Identification of a family of human F-box proteins.

F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) [1]. Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins [2] [3]. In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs. The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1). SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins. The need for high substrate specificity and the large number of known F-box proteins in yeast and worms [2] [4] suggest the existence of a large family of mammalian F-box proteins. Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel. Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs. We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs. We have further characterized representative members of these three classes of F-box proteins.     

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.     

The FBXL family of F-box proteins: variations on a theme

The ubiquitin–proteasome system (UPS) is responsible for the rapid targeting of proteins for degradation at 26S proteasomes and requires the orchestrated action of E1, E2 and E3 enzymes in a well-defined cascade. F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases that determine which proteins are ubiquitinated. To date, around 70 FBPs have been identified in humans and can be subdivided into distinct families, based on the protein-recruiting domains they possess. The FBXL subfamily is defined by the presence of multiple leucine-rich repeat (LRR) protein-binding domains. But how the 22 FBPs of the FBXL family achieve their individual specificities, despite having highly similar structural domains to recruit their substrates, is not clear. Here, we review and explore the FBXL family members in detail highlighting their structural and functional similarities and differences and how they engage their substrates through their LRRs to adopt unique interactomes.     

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.     

The F-box protein family

The F-box is a protein motif of approximately 50 amino acids that functions as a site of protein-protein interaction. F-box proteins were first characterized as components of SCF ubiquitin-ligase complexes (named after their main components, Skp I, Cullin, and an F-box protein), in which they bind substrates for ubiquitin-mediated proteolysis. The F-box motif links the F-box protein to other components of the SCF complex by binding the core SCF component Skp I. F-box proteins have more recently been discovered to function in non-SCF protein complexes in a variety of cellular functions. There are 11 F-box proteins in budding yeast, 326 predicted in Caenorhabditis elegans, 22 in Drosophila, and at least 38 in humans. F-box proteins often include additional carboxy-terminal motifs capable of protein-protein interaction; the most common secondary motifs in yeast and human F-box proteins are WD repeats and leucine-rich repeats, both of which have been found to bind phosphorylated substrates to the SCF complex. The majority of F-box proteins have other associated motifs, and the functions of most of these proteins have not yet been defined.     

cDNA cloning and expression analysis of new members of the mammalian F-box protein family

F-box proteins are critical components of the SCF ubiquitin-protein ligase complex and are involved in substrate recognition and recruitment for ubiquitination and consequent degradation by the proteasome. We have isolated cDNAs encoding a further 10 mammalian F-box proteins. Five of them (FBL3 to FBL7) share structural similarities with Skp2 and contain C-terminal leucine-rich repeats. The other 5 proteins have different putative protein-protein interaction motifs. Specifically, FBS and FBWD4 proteins contain Sec7 and WD40-repeat domains, respectively. The C-terminal region of FBA shares similarity with bacterial protein ApaG while FBG2 shows homology with the F-box protein NFB42. The marked differences in F-box gene expression in human tissues suggest their distinct role in ubiquitin-dependent protein degradation.     

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.     

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。     

Secretion of the galectin family of mammalian carbohydrate-binding proteins

Galectins are cytosolic proteins that lack any signal sequence for transport into the endoplasmic reticulum and are not glycosylated, although several galectins contain consensus sites for N-glycosylation, indicating that these proteins do not traverse the ER-Golgi network. However, there is abundant evidence for the extracellular localisation of some galectins at cell surfaces, in the extracellular matrix and in cell secretions consistent with other evidence for extracellular roles of galectins as modulators of cell adhesion and signalling. How then are galectins secreted if not through the classical secretory pathway? Do all galectins share the same secretory pathway? Can a particular galectin utilise more than one secretory pathway? If galectins play important extracellular roles how is their secretion regulated in relation to function? These are still largely unanswered questions but recent studies are beginning to give glimpses into some novel aspects of the secretion of these intriguing proteins.     

Ubiquitination-mediated degradation of cell cycle-related proteins by F-box proteins

F-box proteins, subunits of SKP1-cullin 1-F-box protein (SCF) type of E3 ubiquitin ligase complexes, have been validated to play a crucial role in governing various cellular processes such as cell cycle, cell proliferation, apoptosis, migration, invasion and metastasis. Recently, a wealth of evidence has emerged that F-box proteins is critically involved in tumorigenesis in part through governing the ubiquitination and subsequent degradation of cell cycle proteins, and dysregulation of this process leads to aberrant cell cycle progression and ultimately, tumorigenesis. Therefore, in this review, we describe the critical role of F-box proteins in the timely regulation of cell cycle. Moreover, we discuss how F-box proteins involve in tumorigenesis via targeting cell cycle-related proteins using biochemistry studies, engineered mouse models, and pathological gene alternations. We conclude that inhibitors of F-box proteins could have promising therapeutic potentials in part through controlling of aberrant cell cycle progression for cancer therapies.     

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.     

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.     

Skp1 stabilizes the conformation of F-box proteins

The SCF ubiquitin ligase complex consists of four components, Skp1, Cul1, ROC1/Rbx1, and a variable subunit F-box protein, which serves as a receptor for target proteins. The F-box proteins consist of an N-terminal ∼40 amino acid F-box domain that binds to Skp1 and the C-terminal substrate-binding domain. We have reported previously that Fbs1 and Fbs2 are N-linked glycoprotein-specific F-box proteins. In addition, other three F-box proteins, Fbg3, Fbg4, and Fbg5, show high homology to Fbs1 and Fbs2, but their functions remain largely unknown. Here we report that Skp1 assists in correct folding of exogenously expressed F-box proteins. Fbs2 as well as Fbg3, Fbg4, and Fbg5 proteins formed SCF complexes but did not bind to N-glycoproteins when exogenously expressed alone. However, co-expression of Fbs2 and Fbg5 with Skp1 facilitated their binding to glycoproteins that reacted with ConA. Furthermore, Skp1 increased the cellular concentrations of F-box proteins by preventing aggregate formation. These observations suggest that Skp1 plays an important role in stabilizing the conformation of these F-box proteins, which increases their expression levels and substrate-binding.     

Roles of F-box proteins in plant hormone responses

The F-box protein is an important component of the E3 ubiquitin ligase Skpl-Cullin-F-box protein complex. It binds specific substrates for ubiquitin-mediated proteolysis. The F-box proteins contain a signature F-box motif at their amino-terminus and some protein-protein interaction motifs at their carboxyterminus, such as Trp-Asp repeats or leucine rich repeats. Many F-box proteins have been identified to be involved in plant hormone response as receptors or important medial components. These breakthrough findings shed light on our current understanding of the structure and function of the various F-box proteins, their related plant hormone signaling pathways, and their roles in regulating plant development.