Ubiquitin-protein Ligase Complex Research Articles

Elongin B promotes breast cancer progression by ubiquitinating tumor suppressor p14/ARF

Elongin B (ELOB), a pivotal element in the ELOB/c-Cullin2/5-SOCS-box E3 ubiquitin-protein ligase complex, plays a significant role in catalyzing the ubiquitination and subsequent degradation of a broad spectrum of target proteins. Notably, it is documented to facilitate these processes. However, the regulatory role of ELOB in breast cancer remains ambiguous. In this study, through bio-informatic analysis of The Cancer Genome Atlas and Fudan University Shanghai Cancer Center database, we demonstrated that ELOB was over-expressed in breast cancer tissues and was related to unfavorable prognosis. Additionally, pathway enrichment analysis illustrated that high expression of ELOB was associated with multiple cancer promoting pathways, like cell cycle, DNA replication, proteasome and PI3K − Akt signaling pathway, indicating ELOB as a potential anticancer target. Then, we confirmed that both in vivo and in vitro, the proliferation of breast cancer cells could be significantly suppressed by the down-regulation of ELOB. Mechanically, immunoprecipitation and in vivo ubiquitination assays prompted that, as the core element of Cullin2-RBX1-ELOB E3 ligase (CRL2) complex, ELOB regulated the ubiquitination and the subsequent degradation of oncoprotein p14/ARF. Moreover, the anticancer efficacy of erasing ELOB could be rescued by simultaneous knockdown of p14/ARF. Finally, through analyzing breast cancer tissue microarrays and western blot of patient samples, we demonstrated that the expression of ELOB in tumor tissues was elevated in compared to adjacent normal tissues. In conclusion, ELOB is identified to be a promising innovative target for the drug development of breast cancer by promoting the ubiquitination and degradation of oncoprotein p14/ARF.Graphical headlightsELOB is highly expressed in breast cancer.High ELOB levels were positively associated with poor prognosis.ELOB promotes p14/ARF degradation as part of the Cullin2-RBX1-ELOB E3 ligase complex.ELOB is a promising biomarker for breast cancer.Graphical

SKping cell cycle regulation: role of ubiquitin ligase SKP2 in hematological malignancies.

SKP2 (S-phase kinase-associated protein 2) is a member of the F-box family of substrate-recognition subunits in the SCF ubiquitin-protein ligase complexes. It is associated with ubiquitin-mediated degradation in the mammalian cell cycle components and other target proteins involved in cell cycle progression, signal transduction, and transcription. Being an oncogene in solid tumors and hematological malignancies, it is frequently associated with drug resistance and poor disease outcomes. In the current review, we discussed the novel role of SKP2 in different hematological malignancies. Further, we performed a limited in-silico analysis to establish the involvement of SKP2 in a few publicly available cancer datasets. Interestingly, our study identified Skp2 expression to be altered in a cancer-specific manner. While it was found to be overexpressed in several cancer types, few cancer showed a down-regulation in SKP2. Our review provides evidence for developing novel SKP2 inhibitors in hematological malignancies. We also investigated the effect of SKP2 status on survival and disease progression. In addition, the role of miRNA and its associated families in regulating Skp2 expression was explored. Subsequently, we predicted common miRNAs against Skp2 genes by using miRNA-predication tools. Finally, we discussed current approaches and future prospective approaches to target the Skp2 gene by using different drugs and miRNA-based therapeutics applications in translational research.

FBXL4 suppresses mitophagy by restricting the accumulation of NIX and BNIP3 mitophagy receptors.

