CiteULike: kieran101's ubiquitin

"HIV-1 Vif induces cell cycle delay via recruitment of the same E3 ubiquitin ligase complex that targets APOBEC3 proteins for degradation"

Jason L Dehart — 2008-07-11T12:06:05-00:00

Journal of virology (2 July 2008)

HIV-1 Vif recruits a Cullin5 ubiquitin ligase that targets APOBEC3 proteins for degradation. Recently, Vif has also been shown to induce cell cycle disturbance in G2. We show that, in contrast with Vpr, expression of Vif does not preclude cell division and, therefore, Vif causes delay, and not arrest, in G2. We also demonstrate that interaction of Vif with the ubiquitin ligase is required for cell cycle disruption, as was previously shown for HIV-1 Vpr. The presence of APOBEC3 D/E, F and G had no influence on Vif-induced alteration of the cell cycle. We conclude that cell cycle delay by Vif is a result of ubiquitination and degradation of a cellular protein that is different from the known APOBEC3 family members.

Assembly with the Cul4A-DDB1-DCAF1 ubiquitin ligase protects HIV-1 Vpr from proteasomal degradation

Erwann Le Rouzic — 2008-06-09T09:34:31-00:00

J. Biol. Chem. (4 June 2008), M710298200.

Many viruses subvert the host ubiquitin-proteasome system to optimize their life cycle. We recently documented such a mechanism for the HIV-1 Vpr protein, which promotes cell cycle arrest by recruiting the DCAF1 adaptor of the Cul4A-DDB1 ubiquitin ligase, a finding now confirmed by several groups. Here we examined the impact of Cul4A-DDB1DCAF1 on Vpr stability. We show that the Vpr Q65R mutant, which is defective in DCAF1 binding, undergoes proteasome-mediated degradation at a higher rate than wild-type Vpr. DCAF1 overexpression stabilizes Vpr wt and leads to its cytoplasmic accumulation, whereas it has no effect on the Vpr Q65R mutant. Conversely siRNA-mediated silencing of DCAF1 decreases the steady state amount of the viral protein. Stabilization by DCAF1, which is conserved by Vpr species from HIV-2 and SIVmac, results in increased G2 arrest and requires the presence of DDB1, indicating that it occurs through assembly of Vpr with a functional Cul4A-DDB1DCAF1 complex. Furthermore in HIV-1 infected cells, the Vpr protein, issued from the incoming viral particle, is destabilized under DCAF1 or DDB1 silencing. Together with our previous findings, our data suggest that Cul4A-DDB1DCAF1 acts at a dual level by providing Vpr with the equipment for the degradation of specific host proteins and by counteracting its proteasome targeting by another cellular E3 ubiquitin ligase. This protection mechanism may represent an efficient way to optimize the activity of Vpr molecules that are delivered by the incoming virus before neosynthesis takes place. Targeting the Vpr-DCAF1 interaction might therefore present therapeutic interest. 10.1074/jbc.M710298200

Hsp90 inhibition results in autophagy-mediated proteasome-independent degradation of I[kappa]B kinase (IKK)

Guoliang Qing — 2008-05-27T12:37:29-00:00

Cell Res, Vol. 16, No. 11. (0000), pp. 895-901.

Unified nomenclature for the COP9 signalosome and its subunits: an essential regulator of development

Xing-Wang Deng — 2008-05-27T12:36:04-00:00

Trends in Genetics, Vol. 16, No. 5. (1 May 2000), pp. 202-203.

Linking of Autophagy to Ubiquitin-Proteasome System Is Important for the Regulation of Endoplasmic Reticulum Stress and Cell Viability

Ding — 2007-08-25T23:20:29-00:00

American Journal of Pathology, Vol. 171, No. 2. (1 August 2007), pp. 513-524.

Cytoplasmic Lipid Droplets Are Sites of Convergence of Proteasomal and Autophagic Degradation of Apolipoprotein B

Yuki Ohsaki — 2008-05-22T16:26:39-00:00

Mol. Biol. Cell, Vol. 17, No. 6. (1 June 2006), pp. 2674-2683.

Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make "ApoB-crescents." ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50-100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12-24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two pathways. 10.1091/mbc.E05-07-0659

NEDD4L Overexpression Rescues the Release and Infectivity of Human Immunodeficiency Virus Type 1 Constructs Lacking PTAP and YPXL Late Domains

Hyo-Young Chung — 2008-05-15T17:16:50-00:00

J. Virol., Vol. 82, No. 10. (15 May 2008), pp. 4884-4897.

The cellular ESCRT pathway functions in membrane remodeling events that accompany endosomal protein sorting, cytokinesis, and enveloped RNA virus budding. In the last case, short sequence motifs (termed late domains) within human immunodeficiency virus type 1 (HIV-1) p6Gag bind and recruit two ESCRT pathway proteins, TSG101 and ALIX, to facilitate virus budding. We now report that overexpression of the HECT ubiquitin E3 ligase, NEDD4L/NEDD4-2, stimulated the release of HIV-1 constructs that lacked TSG101- and ALIX-binding late domains, increasing infectious titers >20-fold. Furthermore, depletion of endogenous NEDD4L inhibited the release of these crippled viruses and led to cytokinesis defects. Stimulation of virus budding was dependent upon the ubiquitin ligase activity of NEDD4L and required only the minimal HIV-1 Gag assembly regions, demonstrating that Gag has ubiquitin-dependent, cis-acting late domain activities located outside of the p6 region. NEDD4L stimulation also required TSG101 and resulted in ubiquitylation of several ESCRT-I subunits, including TSG101. Finally, we found that TSG101/ESCRT-I was required for efficient release of Mason-Pfizer monkey virus, which buds primarily by using a PPXY late domain to recruit NEDD4-like proteins. These observations suggest that NEDD4L and possibly other NEDD4-like proteins can ubiquitylate and activate ESCRT-I to function in virus budding. 10.1128/JVI.02667-07

E3 Ubiquitin Ligases as Regulators of Membrane Protein Trafficking and Degradation

Alessandra D'azzo — 2005-05-10T15:39:26-00:00

Traffic, Vol. 6, No. 6. (June 2005), pp. 429-441.

The novel functions of ubiquitination in signaling.

L Sun — 2007-09-17T12:59:17-00:00

Curr Opin Cell Biol, Vol. 16, No. 2. (April 2004), pp. 119-126.

Ubiquitin is best known for its function in targeting proteins for degradation by the proteasome. Recent studies have revealed several new functions of ubiquitin that are independent of proteasomal degradation. These functions include the novel signaling roles of ubiquitin in DNA repair and the activation of protein kinases such as IkappaB kinase. In both cases, a novel form of polyubiquitin chain linked through lysine-63 of ubiquitin plays an important regulatory role. Monoubiquitination also has signaling roles that are distinct from those of polyubiquitination, as illustrated from the studies of DNA repair. Thus, polyubiquitination and monoubiquitination have emerged as important signaling mechanisms that control diverse physiological and pathological processes.

Distinct monoubiquitin signals in receptor endocytosis

Kaisa Haglund — 2008-05-15T17:07:24-00:00

Trends in Biochemical Sciences, Vol. 28, No. 11. (November 2003), pp. 598-604.

Numerous cellular proteins are post-translationally modified by the addition of the small modifier protein ubiquitin (Ub). The functional consequences of the type of ubiquitination vary, such that polyubiquitinated proteins are targeted for degradation by the proteasome, whereas monoubiquitination is implicated in other cellular functions, including endocytic trafficking and DNA repair. The monoubiquitination of trafficking cargoes, such as receptors and associated proteins, as well as of endocytic accessory Ub-binding proteins, raises the question of the precise function of monoubiquitin signals in the endocytic route. Recent biochemical and genetic evidence shows that multiple monoubiquitination of epidermal growth factor and platelet-derived growth factor receptors provides trafficking and sorting tags that ensure receptor endocytosis and degradation, whereas monoubiquitination of accessory proteins might play a role in regulating their function as Ub-receptors in the endosome.

Signaling through monoubiquitination.

S Sigismund — 2007-05-30T13:26:06-00:00

Curr Top Microbiol Immunol, Vol. 286 (2004), pp. 149-185.

Ubiquitination is a post-translational modification in which a small conserved peptide, ubiquitin, is appended to target proteins in the cell, through a series of complex enzymatic reactions. Recently, a particular form of ubiquitination, monoubiquitination, has emerged as a nonproteolytic reversible modification that controls protein function. In this review, we highlight recent findings on monoubiquitination as a signaling-induced modification, controlled, among others, by pathways originating from active receptor tyrosine kinases. Furthermore, we review the major cellular processes controlled by ubiquitin modification, including membrane trafficking, histone function, transcription regulation, DNA repair, and DNA replication.

