The transcriptional co-activator PCAF regulates cdk2 activity

Cyclin dependent kinases (cdks) regulate cell cycle progression and transcription. We report here that the transcriptional co-activator PCAF directly interacts with cdk2. This interaction is mainly produced during S and G2/M phases of the cell cycle. As a consequence of this association, PCAF inhibits the activity of cyclin/cdk2 complexes. This effect is specific for cdk2 because PCAF does not inhibit either cyclin D3/cdk6 or cyclin B/cdk1 activities. The inhibition is neither competitive with ATP, nor with the substrate histone H1 suggesting that somehow PCAF disturbs cyclin/cdk2 complexes. We also demonstrate that overexpression of PCAF in the cells inhibits cdk2 activity and arrests cell cycle progression at S and G2/M. This blockade is dependent on cdk2 because it is rescued by the simultaneous overexpression of this kinase. Moreover, we also observed that PCAF acetylates cdk2 at lysine 33. As this lysine is essential for the interaction with ATP, acetylation of this residue inhibits cdk2 activity. Thus, we report here that PCAF inhibits cyclin/cdk2 activity by two different mechanisms: (i) by somehow affecting cyclin/cdk2 interaction and (ii) by acetylating K33 at the catalytic pocket of cdk2. These findings identify a previously unknown mechanism that regulates cdk2 activity

Determinants of Consumption of Vegetables among the Spanish Population: A Cross-Sectional Study

The consumption of vegetables is one of the fundamentals of a healthy diet. The purposes of the present study were to describe the frequency of consumption of vegetables in the general Spanish population and to explore the relations between the consumption of vegetables and sex, age, cohabitation circumstances, educational level, and body mass index (BMI). Methods: An analytical cross-sectional study was accomplished based on data from the European Health Survey in Spain (2020). Results: A total of 20,745 (52.1% women) subjects with a median age of 54 years old were included. Only 2.8% of them ate vegetables at least three times a day. The adjusted generalized linear model showed that being a woman increased the odds of consuming vegetables at least three times a day by 1.666 times (p < 0.001). Not cohabiting as a couple decreased the odds by 0.783 (p < 0.001). Having studied at a university increased the odds by 1.812 times (p < 0.001) and possessing a certificate of higher education by 1.408 (p = 0.030). Being overweight decreased the odds by 0.924 (p = 0.006). For every additional year of age, the odds of consuming vegetables at least three times a day increased by 1.3% (p < 0.001). Conclusions: The vast majority of the general Spanish population did not consume an optimal amount of vegetables. Women, people with higher levels of education, and older individuals reported having a more frequent intake of vegetables. Not cohabiting as a couple and being overweight were related to a less frequent intake of vegetables

A CD317/tetherin–RICH2 complex plays a critical role in the organization of the subapical actin cytoskeleton in polarized epithelial cells

CD317/tetherin is a lipid raft–associated integral membrane protein with a novel topology. It has a short N-terminal cytosolic domain, a conventional transmembrane domain, and a C-terminal glycosyl-phosphatidylinositol anchor. We now show that CD317 is expressed at the apical surface of polarized epithelial cells, where it interacts indirectly with the underlying actin cytoskeleton. CD317 is linked to the apical actin network via the proteins RICH2, EBP50, and ezrin. Knocking down expression of either CD317 or RICH2 gives rise to the same phenotype: a loss of the apical actin network with concomitant loss of apical microvilli, an increase in actin bundles at the basal surface, and a reduction in cell height without any loss of tight junctions, transepithelial resistance, or the polarized targeting of apical and basolateral membrane proteins. Thus, CD317 provides a physical link between lipid rafts and the apical actin network in polarized epithelial cells and is crucial for the maintenance of microvilli in such cells

Antagonism of Tetherin Restriction of HIV-1 Release by Vpu Involves Binding and Sequestration of the Restriction Factor in a Perinuclear Compartment