To maintain both mitochondrial quality and quantity, cells selectively remove damaged or excessive mitochondria through mitophagy, which is a specialised form of autophagy. Mitophagy is induced in response to diverse conditions, including hypoxia, cellular differentiation and mitochondrial damage. However, the mechanisms that govern the removal of specific dysfunctional mitochondria under steady-state conditions to fine-tune mitochondrial content are not well understood. Here, we report that SCFFBXL4 , an SKP1/CUL1/F-box protein ubiquitin ligase complex, localises to the mitochondrial outer membrane in unstressed cells and mediates the constitutive ubiquitylation and degradation of the mitophagy receptors NIX and BNIP3 to suppress basal levels of mitophagy. We demonstrate that the pathogenic variants of FBXL4 that cause encephalopathic mtDNA depletion syndrome (MTDPS13) do not efficiently interact with the core SCF ubiquitin ligase machinery or mediate the degradation of NIX and BNIP3. Thus, we reveal a molecular mechanism whereby FBXL4 actively suppresses mitophagy by preventing NIX and BNIP3 accumulation. We propose that the dysregulation of NIX and BNIP3 turnover causes excessive basal mitophagy in FBXL4-associated mtDNA depletion syndrome.

Abstract 4962: Discovery of cancer-specific E3 ligase ligands for targeted protein degradation

Abstract F-box/WD repeat-containing protein 7 (FBXW7) is the substrate recognition component of SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex which mediates the ubiquitination and subsequent proteasomal degradation of target proteins. FBXW7 recognizes and binds phosphor-degrons within target proteins and capture them to SCF complex for ubiquitination. Know FBXW7 substrates include cyclin-E, DISC1, JUN, MYC, NFE2L1, NOTCH2, MCL1, RICTOR, and NOTCH1 released notch intracellular domain (NICD). FBXW7 is frequently mutated in cancer, results in loss of substrate recognition and binding. R465C is a one of the major FBXW7 mutations and is found in over 70,000 cancer patients worldwide. From a high-throughput screen, we discovered compounds that selectively bind to FBXW7 R465C mutant. PROTACs against various protein targets have been made with these mutant specific ligands. Some of these PROTACs could induce targeted protein degradation in cells carrying FBXW7 R465C mutant but not wild-type FBXW7. Targeted protein degradation with these PROTACs could represent a novel therapeutic approach against FBXW7 R465C mutant cancer. Citation Format: Zhaofu Wang, Lynette Zhang, Zhen Chang, Mengxi Zhao, Guobin Li, Fang Qin, Shichao Ma, Yunfeng Li, Zhongguo Zhang, Xinghao Wang, Ying Kong, Shidi Lou, Sheng Chen, Lanzhen Liu, Shuaijie Xu, Hailong Zhang. Discovery of cancer-specific E3 ligase ligands for targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4962.

The effects of phosphate fertilizer on the growth and reproduction of Pardosa pseudoannulata and its potential mechanisms

In fields, the natural enemy spider, Pardosa pseudoannulata, plays important roles in insect pest control. Agrochemicals, such as phosphate fertilizer, disturb the ecosystem and weaken the pest control efficiency of the spider. According to the usual habitat of the spider in soil cracks, the soil-application of phosphate fertilizer was carried out to determine its effects on the growth and reproduction of P. pseudoannulata. Phosphate fertilizer treatment prolonged longevity and increased mortality in subadults. The treatment also negatively affected reproduction of P. pseudoannulata adults even with removing phosphate fertilizer stress before adult emergence, leading to a lower mating rate, fewer eggsacs and eggs per female, and fewer offsprings in the first eggsac. The transcriptomic sequencing analysis revealed the up-regulation of unigenes related to stress resistance and down-regulation of unigenes associated with protein processing and proteasomal degradation in phosphate fertilizer-treated P. pseudoannulata. Decline in proper protein processing by E3 ubiquitin-protein ligase complex and endopeptidase activity might provide a partial explanation for negative effects of phosphate fertilizer on the spider reproduction. The study put a notice on negative effects of phosphate fertilizer on beneficial arthropods, which provide a great potential in the protection of P. pseudoannulata and other predator spider species.

Prognostic Significance and Immunological Role of FBXO5 in Human Cancers: A Systematic Pan-Cancer Analysis.