A New Ticket for Entry into Budding Vesicles--Ubiquitin

Linda Hicke — 2008-05-15T16:54:05-00:00

Cell, Vol. 106, No. 5. (7 September 2001), pp. 527-530.

Protein sorting into multivesicular endosomes

Camilla Raiborg — 2008-05-15T16:49:20-00:00

Current Opinion in Cell Biology, Vol. 15, No. 4. (August 2003), pp. 446-455.

Multivesicular endosomes are important as compartments for receptor downregulation and as intermediates in the formation of secretory lysosomes. Work during the past year has shed light on the molecular mechanisms of protein sorting into multivesicular endosomes and yielded information about the machinery involved in multivesicular endosome formation. Monoubiquitination functions as a signal for sorting transmembrane proteins into intraluminal vesicles of multivesicular endosomes and subsequent delivery to lysosomes. A molecular machinery that contains the ubiquitin-binding protein Hrs/Vps27 appears to be central in this sorting process. Three conserved multisubunit complexes, ESCRT-I, -II and -III, are essential for both sorting and multivesicular endosomes formation. Enveloped RNA viruses such as HIV can redirect these complexes from multivesicular endosomes to the plasma membrane to facilitate viral budding.

Efficient and Specific Rescue of Human Immunodeficiency Virus Type 1 Budding Defects by a Nedd4-Like Ubiquitin Ligase

Yoshiko Usami — 2008-05-15T16:23:02-00:00

J. Virol., Vol. 82, No. 10. (15 May 2008), pp. 4898-4907.

To exit infected cells, human immunodeficiency virus type 1 (HIV-1) exploits the vacuolar protein-sorting pathway by engaging Tsg101 and ALIX through PTAP and LYPxnL late assembly (L) domains. In contrast, less-complex retroviruses often use PPxY L domains to recruit Nedd4 family ubiquitin ligases. Although HIV-1 Gag lacks PPxY motifs, we now show that the budding of various HIV-1 L-domain mutants is dramatically enhanced by ectopic Nedd4-2s, a native isoform with a truncated C2 domain. The effect of Nedd4-2s on HIV-1 budding required a catalytically active HECT domain and was specific, since other Nedd4 family proteins showed little activity and an unrelated retrovirus was not rescued. The residual C2 domain of Nedd4-2s was critical for the enhancement of HIV-1 budding and for the association of Nedd4-2s with Gag, as reflected by its incorporation into virus-like particles. Interestingly, the incorporation of Nedd4-2s also depended on its active site, indicating that the ability to form a thioester with ubiquitin was required. These data suggest a novel mechanism by which HIV-1 Gag can connect to cellular budding machinery. 10.1128/JVI.02675-07

The HIV-1 Vif Protein Mediates Degradation of Vpr and Reduces Vpr-Induced Cell Cycle Arrest

Jiangfang Wang — 2008-05-12T09:22:50-00:00

DNA and Cell Biology, Vol. 27, No. 5. (2008), pp. 267-277.

Prior work has implicated viral protein R (Vpr) in the arrest of human immunodeficiency virus type 1 (HIV-1)-infected cells in the G2 phase of the cell cycle, associated with increased viral replication and host cell apoptosis. We and others have recently shown that virion infectivity factor (Vif ) also plays a role in the G2 arrest of HIV-1-infected cells. Here, we demonstrate that, paradoxically, at early time points postinfection, Vif expression blocks Vpr-mediated G2 arrest, while deletion of Vif from the HIV-1 genome leads to a marked increase in G2 arrest of infected CD4 T-cells. Consistent with this increased G2 arrest, T-cells infected with Vif-deleted HIV-1 express higher levels of Vpr protein than cells infected with wild-type virus. Further, expression of exogenous Vif inhibits the expression of Vpr, associated with a decrease in G2 arrest of both infected and transfected cells. Treatment with the proteasome inhibitor MG132 increases Vpr protein expression and G2 arrest in wild-type, but not Vif-deleted, NL4-3-infected cells, and in cells cotransfected with Vif and Vpr. In addition, Vpr coimmunoprecipitates with Vif in cotransfected cells in the presence of MG132. This suggests that inhibition of Vpr by Vif is mediated at least in part by proteasomal degradation, similar to Vif-induced degradation of APOBEC3G. Together, these data show that Vif mediates the degradation of Vpr and modulates Vpr-induced G2 arrest in HIV-1-infected T-cells.

