ABSTRACT
Autophagy is an essential cellular process affecting virus infections and other diseases and Beclin1 (BECN1) is one of its key regulators. Here, we identified S-phase kinase-associated protein 2 (SKP2) as E3 ligase that executes lysine-48-linked poly-ubiquitination of BECN1, thus promoting its proteasomal degradation. SKP2 activity is regulated by phosphorylation in a hetero-complex involving FKBP51, PHLPP, AKT1, and BECN1. Genetic or pharmacological inhibition of SKP2 decreases BECN1 ubiquitination, decreases BECN1 degradation and enhances autophagic flux. Middle East respiratory syndrome coronavirus (MERS-CoV) multiplication results in reduced BECN1 levels and blocks the fusion of autophagosomes and lysosomes. Inhibitors of SKP2 not only enhance autophagy but also reduce the replication of MERS-CoV up to 28,000-fold. The SKP2-BECN1 link constitutes a promising target for host-directed antiviral drugs and possibly other autophagy-sensitive conditions.
Subject(s)
Autophagy/immunology , Beclin-1/metabolism , Coronavirus Infections/immunology , Middle East Respiratory Syndrome Coronavirus/immunology , S-Phase Kinase-Associated Proteins/metabolism , Animals , Autophagy/drug effects , Chlorocebus aethiops , Coronavirus Infections/virology , Gene Knockdown Techniques , HEK293 Cells , Humans , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Proteolysis/drug effects , S-Phase Kinase-Associated Proteins/antagonists & inhibitors , S-Phase Kinase-Associated Proteins/genetics , Ubiquitination/drug effects , Ubiquitination/immunology , Vero CellsABSTRACT
Cell-cycle progression requires careful regulation to ensure accurate propagation of genetic material to the daughter cells. Although many cell-cycle regulators are evolutionarily conserved in the protozoan parasite Trypanosoma brucei, novel regulatory mechanisms seem to have evolved. Here, we analyse the function of the histone methyltransferase DOT1A during cell-cycle progression. Over-expression of DOT1A generates a population of cells with aneuploid nuclei as well as enucleated cells. Detailed analysis shows that DOT1A over-expression causes continuous replication of the nuclear DNA. In contrast, depletion of DOT1A by RNAi abolishes replication but does not prevent karyokinesis. As histone H3K76 methylation has never been associated with replication control in eukaryotes before, we have discovered a novel function of DOT1 enzymes, which might not be unique to trypanosomes.
Subject(s)
DNA Replication , Histone-Lysine N-Methyltransferase/metabolism , Histones/metabolism , Trypanosoma brucei brucei/enzymology , Trypanosoma brucei brucei/genetics , Aneuploidy , Cell Cycle , Cell Line , Histone Methyltransferases , Histone-Lysine N-Methyltransferase/antagonists & inhibitors , Histone-Lysine N-Methyltransferase/genetics , Methylation , RNA InterferenceABSTRACT
As in most eukaryotic cells, replication is regulated by a conserved group of proteins in the early-diverged parasite Trypanosoma brucei. Only a few components of the replication machinery have been described in this parasite and regulation, sub-nuclear localization and timing of replication are not well understood. We characterized the proliferating cell nuclear antigen in T. brucei (TbPCNA) to establish a spatial and temporal marker for replication. Interestingly, PCNA distribution and regulation is different compared to the closely related parasites Trypanosoma cruzi and Leishmania donovani. TbPCNA foci are clearly detectable during S phase of the cell cycle but in contrast to T. cruzi they are not preferentially located at the nuclear periphery. Furthermore, PCNA seems to be degraded when cells enter G2 phase in T. brucei suggesting different modes of replication regulation or functions of PCNA in these closely related eukaryotes.