F-box protein 5 (FBXO5), an essential subunit of the ubiquitin protein ligase complex, is increasingly recognized to exhibit important biological effects in regulating tumor occurrence and progression. The present research was intended to systematically investigate the latent roles of FBXO5 in prognosis and immunological function across cancers. Pan-cancer analyses of FBXO5 were performed based upon publicly available online databases, mainly including the Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx), UCSC Xena, cBioPortal, and ImmuCellAI, revealing the possible relationships between FBXO5 and prognosis, DNA methylation, tumor microenvironment (TME), infiltration of immune cells, immune-related genes, immune checkpoints, tumor mutation burden (TMB), and microsatellite instability (MSI). The results suggested that FBXO5 was expressed at a high level in numerous tumor cell lines with significant upregulation in most cancers as opposed to normal tissues. Of note, elevated expression of FBXO5 was significantly related to an unfavorable prognosis in many cancer types. Furthermore, DNA methylation and TME were confirmed to display evident correlation with the expression of FBXO5 in several malignancies. Moreover, FBXO5 expression was remarkably positively correlated with the levels of infiltrating Treg cells and Tcm cells in most tumors, but negatively correlated with tumor-infiltrating CD8+ T cells, NK/NKT cells, and Th2 cells. Meanwhile, FBXO5 was demonstrated to be co-expressed with the genes encoding immune activating and suppressive factors, chemokines, chemokine receptors, and major histocompatibility complex (MHC). Immune checkpoints, TMB, and MSI were also overtly associated with FBXO5 dysregulation among diverse kinds of cancers. Additionally, the enrichment analyses showed close relationships between FBXO5 expression and the processes related to cell cycle and immune inflammatory response. These findings provided a detailed comprehension of the oncogenic function of FBXO5. Because of its crucial roles in cancer immunity and tumorigenesis, FBXO5 may serve as a novel prognostic indicator and immunotherapeutic target for various malignancies.

Recent ultra-rare inherited variants implicate new autism candidate risk genes.

Autism is a highly heritable complex disorder in which de novo mutation (DNM) variation contributes significantly to risk. Using whole-genome sequencing data from 3,474 families, we investigate another source of large-effect risk variation, ultra-rare variants. We report and replicate a transmission disequilibrium of private, likely gene-disruptive (LGD) variants in probands but find that 95% of this burden resides outside of known DNM-enriched genes. This variant class more strongly affects multiplex family probands and supports a multi-hit model for autism. Candidate genes with private LGD variants preferentially transmitted to probands converge on the E3 ubiquitin-protein ligase complex, intracellular transport and Erb signaling protein networks. We estimate that these variants are approximately 2.5 generations old and significantly younger than other variants of similar type and frequency in siblings. Overall, private LGD variants are under strong purifying selection and appear to act on a distinct set of genes not yet associated with autism.

Computational search of hybrid human/SARS-CoV-2 dsRNA reveals unique viral sequences that diverge from those of other coronavirus strains

The role of the RNAi/Dicer/Ago system in degrading RNA viruses has been elusive in mammals in the past, which has prompted authors to think that interferon (IFN) synthesis is essential in this clade, relegating the RNAi defense strategy against viral infection as an accessory function. However, recent publications highlight the existence of abundant viral small interference and micro RNAs (VsiRNAs and VmiRNAs) in both cell-line and whole organism based experiments, indicating a contribution of these molecules in host responses and/or viral replication. We explore the theoretical possibility that RNAi triggered by SARS-CoV-2 might degrade some host transcripts in the opposite direction, although this hypothesis seems counterintuitive. The SARS-CoV-2 genome was therefore computationally searched for exact intrapairing within the viral RNA and exact hybrid pairing with the human transcriptome over a minimum of 20 bases in length. Minimal segments of 20-base lengths of SARS-CoV-2 RNA were found based on the theoretical matching with existing complementary strands in the human host transcriptome. Few human genes potentially annealing with SARS-CoV-2 RNA, including mitochondrial deubiquitinase USP30, the subunit of ubiquitin protein ligase complex FBXO21 and two long noncoding RNAs, were retrieved. The hypothesis that viral-originated RNAi might mediate degradation of host transcriptome messages was corroborated by published high throughput sequencing of RNA from infected tissues and cultured cells, clinical observation and phylogenetic comparative analysis, indicating a strong specificity of these SARS-CoV-2 hybrid pairing sequences for human genomes.

