Comprehensive Analysis of Expression, Prognostic Value, and Immune Infiltration for Ubiquitination-Related FBXOs in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most refractory human malignancies. F-box only proteins (FBXO) are the core components of SKP1-cullin 1-F-box E3 ubiquitin ligase, which have been reported to play crucial roles in tumor initiation and progression via ubiquitination-mediated proteasomal degradation. However, the clinical implications and biological functions of FBXOs in PDAC have not been fully clarified. Herein we perform a comprehensive analysis for the clinical values and functional roles of FBXOs in PDAC using different public databases. We found that FBXO1 (CCNF), FBXO20 (LMO7), FBXO22, FBXO28, FBXO32, and FBXO45 (designated six-FBXOs) were robustly upregulated in PDAC tissues, which predicted an adverse prognosis of PDAC patients. There was a significant correlation between the expression levels of six-FBXOs and the clinicopathological features in PDAC. The transcriptional levels of six-FBXOs were subjected to the influence of promoter methylation levels. There were more than 40% genetic alterations and mutations of six-FBXOs, which affected the clinical outcome of PDAC patients. Furthermore, the expression of six-FBXOs was associated with immune infiltrations and activated status, including B cells, CD8+ T cells, CD4+ T cells, NK cells, macrophages, and dendritic cells. The functional prediction revealed that the six-FBXOs were involved in ubiquitination-related pathways and other vital signaling pathways, such as p53, PI3K/Akt, and Hippo pathway. Therefore, six-FBXOs are the promising prognostic biomarkers or potential targets for PDAC diagnosis and treatment.

Fbxo45 Binds SPRY Motifs in the Extracellular Domain of N-Cadherin and Regulates Neuron Migration during Brain Development.

Several events during the normal development of the mammalian neocortex depend on N-cadherin, including the radial migration of immature projection neurons into the cortical plate. Remarkably, radial migration requires the N-cadherin extracellular domain but not N-cadherin-dependent homophilic cell-cell adhesion, suggesting that other N-cadherin-binding proteins may be involved. We used proximity ligation and affinity purification proteomics to identify N-cadherin-binding proteins. Both screens detected MycBP2 and SPRY domain protein Fbxo45, two components of an intracellular E3 ubiquitin ligase. Fbxo45 appears to be secreted by a nonclassical mechanism, not involving a signal peptide and not requiring transport from the endoplasmic reticulum to the Golgi apparatus. Fbxo45 binding requires N-cadherin SPRY motifs that are not involved in cell-cell adhesion. SPRY mutant N-cadherin does not support radial migration in vivo Radial migration was similarly inhibited when Fbxo45 expression was suppressed. The results suggest that projection neuron migration requires both Fbxo45 and the binding of Fbxo45 or another protein to SPRY motifs in the extracellular domain of N-cadherin.

Regulation of KDM2B and Brg1 on Inflammatory Response of Nasal Mucosa in CRSwNP.

Chronic nasal sinusitis with nasal polyps (CRSwNP) is a reversible nasal mucosal remodeling disease caused by persistent inflammation and structural changes in chronic nasal mucosa. Although there have been many studies on the inflammation of the nasal mucosa epithelium, the mechanism remains unclear. Our study found that H3K4me3 histone demethylase KDM2B (also known as JHDM1B) and transcriptional regulator Brg1 (also called SNF2-ß or Smarca4) were significantly decreased in nasal mucosa of CRSwNP patients, and they were positively correlated. Brg1 and KDM2B co-localize in the epithelial cells of nasal mucosa. We used poly(I:C)-treated nasal mucosal epithelial cells (HNECs) to find that the expression of KDM2B and Brg1 was also decreased, and the main expression position transferred from the nucleus to the nuclear membrane. We used small interfering RNA to knock down the expression of KDM2B and Brg1 in nasal epithelial cells. It was interesting to find that the decreased expression of KDM2B and Brg1 produced similar effects to that of poly(I:C)-treated cells, which could promote inflammatory response of nasal mucosal epithelial cells. And Brg1 appears to play a role in KDM2B regulating gene promoters of IL-6 and TNF-α inflammatory. This study shows that KDM2B and Brg1 may have an inhibitory effect on the development of CRSwNP nasal mucosal epithelial inflammation. This study will provide a new perspective for gene targeting therapy of CRSwNPs.