The Vpu accessory protein promotes HIV-1 release by counteracting Tetherin/BST-2, an interferon-regulated restriction factor, which retains virions at the cell-surface. Recent reports proposed β-TrCP-dependent proteasomal and/or endo-lysosomal degradation of Tetherin as potential mechanisms by which Vpu could down-regulate Tetherin cell-surface expression and antagonize this restriction. In all of these studies, Tetherin degradation did not, however, entirely account for Vpu anti-Tetherin activity. Here, we show that Vpu can promote HIV-1 release without detectably affecting Tetherin steady-state levels or turnover, suggesting that Tetherin degradation may not be necessary and/or sufficient for Vpu anti-Tetherin activity. Even though Vpu did not enhance Tetherin internalization from the plasma membrane (PM), it did significantly slow-down the overall transport of the protein towards the cell-surface. Accordingly, Vpu expression caused a specific removal of cell-surface Tetherin and a re-localization of the residual pool of Tetherin in a perinuclear compartment that co-stained with the TGN marker TGN46 and Vpu itself. This re-localization of Tetherin was also observed with a Vpu mutant unable to recruit β-TrCP, suggesting that this activity is taking place independently from β-TrCP-mediated trafficking and/or degradation processes. We also show that Vpu co-immunoprecipitates with Tetherin and that this interaction involves the transmembrane domains of the two proteins. Importantly, this association was found to be critical for reducing cell-surface Tetherin expression, re-localizing the restriction factor in the TGN and promoting HIV-1 release. Overall, our results suggest that association of Vpu to Tetherin affects the outward trafficking and/or recycling of the restriction factor from the TGN and as a result promotes its sequestration away from the PM where productive HIV-1 assembly takes place. This mechanism of antagonism that results in TGN trapping is likely to be augmented by β-TrCP-dependent degradation, underlining the need for complementary and perhaps synergistic strategies to effectively counteract the powerful restrictive effects of human Tetherin

HIV-1 Vpu Neutralizes the Antiviral Factor Tetherin/BST-2 by Binding It and Directing Its Beta-TrCP2-Dependent Degradation

Host cells impose a broad range of obstacles to the replication of retroviruses. Tetherin (also known as CD317, BST-2 or HM1.24) impedes viral release by retaining newly budded HIV-1 virions on the surface of cells. HIV-1 Vpu efficiently counteracts this restriction. Here, we show that HIV-1 Vpu induces the depletion of tetherin from cells. We demonstrate that this phenomenon correlates with the ability of Vpu to counteract the antiviral activity of both overexpressed and interferon-induced endogenous tetherin. In addition, we show that Vpu co-immunoprecipitates with tetherin and β-TrCP in a tri-molecular complex. This interaction leads to Vpu-mediated proteasomal degradation of tetherin in a β-TrCP2-dependent manner. Accordingly, in conditions where Vpu-β-TrCP2-tetherin interplay was not operative, including cells stably knocked down for β-TrCP2 expression or cells expressing a dominant negative form of β-TrCP, the ability of Vpu to antagonize the antiviral activity of tetherin was severely impaired. Nevertheless, tetherin degradation did not account for the totality of Vpu-mediated counteraction against the antiviral factor, as binding of Vpu to tetherin was sufficient for a partial relief of the restriction. Finally, we show that the mechanism used by Vpu to induce tetherin depletion implicates the cellular ER-associated degradation (ERAD) pathway, which mediates the dislocation of ER membrane proteins into the cytosol for subsequent proteasomal degradation. In conclusion, we show that Vpu interacts with tetherin to direct its β-TrCP2-dependent proteasomal degradation, thereby alleviating the blockade to the release of infectious virions. Identification of tetherin binding to Vpu provides a potential novel target for the development of drugs aimed at inhibiting HIV-1 replication

La HDAC3 regula l’estabilitat de la ciclina A

[cat] El cicle cel•lular consta d’una sèrie de fases que donen lloc a dues cèl•lules filles genèticament idèntiques. Les diferents fases del cicle cel•lular (G1, S, G2, la mitosi i la citocinesi) són regulades per diferents complexes CDKs/ciclines. Aquests complexes estan formats per una subunitat catalítica, CDK i una subunitat reguladora, la ciclina. Els complexes CDK4-6/ciclina D regulen la transició G1/S, els complexes CDK2/ciclina A-E regulen la fase S i els complexes CDK1/ciclina A-B regulen la fase M. En aquesta tesi s’ha estudiat la regulació d’una d’aquestes ciclines, la ciclina A. Aquesta proteïna realitza funcions durant la fase S i la fase G2/M del cicle cel•lular, com per exemple, la seva implicació en l’inici i progressió de la replicació del DNA, evitar la re-replicació, regular la formació dels centrosomes, la condensació dels cromosomes i el trencament de l’embolcall nuclear. També, però s’han descrit funcions independents de CDKs, com per exemple la regulació de la invasió cel•lular a través de la via de senyalització de RhoA. La inhibició de la ciclina A provoca un retard en la metafase i anafase, causant un retard en la separació de les cromàtides germanes. En canvi, la sobreexpressió de la ciclina A provoca un avançament en la fase S. De la mateixa manera, s’han trobat nivells elevats de la ciclina A en certs tumors de mama, colon, pròstata,… A més, l’obtenció de ratolins knockout de ciclina A causa letalitat embrionària. En resum l’alteració de l’expressió de la ciclina A causa un desajustament durant el cicle cel•lular. Així doncs, els mecanismes que controlen la regulació de la ciclina A són importants pell bon funcionament de la proteïna a cada fase del cicle cel•lular. La ciclina A es degrada durant la prometafase. La causa de la seva degradació és l’acetilació per l’acetilasa P/CAF a quatre residus de lisina, els quals causen la seva ubiquitinització i posterior degradació per la via del proteasoma. El balanç acetilació/deacetilació està controlat per l’acció oposada d’acetilases i deacetilases. L’actual treball ha demostrat que la ciclina A interacciona amb la HDAC3, un proteïna que pertany a la classe I de la família de les HDACs clàssiques. Hem comprovat la interacció directa de la ciclina A i la HDAC3 a través del domini N-terminal de la ciclina A, el qual és important per a la degradació de la ciclina A. A més, aquestes proteïnes interaccionen durant les fases G1/S i G2/M. La sobreexpressió de la HDAC3 causa una disminució de l’acetilació de la ciclina A, així com la deleció de la HDAC3, provoca un increment en el seu estat d’acetilació, i per tant, una disminució de la vida mitra de la ciclina A. Finalment, hem comprovat que la HDAC3, a la vegada, també es degrada durant la mitosi, i estudis inicials en indiquen que possiblement la fosforilació de la HDAC3 regularia la seva estabilitat