Genome-wide siRNA screening reveals that DCAF4-mediated ubiquitination of optineurin stimulates autophagic degradation of Cu,Zn-superoxide dismutase

Cu, Zn superoxide dismutase (SOD1) is one of the genes implicated in the devastating neurodegenerative disorder amyotrophic lateral sclerosis (ALS). Although the precise mechanisms of SOD1 mutant (SOD1mut)-induced motoneuron toxicity are still unclear, defects in SOD1 proteostasis are known to have a critical role in ALS pathogenesis. We previously reported that the SOD1mut adopts a conformation that exposes a Derlin-1-binding region (DBR) and that DBR-exposed SOD1 interacts with Derlin-1, leading to motoneuron death. We also found that an environmental change, i.e. zinc depletion, induces a conformational change in WT SOD1 (SOD1WT) to the DBR-exposed conformation, suggesting the presence of an equilibrium state between the DBR-masked and DBR-exposed states even with SOD1WT Here, we conducted a high-throughput screening based on time-resolved FRET to further investigate the SOD1WT conformational change, and we used a genome-wide siRNA screen to search for regulators of SOD1 proteostasis. This screen yielded 30 candidate genes that maintained an absence of the DBR-exposed SOD1WT conformation. Among these genes was one encoding DDB1- and CUL4-associated factor 4 (DCAF4), a substrate receptor of the E3 ubiquitin-protein ligase complex. Of note, we found that DCAF4 mediates the ubiquitination of an ALS-associated protein and autophagy receptor, optineurin (OPTN), and facilitates autophagic degradation of DBR-exposed SOD1. In summary, our screen identifies DCAF4 as being required for proper proteostasis of DBR-exposed SOD1, which may have potential relevance for the development of therapies for managing ALS.

FKBP51 and FKBP12.6-Novel and tight interactors of Glomulin.

The protein factor Glomulin (Glmn) is a regulator of the SCF (Skp1-CUL1-F-box protein) E3 ubiquitin-protein ligase complex. Mutations of Glmn lead to glomuvenous malformations. Glmn has been reported to be associated with FK506-binding proteins (FKBP). Here we present in vitro binding analyses of the FKBP—Glmn interaction. Interestingly, the previously described interaction of Glmn and FKBP12 was found to be comparatively weak. Instead, the closely related FKBP12.6 and FKBP51 emerged as novel binding partners. We show different binding affinities of full length and truncated FKBP51 and FKBP52 mutants. Using FKBP51 as a model system, we show that two amino acids lining the FK506-binding site are essential for binding Glmn and that the FKBP51-Glmn interaction is blocked by FKBP ligands. This data suggest FKBP inhibition as a pharmacological approach to regulate Glmn and Glmn-controlled processes.

Fbxo6 confers drug-sensitization to cisplatin via inhibiting the activation of Chk1 in non-small cell lung cancer.

Fbxo6 (also called FBG2) is a critical component of the evolutionarily conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box). Previous studies have demonstrated that Fbxo6 facilitates the growth and proliferation but inhibits the apoptosis and invasion of gastric cancer cells. However, the role of Fbxo6 in non-small cell lung cancer (NSCLC) is still not clear. Our results revealed that Fbxo6 expression is correlated with early TNM stage and favorable overall survival of NSCLC patients. Further in vitro experiments showed that Fbxo6 inhibits proliferation, facilitates apoptosis and promotes the sensitivity of cisplatin via decreased expression and phosphorylation of Chk1. Thus, Fbxo6 may be a useful prognosis marker and therapeutic target to overcome the chemoresistance of cisplatin-based chemotherapy agents in NSCLC patients.