Novel Role of FBXW7 Circular RNA in Repressing Glioma Tumorigenesis.

Background: Circular RNAs (circRNAs) are RNA transcripts that are widespread in the eukaryotic genome. Recent evidence indicates that circRNAs play important roles in tissue development, gene regulation, and carcinogenesis. However, whether circRNAs encode functional proteins remains elusive, although translation of several circRNAs was recently reported. Methods: CircRNA deep sequencing was performed by using 10 pathologically diagnosed glioblastoma samples and their paired adjacent normal brain tissues. Northern blotting, Sanger sequencing, antibody, and liquid chromatograph Tandem Mass Spectrometer were used to confirm the existence of circ-FBXW7 and its encoded protein in in two cell lines. Lentivirus-transfected stable U251 and U373 cells were used to assess the biological functions of the novel protein invitro and invivo (five mice per group). Clinical implications of circ-FBXW7 were assessed in 38 pathologically diagnosed glioblastoma samples and their paired periphery normal brain tissues by using quantitative polymerase chain reaction (two-sided log-rank test). Results: Circ-FBXW7 is abundantly expressed in the normal human brain (reads per kilobase per million mapped reads [RPKM] = 9.31). The spanning junction open reading frame in circ-FBXW7 driven by internal ribosome entry site encodes a novel 21-kDa protein, which we termed FBXW7-185aa. Upregulation of FBXW7-185aa in cancer cells inhibited proliferation and cell cycle acceleration, while knockdown of FBXW7-185aa promoted malignant phenotypes invitro and invivo. FBXW7-185aa reduced the half-life of c-Myc by antagonizing USP28-induced c-Myc stabilization. Moreover, circ-FBXW7 and FBXW7-185aa levels were reduced in glioblastoma clinical samples compared with their paired tumor-adjacent tissues (P < .001). Circ-FBXW7 expression positively associated with glioblastoma patient overall survival (P = .03). Conclusions: Endogenous circRNA encodes a functional protein in human cells, and circ-FBXW7 and FBXW7-185aa have potential prognostic implications in brain cancer.

Asparagine reduces the mRNA expression of muscle atrophy markers via regulating protein kinase B (Akt), AMP-activated protein kinase α, toll-like receptor 4 and nucleotide-binding oligomerisation domain protein signalling in weaning piglets after lipopolysaccharide challenge.

Pro-inflammatory cytokines are critical in mechanisms of muscle atrophy. In addition, asparagine (Asn) is necessary for protein synthesis in mammalian cells. We hypothesised that Asn could attenuate lipopolysaccharide (LPS)-induced muscle atrophy in a piglet model. Piglets were allotted to four treatments (non-challenged control, LPS-challenged control, LPS+0·5 % Asn and LPS+1·0 % Asn). On day 21, the piglets were injected with LPS or saline. At 4 h post injection, piglet blood and muscle samples were collected. Asn increased protein and RNA content in muscles, and decreased mRNA expression of muscle atrophy F-box (MAFbx) and muscle RING finger 1 (MuRF1). However, Asn had no effect on the protein abundance of MAFbx and MuRF1. In addition, Asn decreased muscle AMP-activated protein kinase (AMPK) α phosphorylation, but increased muscle protein kinase B (Akt) and Forkhead Box O (FOXO) 1 phosphorylation. Moreover, Asn decreased the concentrations of TNF-α, cortisol and glucagon in plasma, and TNF-α mRNA expression in muscles. Finally, Asn decreased mRNA abundance of muscle toll-like receptor (TLR) 4 and nucleotide-binding oligomerisation domain protein (NOD) signalling-related genes, and regulated their negative regulators. The beneficial effects of Asn on muscle atrophy may be associated with the following: (1) inhibiting muscle protein degradation via activating Akt and inactivating AMPKα and FOXO1; and (2) decreasing the expression of muscle pro-inflammatory cytokines via inhibiting TLR4 and NOD signalling pathways by modulation of their negative regulators.

Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.

Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.

Differential proteomic profiling in human spermatozoa that did or did not result in pregnancy via IVF and AID.

PURPOSE: To identify biochemical markers in men with idiopathic infertility and normal sperm counts. EXPERIMENTAL DESIGN: We obtained proteomic profiling proteins in human spermatozoa following successful or unsuccessful pregnancy via assisted reproductive technology (ART) using 6-plex tandem mass tag (TMT) isobaric mass spectrometry. Our study design consisted of two groups: 1. The semen of 6 men whose sperm resulted in a clinical pregnancy following ART and 6 men whose semen did not result in a clinical pregnancy following ART. The results of differentiated mass spectrometry were validated by Western blotting. RESULTS AND DISCUSSION: A total of 2,045 proteins were detected in our cohort. 21 proteins were found to be differentially expressed (>1.2-fold) in men whose sperm resulted in a clinical pregnancy and those that did not. Using the results of bioinformatics analysis and Western Blotting, three proteins (A2LD1, ATP1B3 and FBXO2) were shown to have the same differential pattern (p<0.05) that was observed in the mass spectrometry analysis. CONCLUSIONS AND CLINICAL RELEVANCE: Proteomics may help identity a select cohort of men with abnormal semen parameters and aide infertility diagnoses.

Cloning and characterization of a novel oocyte-specific gene encoding an F-Box protein in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Oocyte-specific genes play critical roles in oogenesis, folliculogenesis and early embryonic development. The objectives of this study were to characterize the expression of a novel oocyte-specific gene encoding an F-box protein during ovarian development in rainbow trout, and identify its potential interacting partners in rainbow trout oocytes. METHODS: Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, a novel transcript represented by ESTs only from the oocyte library was identified. The complete cDNA sequence for the novel gene (named fbxoo) was obtained by assembling sequences from an EST clone and a 5'RACE product. The expression and localization of fbxoo mRNA and protein in ovaries of different developmental stages were analyzed by quantitative real time PCR, immunoblotting, in situ hybridization and immunohistochemistry. Identification of Fbxoo binding proteins was performed by yeast two-hybrid screening. RESULTS: fbxoo mRNA is specifically expressed in mature oocytes as revealed by tissue distribution analysis. The fbxoo cDNA sequence is 1,996 bp in length containing an open reading frame, which encodes a predicted protein of 514 amino acids. The novel protein sequence does not match any known protein sequences in the NCBI database. However, a search of the Pfam protein database revealed that the protein contains an F-box motif at the N-terminus, indicating that Fbxoo is a new member of the F-box protein family. The expression of fbxoo mRNA and protein is high in ovaries at early pre-vitellogenesis stage, and both fbxoo mRNA and protein are predominantly expressed in early pre-vitellogenic oocytes. Several proteins including tissue inhibitor of metalloproteinase 2 (Timp2) were identified as potential Fbxoo protein binding partners. CONCLUSIONS: Results suggest that the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout.

Clear cell carcinomas of the ovary: a multi-institutional study of 129 cases in Korea with prognostic significance of Emi1 and Galectin-3.

Accurate diagnosis of ovarian clear cell carcinoma (CCC) is important because of its poor prognosis with chemoresistance and a high recurrent rate. The clinicopathologic characteristics and prognostic significance of the cell cycle regulator [early mitotic inhibitor-1 (Emi1)] and galactoside-binding protein (Galectin-3) were evaluated. Among 155 CCCs from 18 hospitals in Korea between 1995 and 2006, 129 pure CCCs were selected with consensus using immunohistochemical stains for hepatocyte nuclear factor-1ß, Wilms' tumor protein, and estrogen receptor. The expressions of Emi1, Galectin-3, p53, and Ki-67 labeling index were analyzed with clinicopathologic parameters and the patient's survival. The mean age of the patients was 49.6 yr; the tumors were bilateral in 10.9%, and the average size was 12 cm. Adenofibromatous component was found in 7%, and endometriosis in 48.1% of the cases. Psammoma body was seen in 16.3%. Disease-free survival and overall survival rates were 78.3% and 79.1%, respectively. The International Federation of Obstetrics and Gynecology (FIGO) stage was the most important prognostic indicator. Emi1 expression (>5%) was seen in 23.3% of CCCs, and associated with high FIGO grades and poor overall survival (P<0.05). High Galectin-3 (≥80%) expression was seen in 59.7% of CCCs, and associated with FIGO stages III and IV, and high Ki-67 labeling index. High Ki-67 labeling index (≥50%) and p53 expression (≥50%) were seen in 27.1% and 18.6% of CCCs, respectively, but there was no clinicopathologic and prognostic significance. On the basis of the fact that the expression of Emi1 in CCC was correlated with a high histologic grade and worse overall survival, target therapy using inhibitors of Emi1 may be tried in the management of CCC patients with Emi1 expression.

