Masayoshi Yada

Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7

The F‐box protein Skp2 mediates c‐Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c‐Myc is largely dependent on phosphorylation of threonine‐58 and serine‐62 in MB1, residues that are often mutated in cancer. We now show that the F‐box protein Fbw7 interacts with and thereby destabilizes c‐Myc in a manner dependent on phosphorylation of MB1. Whereas wild‐type Fbw7 promoted c‐Myc turnover in cells, an Fbw7 mutant lacking the F‐box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c‐Myc. Accumulation of c‐Myc was also apparent in mouse Fbw7−/− embryonic stem cells. These observations suggest that two F‐box proteins, Fbw7 and Skp2, differentially regulate c‐Myc stability by targeting MB1 and MB2, respectively.

Degradation of p57Kip2 mediated by SCFSkp2-dependent ubiquitylation

The abundance of the cyclin-dependent kinase (CDK) inhibitor p57Kip2, an important regulator of cell cycle progression, is thought to be controlled by the ubiquitin-proteasome pathway. The Skp1/Cul1/F-box (SCF)-type E3 ubiquitin ligase complex SCFSkp2 has now been shown to be responsible for regulating the cellular level of p57Kip2 by targeting it for ubiquitylation and proteolysis. The elimination of p57Kip2 was impaired in Skp2-/- cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Skp2 interacts with p57Kip2 in vivo. Overexpression of WT Skp2 promoted degradation of p57Kip2, whereas expression of a dominant negative mutant of Skp2 prolonged the half-life of p57Kip2. Mutation of the threonine residue (Thr-310) of human p57Kip2 that is conserved between the COOH-terminal QT domains of p57Kip2 and p27Kip1 prevented the effect of Skp2 on the stability of p57Kip2, suggesting that phosphorylation at this site is required for SCFSkp2-mediated ubiquitylation. Finally, the purified recombinant SCFSkp2 complex mediated p57Kip2 ubiquitylation in vitro in a manner dependent on the presence of the cyclin E-CDK2 complex. These observations thus demonstrate that the SCFSkp2 complex plays an important role in cell-cycle progression by determining the abundance of p57Kip2 and that of the related CDK inhibitor p27Kip1.

Molecular clearance of ataxin‐3 is regulated by a mammalian E4

Insoluble aggregates of polyglutamine‐containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin‐3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin‐3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin‐3. Expression of E4B promoted degradation of a pathological form of ataxin‐3. In contrast, a dominant‐negative mutant of E4B inhibited degradation of this form of ataxin‐3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin‐3 mutant. These observations suggest that E4 is a rate‐limiting factor in the degradation of pathological forms of ataxin‐3, and that targeted expression of E4B is a potential gene therapy for SCA3.

Interaction of U‐box‐type ubiquitin‐protein ligases (E3s) with molecular chaperones

Members of the U‐box family of proteins constitute a class of ubiquitin‐protein ligases (E3s) distinct from the HECT‐type and RING finger‐containing E3 families. Two representative mammalian U‐box proteins, UFD2a and CHIP, interact with the molecular chaperones VCP and either Hsp90 or Hsc70, respectively, and are implicated in the degradation of damaged proteins. We have now investigated the roles of mammalian U‐box proteins by performing a comprehensive screen for molecules that interact with these proteins in the yeast two‐hybrid system. All mammalian U‐box proteins tested were found to interact with molecular chaperones or cochaperones, including Hsp90, Hsp70, DnaJc7, EKN1, CRN, and VCP. These observations suggest that the function of U box‐type E3s is to mediate the degradation of unfolded or misfolded proteins in conjunction with molecular chaperones as receptors that recognize such abnormal proteins.

Preferential interaction of TIP120A with Cul1 that is not modified by NEDD8 and not associated with Skp1.

The SCF complex, which consists of the invariable components Skp1, Cul1, and Rbx1 as well as a variable F-box protein, functions as an E3 ubiquitin ligase. The mechanism by which the activity of this complex is regulated, however, has been unclear. The application of tandem affinity purification has now resulted in the identification of a novel Cul1-binding protein: TATA-binding protein-interacting protein 120A (TIP120A, also called CAND1). Immunoprecipitation, immunoblot, and immunofluorescence analyses with mammalian cells revealed that TIP120A physically associates with Cul1 in the nucleus and that this interaction is mediated by a central region of Cul1 distinct from its binding sites for Skp1 and Rbx1. Furthermore, TIP120A was shown to interact selectively with Cul1 that is not modified by NEDD8. The Cul1-TIP120A complex does not include Skp1, raising the possibility that TIP120A competes with Skp1 for binding to Cul1. These observations thus suggest that TIP120A may function as a negative regulator of the SCF complex by binding to nonneddylated Cul1 and thereby preventing assembly of this ubiquitin ligase.

Multiple Skp1-related proteins in Caenorhabditis elegans: diverse patterns of interaction with Cullins and F-box proteins.