Identification and structural characterization of deleterious non-synonymous single nucleotide polymorphisms in the human SKP2 gene

In SCF (Skp, Cullin, F-box) ubiquitin-protein ligase complexes, S-phase kinase 2 (SKP2) is one of the major players of F-box family, that is responsible for the degradation of several important cell regulators and tumor suppressor proteins. Despite of having significant evidence for the role of SKP2 on tumorgenesis, there is a lack of available data regarding the effect of non-synonymous polymorphisms. In this communication, the structural and functional consequences of non-synonymous single nucleotide polymorphisms (nsSNPs) of SKP2 have been reported by employing various computational approaches and molecular dynamics simulation. Initially, several computational tools like SIFT, PolyPhen-2, PredictSNP, I-Mutant 2.0 and ConSurf have been implicated in this study to explore the damaging SNPs. In total of 172 nsSNPs, 5 nsSNPs were identified as deleterious and 3 of them were predicted to be decreased the stability of protein. Guided from ConSurf analysis, P101L (rs761253702) and Y346C (rs755010517) were categorized as the highly conserved and functional disrupting mutations. Therefore, these mutations were subjected to three dimensional model building and molecular dynamics simulation study for the detailed structural consequences upon the mutations. The study revealed that P101L and Y346C mutations increased the flexibility and changed the structural dynamics. As both these mutations are located in the most functional regions of SKP2 protein, these computational insights might be helpful to consider these nsSNPs for wet-lab confirmatory analysis as well as in rationalizing future population based studies and structure based drug design against SKP2.

Casein kinase II phosphorylation of cyclin F at serine 621 regulates the Lys48-ubiquitylation E3 ligase activity of the SCF(cyclin F) complex.

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.

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48-linked ubiquitylation and defective autophagy.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.

MAGEA1 interacts with FBXW7 and regulates ubiquitin ligase-mediated turnover of NICD1 in breast and ovarian cancer cells

Melanoma-associated antigen A1 (MAGEA1) is member of the MAGE gene family that is expressed in male germ line cells and placenta under normal physiological conditions. Although MAGEA1's expression levels have been evaluated as one of the cancer testis (CT) antigens for immunotherapy in melanoma and several other cancers, its functional role and signaling mechanisms are largely unknown. In this study, we examined the functional involvement and signaling mechanisms of MAGEA1 in breast and ovarian cancer cells. Inhibitory effects of MAGEA1 on cell proliferation and migration were observed using both gain-of- (MAGEA1 overexpression) and loss-of- (siRNA interference) function approaches in breast (MCF-7 and MDA-MB-231) and ovarian (SKOV3 and SKOV3ip) cancer cell lines. We revealed a novel interaction between MAGEA1 and the intracellular segment of NOTCH1 receptor (NICD1). MAGEA1 reduced NICD1's stability by promoting the ubiquitin modification of NICD1. MAGEA1 also interacted with FBXW7, subunit of E3 ubiquitin protein ligase complex SCF, and the latter was functionally involved in NICD1 ubiquitination and degradation. In addition, siRNA interference of FBXW7 reversed the inhibitory effect of MAGEA1 on migration and proliferation of MCF-7 and MDA-MB-231 cells. These newly discovered MAGEA1-NICD1 and MAGEA1-FBXW7 interactions have potential clinical implications in breast and ovarian cancer treatment.

Associations and prognostic significance of p27Kip1, Jab1 and Skp2 in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is a primary tumor arising in lymph nodes and lymphoid tissue. The incidence of NHL is increasing at an annual rate of 3%. The human Jun activation domain-binding protein 1/COP9 signalosome subunit 5 (Jab1/CSN5) is a negative regulator of the cell cycle inhibitor p27Kip1 and abnormal expression of Jab1 is correlated with reduced p27 expression and associated with advanced tumor stage and poor prognosis in several human cancers. F-box protein S-phase kinase-interacting protein-2 (Skp2), the substrate recognition subunit of the Skp1-Cul1-F-box protein ubiquitin protein ligase complex, is required for the ubiquitination and consequent degradation of p27. The Skp2 protein is overexpressed in several human cancers and is associated with the degree of differentiation and the prognosis. The aim of the present study was to investigate the expression status of p27Kip1, Jab1 and Skp2 by immunohistochemistry, and assess their prognostic significance in patients with NHL. Immunohistochemical analysis revealed an inverse association between Jab1 and p27 in NHL tissue samples. Kaplan-Meier analysis demonstrated that Jab1 overexpression, Skp2 overexpression and low p27 expression were significantly associated with poor prognosis. Among clinicopathological parameters, overexpression of Jab1 was significantly associated with tumor size and International Prognostic Index (IPI), whereas Skp2 expression was significantly associated with metastasis and IPI. These findings suggest that the overexpression of Jab1 or Skp2 plays an important role in the pathogenesis of NHL. Thus, the expression of p27Kip1, Jab1 and Skp2 provided a clinical reference for the treatment of NHL.