A failure of meiotic chromosome segregation in a fbh1Delta mutant correlates with persistent Rad51-DNA associations.

The F-box DNA helicase Fbh1 constrains homologous recombination in vegetative cells, most likely through an ability to displace the Rad51 recombinase from DNA. Here, we provide the first evidence that Fbh1 also serves a vital meiotic role in fission yeast to promote normal chromosome segregation. In the absence of Fbh1, chromosomes remain entangled or segregate unevenly during meiosis, and genetic and cytological data suggest that this results in part from a failure to efficiently dismantle Rad51 nucleofilaments that form during meiotic double-strand break repair.

Identification of FBXO25-interacting proteins using an integrated proteomics approach.

FBXO25 is one of the 68 human F-box proteins that serve as specificity factors for a family of ubiquitin ligases composed of s-phase-kinase associated protein 1, really interesting new gene-box 1, Cullin 1, and F-box protein (SCF1) that are involved in targeting proteins for destruction across the ubiquitin proteasome system. We recently reported that the FBXO25 protein accumulates in novel subnuclear structures named FBXO25-associated nuclear domains (FAND). Combining two-step affinity purification followed by MS with a classical two-hybrid screen, we identified 132 novel potential FBXO25 interacting partners. One of the identified proteins, beta-actin, physically interacts through its N-terminus with FBXO25 and is enriched in the FBXO25 nuclear compartments. Inhibitors of actin polymerization promote a significant disruption of FAND, indicating that they are compartments influenced by the organizational state of actin in the nucleus. Furthermore, FBXO25 antibodies interfered with RNA polymerase II transcription in vitro. Our results open new perspectives for the understanding of this novel compartment and its nuclear functions.

[Effects of UO-126 on proliferation and fbw7 expression of HeLa cells].

AIM: To observe the effects of UO-126 on the expression of F-box and WD repeat domain-containing protein 7(FBW7)and on the proliferation of human cervical cancer cell lines (HeLa cells). METHODS: HeLa cells were treated with different concentrations of UO-126, MTT assay was used to observe the proliferation of HeLa cells. Immunofluorescence showed the location and expression of FBW7 in HeLa cells. The mRNA and protein expression of FBW7 were detected by RT-PCR and Western blot before and after mitogen-activated protein kinases (MAPK)signal was blocked by UO-126 a MAPK inhibitor. RESULTS: MTT results showed that the concentration range of MAPK signaling pathway inhibitor UO-126 inhibited the proliferation of HeLa cells in a concentration-and time-dependent manner(P<0.05). Immunofluorescence showed that the expression of positive FBW7 had increased after HeLa cells were treated with UO-126. RT-PCR and Western blot exhibited that the FBW7 mRNA and protein expression had significantly increased before and after HeLa cells were treated with UO-126(P<0.05). CONCLUSION: UO-126 could inhibit HeLa cells proliferation, FBW7 lied downstream of MAPK signaling pathway.

An F-box gene, CPR30, functions as a negative regulator of the defense response in Arabidopsis.