HIV-1 Vpr-mediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase.

JP Belzile — 2008-05-12T09:21:57-00:00

PLoS pathogens, Vol. 3, No. 7. (July 2007)

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has been shown to cause G2 cell cycle arrest in human cells by inducing ATR-mediated inactivation of p34cdc2, but factors directly engaged in this process remain unknown. We used tandem affinity purification to isolate native Vpr complexes. We found that damaged DNA binding protein 1 (DDB1), viral protein R binding protein (VPRBP), and cullin 4A (CUL4A)--components of a CUL4A E3 ubiquitin ligase complex, DDB1-CUL4A(VPRBP)--were able to associate with Vpr. Depletion of VPRBP by small interfering RNA impaired Vpr-mediated induction of G2 arrest. Importantly, VPRBP knockdown alone did not affect normal cell cycle progression or activation of ATR checkpoints, suggesting that the involvement of VPRBP in G2 arrest was specific to Vpr. Moreover, leucine/isoleucine-rich domain Vpr mutants impaired in their ability to interact with VPRBP and DDB1 also produced strongly attenuated G2 arrest. In contrast, G2 arrest-defective C-terminal Vpr mutants were found to maintain their ability to associate with these proteins, suggesting that the interaction of Vpr with the DDB1-VPRBP complex is necessary but not sufficient to block cell cycle progression. Overall, these results point toward a model in which Vpr could act as a connector between the DDB1-CUL4A(VPRBP) E3 ubiquitin ligase complex and an unknown cellular factor whose proteolysis or modulation of activity through ubiquitination would activate ATR-mediated checkpoint signaling and induce G2 arrest.

The Role of Ubiquitin in Retroviral Egress

Martin-Serrano — 2007-09-14T14:46:14-00:00

Traffic, Vol. 8, No. 10. (October 2007), pp. 1297-1303.

A ubiquitin-like system mediates protein lipidation.

Y Ichimura — 2008-04-10T13:26:47-00:00

Nature, Vol. 408, No. 6811. (23 November 2000), pp. 488-492.

Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.

Vif Overcomes the Innate Antiviral Activity of APOBEC3G by Promoting Its Degradation in the Ubiquitin-Proteasome Pathway

Andrew Mehle — 2008-04-10T08:22:43-00:00

J. Biol. Chem., Vol. 279, No. 9. (27 February 2004), pp. 7792-7798.

Viruses must overcome diverse intracellular defense mechanisms to establish infection. The Vif (virion infectivity factor) protein of human immunodeficiency virus 1 (HIV-1) acts by overcoming the antiviral activity of APOBEC3G (CEM15), a cytidine deaminase that induces G to A hypermutation in newly synthesized viral DNA. In the absence of Vif, APOBEC3G incorporation into virions renders HIV-1 non-infectious. We report here that Vif counteracts the antiviral activity of APOBEC3G by targeting it for destruction by the ubiquitin-proteasome pathway. Vif forms a complex with APOBEC3G and enhances APOBEC3G ubiquitination, resulting in reduced steady-state APOBEC3G levels and a decrease in protein half-life. Furthermore, Vif-dependent degradation of APOBEC3G is blocked by proteasome inhibitors or ubiquitin mutant K48R. A mutation of highly conserved cysteines or the deletion of a conserved SLQ(Y/F)LA motif in Vif results in mutants that fail to induce APOBEC3G degradation and produce non-infectious HIV-1; however, mutations of conserved phosphorylation sites in Vif that impair viral replication do not affect APOBEC3G degradation, suggesting that Vif is important for other functions in addition to inducing proteasomal degradation of APOBEC3G. Vif is monoubiquitinated in the absence of APOBEC3G but is polyubiquitinated and rapidly degraded when APOBEC3G is coexpressed, suggesting that coexpression accelerates the degradation of both proteins. These results suggest that Vif functions by targeting APOBEC3G for degradation via the ubiquitin-proteasome pathway and implicate the proteasome as a site of dynamic interplay between microbial and cellular defenses. 10.1074/jbc.M313093200