A combination of the F-box motif and kelch repeats defines a large Arabidopsis family of F-box proteins

In the sequences released by the Arabidopsis Genome Initiative (AGI), we have discovered a new large gene family (48 genes as of July 2000). A detailed computational and biochemical analysis of the predicted gene products reveals a novel family of plant F-box proteins, where the amino (N)-terminal F-box motif is followed by four kelch repeats and a characteristic carboxy-terminal domain. F-box proteins are an expanding family of eukaryotic proteins, which have been shown in some cases to be critical for the controlled degradation of cellular regulatory proteins via the ubiquitin pathway. The F-box motif of the At5g48990 gene product, a member of the family, was shown to be functionally active by its ability to mediate the in vitro interaction between At5g48990 and ASK1 proteins. F-box proteins specifically recruit the targets to be ubiquitinated, mainly through protein-protein interaction modules such as WD-40 domains or leucine-rich repeats (LRRs). The kelch repeats of the family described here form a potential protein-protein interaction domain, as molecular modelling of the kelch repeats according to the galactose oxidase crystal structure (the only solved structure containing kelch repeats) predicts a -propeller. The identification of this family of F-box proteins greatly expands the field of plant F-box proteins and suggests that controlled degradation of cellular proteins via the ubiquitin pathway could play a critical role in multiple plant cellular processes.     

F-box proteins everywhere

The ubiquitin proteasome system is a key regulator of many biological processes in all eukaryotes. This mechanism employs several types of enzymes, the most important of which are the ubiquitin E3 ligases that catalyse the attachment of polyubiquitin chains to target proteins for their subsequent degradation by the 26S proteasome. Among the E3 families, the SCF is the best understood; it consists of a multi-protein complex in which the F-box protein plays a crucial role by recruiting the target substrate. Strikingly, nearly 700 F-box proteins have been predicted in Arabidopsis, suggesting that plants have the capacity to assemble a multitude of SCF complexes, possibly controlling the stability of hundreds of substrates involved in a plethora of biological processes. Interestingly, viruses and even pathogenic bacteria have also found ways to hijack the plant SCF and to reprogram it for their own purposes.     

A new subfamily of structurally related human F-box proteins

F-box proteins, a critical component of the evolutionary conserved ubiquitin-protein ligase complex SCF (Skp1/Cdc53-Cullin1/F-box), recruit substrates for ubiquitination and consequent degradation through their specific protein-protein interaction domains. Here, we report the identification of full-length cDNAs encoding three novel human F-box proteins named FBG3, FBG4 and FBG5 which display similarity with previously identified NFB42 (FBX2) and FBG2 (FBX6) proteins. All five proteins are characterized by an approximately 180-amino-acid (aa) conserved C-terminal domain and thus constitute a third subfamily of mammalian F-box proteins. Analysis of genomic organization of the five FBG genes revealed that all of them consist of six exons and five introns. FBG1, FBG2 and FBG3 genes are located in tandem on chromosome 1p36, and FBG4 and FBG5 are mapped to chromosome 19q13. FBG genes are expressed in a limited number of human tissues including kidney, liver, brain and muscle tissues. Expression of rat FBG2 gene was found related to differentiation/proliferation status of hepatocytes. Specifically, FBG2 mRNA was expressed in foetal liver, decreased after birth and re-accumulated in adult liver. Expression of FBG2 was strongly inhibited in hepatoma cells by okadaic acid.     

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.     

Ectromelia virus encodes a novel family of F-box proteins that interact with the SCF complex