Arabidopsis gain-of-resistance mutants, which show HR-like lesion formation and SAR-like constitutive defense responses, were used well as tools to unravel the plant defense mechanisms. We have identified a novel mutant, designated constitutive expresser of PR genes 30 (cpr30), that exhibited dwarf morphology, constitutive resistance to the bacterial pathogen Pseudomonas syringae and the dramatic induction of defense-response gene expression. The cpr30-conferred growth defect morphology and defense responses are dependent on ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), PHYTOALEXIN DEFICIENT 4 (PAD4), and NONRACE-SPECIFIC DISEASE RESISTANCE 1 (NDR1). Further studies demonstrated that salicylic acid (SA) could partially account for the cpr30-conferred constitutive PR1 gene expression, but not for the growth defect, and that the cpr30-conferred defense responses were NPR1 independent. We observed a widespread expression of CPR30 throughout the plant, and a localization of CPR30-GFP fusion protein in the cytoplasm and nucleus. As an F-box protein, CPR30 could interact with multiple Arabidopsis-SKP1-like (ASK) proteins in vivo. Co-localization of CPR30 and ASK1 or ASK2 was observed in Arabidopsis protoplasts. Based on these results, we conclude that CPR30, a novel negative regulator, regulates both SA-dependent and SA-independent defense signaling, most likely through the ubiquitin-proteasome pathway in Arabidopsis.

Fbxw15/Fbxo12J is an F-box protein-encoding gene selectively expressed in oocytes of the mouse ovary.

In recent years, several factors required for follicular assembly and/or early growth of newly formed primordial follicles have been characterized, but additional factors likely remain to be identified. We have used cDNA arrays to compare gene expression in the neonatal mouse ovary at 48 h (when primordial follicles are being assembled) and at 96 h (when early follicular growth is taking place) after birth to that of ovaries collected <24 h after birth (when follicles have not yet been formed). Segregating genes according to their pattern of expression revealed the presence of one cluster of 24 genes for which expression consistently increased at 48 and 96 h. The top increaser in this cluster encodes a approximately 1.5-kb mRNA containing an open reading frame of 1401 bp that encodes a protein of 466 amino acids. The predicted 52.3-kDa protein is a member of the F-box-only (FBXO) protein family, termed FBXW15 or FBXO12J. It has a cytoplasmic localization that includes the endoplasmic reticulum. Expression of Fbxw15/Fbxp12J mRNA is oocyte-specific; the mRNA is first detected on Gestational Day 18, decreasing thereafter to minimal levels on the day of birth. The prevalence of Fbxw15/Fbxp12J mRNA increases again at 48 and 96 h after birth, coinciding with the time of follicular assembly and the initiation of early follicular growth, respectively. The specific expression of Fbxw15/Fbxp12J in oocytes and its developmental pattern of expression suggest a role for this gene in the regulation of oocyte physiology.

FBXO40, a gene encoding a novel muscle-specific F-box protein, is upregulated in denervation-related muscle atrophy.

F-box proteins are key components of SCF (Skp1-Cullin1-F-box protein) complexes, which exert E3 ubiquitin ligase activity and participate in cell cycle and signal transduction. F-box proteins interact with Skp1 through the F-box domain and with proteins to be ubiquitinated through other interaction domains. We have characterized a novel muscle-specific F-box protein, FBXO40, the expression of which decreases in the dystrophic muscle of Limb-girdle muscular dystrophy (LGMD) patient. During the development of skeletal muscle, FBXO40 can only be detected at postnatal stage from about 2 weeks after birth. By overexpressing in C2C12 cells, FBXO40 localized in cytoplasm. Most importantly, the expression of FBXO40 can be upregulated in skeletal muscle from denervation- but not starvation-related muscle atrophy. All our data suggest that FBXO40 may function as a regulator involved in the postnatal myogenesis.

A neural-specific F-box protein Fbs1 functions as a chaperone suppressing glycoprotein aggregation.

Fbs1 is an F-box protein present abundantly in the nervous system. Similar to the ubiquitously expressed Fbs2, Fbs1 recognizes N-glycans at the innermost position as a signal for unfolded glycoproteins, probably in the endoplasmic reticulum-associated degradation pathway. Here, we show that the in vivo majority of Fbs1 is present as Fbs1-Skp1 heterodimers or Fbs1 monomers but not SCF(Fbs1) complex. The inefficient SCF complex formation of Fbs1 and the restricted presence of SCF(Fbs1) bound on the endoplasmic reticulum membrane were due to the short linker sequence between the F-box domain and the sugar-binding domain. In vitro, Fbs1 prevented the aggregation of the glycoprotein through the N-terminal unique sequence of Fbs1. Our results suggest that Fbs1 assists clearance of aberrant glycoproteins in neuronal cells by suppressing aggregates formation, independent of ubiquitin ligase activity, and thus functions as a unique chaperone for those proteins.