BACKGROUND The ubiquitin-proteasome pathway of proteolysis controls the abundance of specific regulatory proteins. The SCF complex is a type of ubiquitin-protein ligase (E3) that contributes to this pathway in many biological systems. In yeast and mammals, the SCF complex consists of common components, including Skp1, Cdc53/Cul1, and Rbx1, as well as variable components known as F-box proteins. Whereas only one functional Skp1 gene is present in the human genome, the genome of Caenorhabditis elegans has now been shown to contain at least 21 Skp1-related (skr) genes. The biochemical properties, expression, and function of the C. elegans SKR proteins were examined. RESULTS Of the 17 SKR proteins examined, eight (SKR-1, -2, -3, -4, -7, -8, -9, and -10) were shown to interact with C. elegans CUL1 by yeast two-hybrid analysis or a coimmunoprecipitation assay in mammalian cells. Furthermore, SKR proteins exhibited diverse binding specificities for C. elegans F-box proteins. The tissue specificity of expression of the CUL1-interacting SKR proteins was also varied. Suppression of skr-1 or skr-2 genes by double-stranded RNA interference resulted in embryonic death, whereas that of skr-7, -8, -9, or -10 was associated with slow growth and morphological abnormalities. CONCLUSIONS The multiple C. elegans SKR proteins exhibit marked differences in their association with Cullins and F-box proteins, in tissue specificity of expression, and in phenotypes associated with functional suppression by RNAi. At least eight of the SKR proteins may, like F-box proteins, act as variable components of the SCF complex in C. elegans.

Mammalian E4 Is Required for Cardiac Development and Maintenance of the Nervous System

ABSTRACT Ubiquitin conjugation typically requires three classes of enzyme: E1, E2, and E3. A fourth type of enzyme (E4), however, was recently shown to be required for the degradation of certain types of substrate in yeast. We previously identified UFD2a (also known as E4B) as an E4 in mammals. UFD2a is exclusively expressed in cardiac muscle during mouse embryonic development, but it is abundant in neurons of adult mice and is implicated in the pathogenesis of neurodegenerative disease. The precise physiological function of this enzyme has remained largely unknown, however. Here, we show that mice lacking UFD2a die in utero, manifesting marked apoptosis in the developing heart. Polyubiquitylation activity for an E4 substrate was greatly reduced in Ufd2a−/− mouse embryonic fibroblasts. Furthermore, Ufd2a +/− mice displayed axonal dystrophy in the nucleus gracilis, as well as degeneration of Purkinje cells accompanied by endoplasmic reticulum stress. These animals also developed a neurological disorder. UFD2a thus appears to be essential for the development of cardiac muscle, as well as for the protection of spinocerebellar neurons from degeneration induced by endoplasmic reticulum stress.

Fbxw7 contributes to tumor suppression by targeting multiple proteins for ubiquitin-dependent degradation

Fbxw7 (also known as Sel‐10, hCdc4 or hAgo) is the F‐box protein component of a Skp1–Cul1–F‐box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin‐mediated degradation of cyclin E, c‐Myc, Aurora‐A, Notch and c‐Jun, all of which appear to function as cell‐cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7‐mutant cancer cells. In the present study, we examined the abundance of cyclin E, c‐Myc and Aurora‐A in seven cancer cell lines, which harbor wild‐type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7‐mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c‐Myc and Aurora‐A in the Fbxw7‐mutant cells. In contrast, a decrease in the expression of cyclin E, c‐Myc or Aurora‐A by RNA interference significantly suppressed the rate of proliferation and anchorage‐independent growth of the Fbxw7‐mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c‐Myc and Aurora‐A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development. (Cancer Sci 2006; 97: 729–736)

Expression profile of lipid metabolism‐associated genes in hepatitis C virus‐infected human liver

Aim:  Recent studies have shown that lipid metabolic pathways are required for the entry, replication and secretion of hepatitis C virus (HCV). Although little is known about the life cycle of HCV in humans, the activation of cholesterol and fatty acid biosynthesis may be critical for HCV proliferation.

Expression profiles of genes associated with viral entry in HCV‐infected human liver

Recent studies have demonstrated that several cellular factors are involved in entry of hepatitis C virus (HCV) into host cells. Detailed gene expression profiles of these factors in HCV‐infected livers have not been reported for humans. Transcriptional levels of LDL receptor (LDLR), CD81, scavenger receptor class B type I (SR‐BI), claudin‐1, and occludin genes in liver samples from patients with chronic hepatitis C were investigated. Serum levels of LDL‐cholesterol (LDL‐C) and HCV core antigen were also evaluated, and expression of claudin‐1 and occludin were immunohistochemically analyzed. Compared with normal liver, transcription of LDLR and claudin‐1 genes was significantly suppressed (P < 0.0001) and occludin transcription was significantly up‐regulated in HCV‐infected livers (P < 0.0001). Significant positive correlations were found for LDLR versus occludin, LDLR versus claudin‐1, occludin versus claudin‐1, and CD81 versus SR‐BI in HCV‐infected (P = 0.0012, P < 0.0001, P = 0.0004, and P < 0.0001, respectively) and normal livers (P < 0.0001, P = 0.0051, P < 0.0001, and P < 0.0001, respectively). Positive correlation was observed between serum levels of HCV core antigen and LDL‐C (P = 0.0147), with their levels negatively correlated to LDLR (P = 0.0270 and P = 0.0021, respectively). Immunohistochemically, hepatocellular expression of claudin‐1 and occludin was increased in HCV‐infected livers. Different levels of expression were demonstrated at the mRNA and protein levels for occludin and claudin‐1 in HCV‐infected and normal livers. Correlation of elements associated with viral entry was comparable in HCV‐infected and normal livers. J. Med. Virol. 83:921–927, 2011.