Poxviruses are notorious for encoding multiple proteins that regulate cellular signaling pathways, including the ubiquitin-proteasome system. Bioinformatics indicated that ectromelia virus, the causative agent of lethal mousepox, encoded four proteins, EVM002, EVM005, EVM154, and EVM165, containing putative F-box domains. In contrast to cellular F-box proteins, the ectromelia virus proteins contain C-terminal F-box domains in conjunction with N-terminal ankyrin repeats, a combination that has not been previously reported for cellular proteins. These observations suggested that the ectromelia virus F-box proteins interact with SCF (Skp1, cullin-1, and F-box) ubiquitin ligases. We focused our studies on EVM005, since this protein had only one ortholog in cowpox virus. Using mass spectrometry, we identified cullin-1 as a binding partner for EVM005, and this interaction was confirmed by overexpression of hemagglutinin (HA)-cullin-1. During infection, Flag-EVM005 and HA-cullin-1 colocalized to distinct cellular bodies. Significantly, EVM005 coprecipitated with endogenous Skp1, cullin-1, and Roc1 and associated with conjugated ubiquitin, suggesting that EVM005 interacted with the components of a functional ubiquitin ligase. Interaction of EVM005 with cullin-1 and Skp1 was abolished upon deletion of the F-box, indicating that the F-box played a crucial role in interaction with the SCF complex. Additionally, EVM002 and EVM154 interacted with Skp1 and conjugated ubiquitin, suggesting that ectromelia virus encodes multiple F-box-containing proteins that regulate the SCF complex. Our results indicate that ectromelia virus has evolved multiple proteins that interact with the SCF complex.     

Identification of a family of human centromere proteins using autoimmune sera from patients with scleroderma

We have examined preimmune serum samples from a patient who progressively developed the symptoms of scleroderma CREST over a period of several years. During this period, anti-centromere antibodies (recognized by indirect immunofluorescence) appeared in the serum. Concomitant with the appearance of the anti-centromere antibodies, antibody species recognizing three chromosomal antigens in immunoblots of SDS polyacrylamide gels appeared in the patient's serum. These antigens migrate with electrophoretic mobilities corresponding to M_r=17, 80, and 140 kilodaltons (kd). Affinity-eluted antibody fractions recognizing the antigens have been prepared from sera of three other patients. Indirect immunofluorescence labeling of mitotic cells using these antibody fractions demonstrates that the antigens are centromere components. We designate them CENP (CENtromere Protein) — A (17kd), CENP-B (80kd), and CENP-C (140kd). The three CENP antigens share antigenic determinants. Immunoblotting experiments show that these patients make antibody species recognizing at least three distinct epitopes on CENP-B and two on CENP-C. Sera from different patients contain different mixtures of the antibody species.     

F-box蛋白家族及其功能

F-box蛋白是一类广泛存在于真核生物中,含有F-box结构域的蛋白家族,在泛素-蛋白酶体途径（ubiquitin-proteasome pathway, UPP）中因特异识别底物蛋白而参与细胞周期调控、转录调控、细胞凋亡、细胞信号转导等生命活动。另外,F-box蛋白还通过其他作用方式参与了体内众多生化过程。本文综述了F-box蛋白的结构和作用途径,以及该蛋白参与的多种生理功能,展示了F-box蛋白家族在生命活动中具有广泛而重要的作用,并指出了F-box蛋白将为阐明疾病发生机制提供新的线索,可望成为疾病治疗中药物作用的靶向位点。     

Identification of centrosomal proteins in a human lymphoblastic cell line.

Highly enriched preparations of centrosomes from human T-lymphoblasts KE 37 were analyzed for their protein content. The specific pattern of polypeptides was characterized by an abundant subset of high mol. wt proteins and a major group of proteins with mol. wt ranging from 50 to 65 kd. Several immunoreactive proteins were identified, using a rabbit serum spontaneously reacting with human centrosomes. They include a family of high mol. wt ranging from 180 to 250 kd, a 130-kd protein and a 60-65 kd doublet. These antigens have the following properties: they are localized within the pericentriolar material; their abundance, as judged by centrosome labelling, changes significantly during the cell cycle, the maximum being observed at the pole of the metaphasic spindle; in Taxol-treated cells where the centrosome is no longer acting as a nucleating center, they redistribute at one end of the microtubule arrays in both mitotic and interphasic cells, as expected for nucleating, or capping, proteins. All these properties are compatible with their involvement in microtubule nucleation.     

