the causal agent of the African sleeping sickness. Depletion of trypanosomal SCFC components TbRBX1, TbSKP1, and TbCDC34 by RNAi resulted in decreased growth rate and contrasting cell cycle abnormalities for both procyclic (PCF) and bloodstream (BSF) forms. Depletion of TbRBX1 in PCF cells interfered with kinetoplast replication, whilst depletion of TbSKP1 arrested PCF and BSF cells in the G1/S transition. Silencing of TbCDC34 in BSF cells resulted in a block in cytokinesis and caused rapid clearance of parasites from infected mice. We also show that TbCDC34 is able to conjugate ubiquitin in vitro and in vivo, and that its activity is necessary for T. brucei infection progression in mice. This study reveals that different components of a putative SCFC have contrasting phenotypes once depleted from the cells, and that TbCDC34 is essential for trypanosome replication, making it a potential target for therapeutic intervention.

Rice black streaked dwarf virus P7-2 forms a SCF complex through binding to Oryza sativa SKP1-like proteins, and interacts with GID2 involved in the gibberellin pathway.

As a core subunit of the SCF complex that promotes protein degradation through the 26S proteasome, S-phase kinase-associated protein 1 (SKP1) plays important roles in multiple cellular processes in eukaryotes, including gibberellin (GA), jasmonate, ethylene, auxin and light responses. P7-2 encoded by Rice black streaked dwarf virus (RBSDV), a devastating viral pathogen that causes severe symptoms in infected plants, interacts with SKP1 from different plants. However, whether RBSDV P7-2 forms a SCF complex and targets host proteins is poorly understood. In this study, we conducted yeast two-hybrid assays to further explore the interactions between P7-2 and 25 type I Oryza sativa SKP1-like (OSK) proteins, and found that P7-2 interacted with eight OSK members with different binding affinity. Co-immunoprecipitation assay further confirmed the interaction of P7-2 with OSK1, OSK5 and OSK20. It was also shown that P7-2, together with OSK1 and O. sativa Cullin-1, was able to form the SCF complex. Moreover, yeast two-hybrid assays revealed that P7-2 interacted with gibberellin insensitive dwarf2 (GID2) from rice and maize plants, which is essential for regulating the GA signaling pathway. It was further demonstrated that the N-terminal region of P7-2 was necessary for the interaction with GID2. Overall, these results indicated that P7-2 functioned as a component of the SCF complex in rice, and interaction of P7-2 with GID2 implied possible roles of the GA signaling pathway during RBSDV infection.