Histone deacetylase 3 regulates cyclin a stability

PCAF and GCN5 acetylate cyclin A at specific lysine residues targeting it for degradation at mitosis. We report here that histone deacetylase 3 (HDAC3) directly interacts with and deacetylates cyclin A. HDAC3 interacts with a domain included in the first 171 aa of cyclin A, a region involved in the regulation of its stability. In cells, overexpression of HDAC3 reduced cyclin A acetylation whereas the knocking down of HDAC3 increased its acetylation. Moreover, reduction of HDAC3 levels induced a decrease of cyclin A that can be reversed by proteasome inhibitors. These results indicate that HDAC3 is able to regulate cyclin A degradation during mitosis via proteasome. Interestingly, HDAC3 is abruptly degraded at mitosis also via proteasome thus facilitating cyclin A acetylation by PCAF/GCN5, which will target cyclin A for degradation. Because cyclin A is crucial for S phase progression and mitosis entry, the knock down of HDAC3 affects cell cycle progression specifically at both, S phase and G2/M transition. In summary we propose here that HDAC3 regulates cyclin A stability by counteracting the action of the acetylases PCAF/GCN5.This work was supported by Grants SAF2009-07769 from the Ministerio de Ciencia e Innovación of Spain and Reticc RD06/0020/0010 from the Istituto de Salud Carlos III.Peer Reviewe

Ubiquitin-Proteasome System, Dynamics and Targeting Acetylation of cyclin A: a new cell cycle regulatory mechanism

Abstract Cyclin A must be degraded at prometaphase in order to allow mitosis progression. Nevertheless, the signals that trigger cyclin A degradation at mitosis have been largely elusive. In the present paper, we review the status of cyclin A degradation in the light of recent evidence indicating that acetylation plays a role in cyclin A stability. leading to its ubiquitylation by the anaphase-promoting factor/cyclosome and its subsequent degradation via proteasome. Interestingly, these four lysine residues in cyclin A also participate in the regulation of cyclin A-Cdk (cyclin-dependent kinase) activity by modulating its interaction with Cdks. Degradation of cyclins A and B is needed for mitosis progression Cell cycle progression is governed by the family of Cdks (cyclin-dependent kinases) [1]. Their activities are regulated by binding to regulatory subunits called cyclins, by phosphorylation at specific sites and by binding to inhibitory proteins [2]. During cell cycle, specific pairs of cyclin-Cdks are formed and activated. Cdk1 together with cyclins A and B governs G 2 /M transition. G 1 progression is under the control of cyclin D-Cdk4/6. Cyclin E-Cdk2 triggers DNA synthesis and cyclin A-Cdk2 drives S-phase progression Key words: acetylation, cAMP-response-element-binding protein (CREB), p300/CREB-binding protein-associated factor (PCAF), cyclin A, degradation, ubiquitylation. Abbreviations used: APC/C, anaphase-promoting complex/cyclosome; Cdc20, cell division cycle 20; Cdk, cyclin-dependent kinase; Cks, Cdk subunit; CREB, cAMP-response-element-binding protein; cycA 4R, cyclin A in which Lys 54 , Lys 68 , Lys 95 and Lys 112 were replaced by arginine residues; cycA 4Q, cyclin A in which Lys 54 , Lys 68 , Lys 95 and Lys 112 were replaced by glutamine residues; GCN5, general control non-derepressible 5; HDAC, histone deacetylase; PCAF, p300/CREB-binding protein-associated factor; WT, wild-type