Identification of F-box proteins that are involved in resistance to methylmercury in Saccharomyces cerevisiae

We searched for F-box proteins that might be related to the mechanism that protects Saccharomyces cerevisiae against the toxic effects of methylmercury. We found that overexpression of Hrt3 and of Ylr224w rendered yeast cells resistant to methylmercury. Yeast cells that overexpressed Hrt3 and Ylr224w were barely resistant to methylmercury in the presence of a proteasome inhibitor. Our results suggest the existence of some protein(s) that enhances the toxicity of methylmercury in yeast cells and, also, that overexpression of Hrt3 or Ylr224w can confer resistance to methylmercury by enhancing the polyubiquitination of this protein(s) and its degradation in proteasomes.     

Five human genes encoding F-box proteins: chromosome mapping and analysis in human tumors

Members of the F-box protein (Fbp) family are characterized by an approximately 40 amino acid F-box motif. SCF complexes (formed by Skp1, cullin, and one of many Fbps) act as protein-ubiquitin ligases that control the G(1)/S transition of the eukaryotic cell cycle. The substrate specificity of SCF complexes is determined by the presence of different Fbp subunits that recruit specific substrates for ubiquitination. Unchecked degradation of cellular regulatory proteins has been observed in certain tumors and it is possible that deregulated ubiquitin ligases play a role in the altered degradation of cell cycle regulators. We have recently identified a family of human Fbps. As a first step aimed at determining if FBP genes could be involved in human neoplasia, we have mapped the chromosome positions of 5 FBP genes by fluorescence in situ hybridization (FISH) to 10q24 (BTRC alias beta-TRCP/FBW1a), 9q34 (FBXW2 alias FBW2), 13q22 (FBXL3A alias FBL3a), 5p12 (FBXO4 alias FBX4) and 6q25-->q26 (FBXO5 alias FBX5). Since most of these are chromosomal loci frequently altered in tumors, we have screened 42 human tumor cell lines and 48 human tumor samples by Southern hybridization and FISH. While no gross alterations of the genes encoding beta-Trcp/Fbw1a, Fbw2, Fbx4 and Fbx5 were found, heterozygous deletion of the FBXL3A gene was found in four of 13 small cell carcinoma cell lines. This is the first evaluation of genes encoding Fbps in human tumors.     

Identification and characterization of a family of Rab11-interacting proteins

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.     

F-box proteins that contain sugar-binding domains

F-box proteins are the substrate-recognition subunits of the SCF (Skp1-Cul1-F-box protein) complex, which is the largest known class of E3 ubiquitin ligases. They play important roles in ubiquitin-dependent proteolysis in eukaryotes. The human genome contains about 70 genes for F-box proteins, and at least five homologous F-box proteins containing a conserved motif in their C-termini are thought to recognize sugar chain of N-linked glycoproteins. Among theses, Fbs1 and Fbs2 are perhaps involved in the endoplasmic reticulum-associated degradation pathway. In this review, I focus on the in vivo function of Fbs1 and homologous proteins, novel intracellular oligosaccharide recognition molecules involved in the quality control system.     

Identification of a novel family of G protein-coupled receptor associated sorting proteins

During the past few years several new interacting partners for G protein-coupled receptors (GPCRs) have been discovered, suggesting that the activity of these receptors is more complex than previously anticipated. Recently, candidate G protein-coupled receptor associated sorting protein (GASP-1) has been identified as a novel interacting partner for the delta opioid receptor and has been proposed to determine the degradative fate of this receptor. We show here that GASP-1 associates in vitro with other opioid receptors and that the interaction domain in these receptors is restricted to a small portion of the carboxyl-terminal tail, corresponding to helix 8 in the three-dimensional structure of rhodopsin. In addition, we show that GASP-1 interacts with COOH-terminus of several other GPCRs from subfamilies A and B and that two conserved residues within the putative helix 8 of these receptors are critical for the interaction with GASP-1. In situ hybridization and northern blot analysis indicate that GASP-1 mRNA is mainly distributed throughout the central nervous system, consistent with a potential interaction with numerous GPCRs in vivo. Finally, we show that GASP-1 is a member of a novel family comprising at least 10 members, whose genes are clustered on chromosome X. Another member of the family, GASP-2, also interacts with the carboxyl-terminal tail of several GPCRs. Therefore, GASP proteins may represent an important protein family regulating GPCR physiology.