Abstract 2868: Mechanisms of transcriptional regulation of FOXO3 by cofactors upon PI3K/AKT/mTOR inhibition

Abstract The PI3K/AKT/mTOR pathway is frequently deregulated in breast cancer. Specific inhibition of its key signaling nodes results in cell death and tumor growth arrest, but also in activation of feedback loops that ultimately limit therapeutic activity. The subcellular localization and activity of Forkhead box O3 (FOXO3) transcription factor is heavily regulated by the PI3K pathway. Phosphorylation of FOXO3 by AKT inhibits its transcriptional functions leading to cell survival and growth. Although our understanding of the transcription function of FOXO3 downstream of the PI3K pathway has increased significantly in recent years, it is still unclear how FOXO3 acts in concert with other cofactors of the transcription machinery. We conjectured that inhibition of the PI3K/AKT/mTOR signaling by specific agents targeting the pathway at different nodes leads to unique FOXO3-mediated transcriptional programs unveiling a complex network of regulation. We therefore sought to identify proteins that differentially co-immunoprecipitate with FOXO3 upon PI3K, AKT or mTOR blockade. To target the pathway at different levels, we used BYL719, a PI3Kα inhibitor; MK2206, an allosteric AKT inhibitor; and RAD001, an allosteric mTORC1 inhibitor. Upon treatment, we immunopreciptated endogenous FOXO3 and searched for FOXO3 interactors by mass spectrometry analysis. In parallel RNA samples from each treatment were analyzed using gene expression microarrays. Gene expression analyses revealed that each specific compound originated distinct transcriptional signatures. Accordingly, mass spectrometry analysis revealed novel FOXO3 co-interactors engaging in different transcriptional complexes at each treatment conditions. We identified some of these proteins as CUL1, the core component of the SCF (SKP1-CUL1-F-BOX protein) E3 ubiquitin-protein ligase complex, the cyclin-dependent kinase 5 (CDK5), the upstream binding transcription factor (UBTF), and the core component of RNA polymerase I POLR1C (polymerase RNA I polypeptide C). We performed coimmunoprecipitation analyses to confirm that these cofactors are bona fide FOXO3-interacting proteins. We then analyzed the effects of overexpressing these proteins on FOXO3 transcriptional activity. Luciferase assays demonstrated an increase in FOXO3 transcriptional activity when CUL1, UBTF, CDK5 or POLR1C were overexpressed. The overexpression of cofactors also induced the expression of FOXO3 target genes such as TRAIL or IRS2. Furthermore, overexpression of these co-interactors enhanced the binding of FOXO3 to its target gene promoters. We are now engaged in both elucidating how these cofactors can modulate the transcriptional activity of FOXO3 upon PI3K/AKT/mTOR inhibition and understanding the therapeutic implications of these FOXO3-containing complexes. Citation Format: Vasilis Hristidis, James Cownie, Ana Bosch, Joanne Soong, Pau Castel, Maurizio Scaltriti, Baselga José. Mechanisms of transcriptional regulation of FOXO3 by cofactors upon PI3K/AKT/mTOR inhibition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2868.

Computational Framework for Prediction of Peptide Sequences That May Mediate Multiple Protein Interactions in Cancer-Associated Hub Proteins