Multiple pathways regulate intracellular shuttling of MoKA, a co-activator of transcription factor KLF7.

MoKA is a novel F-box containing protein that interacts with and stimulates the activity of transcription factor KLF7, a regulator of neuronal differentiation. MoKA accumulates throughout the cell and predominantly in the cytosol, consistent with the presence of several putative nuclear localization and export signals (NLSs and NESs). The present study was designed to refine the identity and location of the sequences responsible for MoKA intracellular shuttling and transcriptional activity. Forced expression of fusion proteins in mammalian cells demonstrated that only one of three putative NLSs potentially recognized by karyopherin receptors is involved in nuclear localization of MoKA. By contrast, three distinct sequences were found to participate in mediating cytoplasmic accumulation. One of them is structurally and functionally related to the leucine-rich export signal that interacts with the exportin 1 (CRM1) receptor. The other two export signals instead display either a novel leucine-rich sequence or an undefined peptide motif, and both appear to act through CRM1-independent pathways. Finally, transcriptional analyses using the chimeric GAL4 system mapped the major activation domain of MoKA to a highly acidic sequence that resides between the NLS and NES clusters.

The wound response mutant suppressor of prosystemin-mediated responses6 (spr6) is a weak allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1).

The systemic defense response of tomato plant in response to insect attack and wounding is regulated by the 18 amino acid peptide systemin and the phytohormone jasmonic acid (JA). Recent genetic analyses based mainly on spr (suppressors of prosystemin-mediated responses) mutant screens have led to the hypothesis that systemin acts at, or near, the site of wounding to amplify the production of JA, which in turn functions as a mobile signal to promote the systemic defense response. In order to identify more components involved in the systemin/JA-signaled defense response, we carried out a larger scale screen for new spr mutants in tomato. Here we describe the characterization of spr6, a mutant impaired in wound- and systemin-induced defense gene expression. Using a candidate gene approach based on genetic linkage, we demonstrate that spr6 is allelic to jai1-1, which is a loss-of-function allele of the tomato homolog of CORONATINE-INSENSITIVE1 (COI1), an F-box protein that is required for JA-signaled processes in Arabidopsis. We show several aspects of the spr6 mutant phenotype distinct from that of jai1-1. First, the responsiveness of spr6 plants to exogenous JA shows a dosage dependency, i.e. it is more sensitive to JA than jai1-1 while less sensitive to JA than the wild-type. Secondly, unlike the sterile jai1-1, the spr6 plant displays normal fertility and seed set and thus can be maintained as a pure line and does not require selection. Therefore, spr6 provides a valuable tool, which can complement the limitations of jai1-1, to study JA signaling in tomato. The gene identification process of Spr6 we described herein represents an example showing the convenience of a candidate gene approach, based on genetic linkage, to identify gene functions of genetic loci defined by tomato wound response mutants.

Characterization of FBX25, encoding a novel brain-expressed F-box protein.

F-box proteins (FBPs) confer substrate specificity to the SCF-type (Skp1/Cul1/FBP) of ubiquitin ligase complexes through their F-box. Multiple FBPs have been predicted, but experimental evidence is lagging. We report on the predicted human FBP hFBX25 which we found to be disrupted in a mentally retarded translocation carrier suffering from epileptic seizures. We investigated hFBX25's genomic organization and established hFBX25 as an FBP by verifying its interaction with Skp1 and Cul1. In the process, we identified an atypical serine residue in the F-box which is crucial for the hFBX25-Skp1 binding. We determined hFBX25's subcellular localization. We found strong transcription in human brain. In mouse embryonic sections, mFbx25 shows predominantly neuronal expression and in adult mouse brain, expression is confined to the hippocampus, the cerebral cortex and the Purkinje cell layer. Interestingly, aberrations in the ubiquitin pathway have been linked to neurological conditions.