A considerable proportion of protein-protein interactions (PPIs) in the cell are estimated to be mediated by very short peptide segments that approximately conform to specific sequence patterns known as linear motifs (LMs), often present in the disordered regions in the eukaryotic proteins. These peptides have been found to interact with low affinity and are able bind to multiple interactors, thus playing an important role in the PPI networks involving date hubs. In this work, PPI data and de novo motif identification based method (MEME) were used to identify such peptides in three cancer-associated hub proteins—MYC, APC and MDM2. The peptides corresponding to the significant LMs identified for each hub protein were aligned, the overlapping regions across these peptides being termed as overlapping linear peptides (OLPs). These OLPs were thus predicted to be responsible for multiple PPIs of the corresponding hub proteins and a scoring system was developed to rank them. We predicted six OLPs in MYC and five OLPs in MDM2 that scored higher than OLP predictions from randomly generated protein sets. Two OLP sequences from the C-terminal of MYC were predicted to bind with FBXW7, component of an E3 ubiquitin-protein ligase complex involved in proteasomal degradation of MYC. Similarly, we identified peptides in the C-terminal of MDM2 interacting with FKBP3, which has a specific role in auto-ubiquitinylation of MDM2. The peptide sequences predicted in MYC and MDM2 look promising for designing orthosteric inhibitors against possible disease-associated PPIs. Since these OLPs can interact with other proteins as well, these inhibitors should be specific to the targeted interactor to prevent undesired side-effects. This computational framework has been designed to predict and rank the peptide regions that may mediate multiple PPIs and can be applied to other disease-associated date hub proteins for prediction of novel therapeutic targets of small molecule PPI modulators.

CCNF mutations in amyotrophic lateral sclerosis and frontotemporal dementia.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are overlapping, fatal neurodegenerative disorders in which the molecular and pathogenic basis remains poorly understood. Ubiquitinated protein aggregates, of which TDP-43 is a major component, are a characteristic pathological feature of most ALS and FTD patients. Here we use genome-wide linkage analysis in a large ALS/FTD kindred to identify a novel disease locus on chromosome 16p13.3. Whole-exome sequencing identified a CCNF missense mutation at this locus. Interrogation of international cohorts identified additional novel CCNF variants in familial and sporadic ALS and FTD. Enrichment of rare protein-altering CCNF variants was evident in a large sporadic ALS replication cohort. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase complex (SCFCyclin F). Expression of mutant CCNF in neuronal cells caused abnormal ubiquitination and accumulation of ubiquitinated proteins, including TDP-43 and a SCFCyclin F substrate. This implicates common mechanisms, linked to protein homeostasis, underlying neuronal degeneration.

Abstract 3792: SKP2 supports cell proliferation and is regulated by Notch signaling in myoblasts and embryonal rhabdomyosarcoma

Abstract Background: Rhabdomyosarcoma (RMS) in a pediatric tumor of myogenic origin. It includes two subtypes: embryonal and alveolar. Embryonal RMS (ERMS) cells express key myogenic factors such as MyoD and Myogenin, but proliferate indefinitely and have lost the ability to terminally differentiate into skeletal myofibers. Differently from the alveolar tumors bearing specific chromosomal translocations, ERMS has cytogenetic aberrations and molecular deregulations of pathways regulating senescence, proliferation and differentiation. It has been shown that SKP2, an F-box protein and a component of the ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box), is over-expressed in RMS primary samples and correlates with a dismal outcome. Therefore, we sought here to investigate the regulation of SKP2 and its role in ERMS. Methods: We modulated SKP2 expression through silencing, by using a siRNA validated in the literature, and forcing its expression through retroviral infection. In parallel, we investigate the effect of Notch signaling modulation on SKP2 expression. Results: SKP2 silencing resulted in cell cycle slowdown in both normal myoblasts and ERMS cells. Down-regulation of Notch1 led to SKP2 reduction while that of Notch3 supported SKP2 expression. Cosilencing SKP2 and Notch3 gave raise to myoblast-like structure formation in ERMS and facilitated myoblasts fusion. Finally, using a SKP2 inhibitor ERMS cell proliferation was completely blocked. Conclusion: Altogether, these preliminary experiments suggest that SKP2 could be regulated by Notch signaling in ERMS and that its inhibition hampers tumor cell proliferative capability. Note: This abstract was not presented at the meeting. Citation Format: Rossella Rota, Laura Adesso, Beatrice Conti, Roberta Ciarapica, Lavinia Raimondi, Maria De Salvo, Sonia Rodriguez, Nadia Carlesso, Lucio Miele, Franco Locatelli. SKP2 supports cell proliferation and is regulated by Notch signaling in myoblasts and embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2015-3792