Autoacetylation of the MYST Lysine Acetyltransferase MOF Protein

The MYST family of histone acetyltransferases (HATs) plays critical roles in diverse cellular processes, such as the epigenetic regulation of gene expression. Lysine autoacetylation of the MYST HATs has recently received considerable attention. Nonetheless, the mechanism and function of the autoacetylation process are not well defined. To better understand the biochemical mechanism of MYST autoacetylation and the impact of autoacetylation on the cognate histone acetylation, we carried out detailed analyses of males-absent-on-the-first (MOF), a key member of the MYST family. A number of mutant MOF proteins were produced with point mutations at several key residues near the active site of the enzyme. Autoradiography and immunoblotting data showed that mutation of these residues affects the autoacetylation activity and HAT activity of MOF by various degrees demonstrating that MOF activity is highly sensitive to the chemical changes in those residues. We produced MOF protein in the deacetylated form by using a nonspecific lysine deacetylase. Interestingly, both the autoacetylation activity and the histone acetylation activity of the deacetylated MOF were found to be very close to that of wild-type MOF, suggesting that autoacetylation of MOF only marginally modulates the enzymatic activity. Also, we found that the autoacetylation rates of MOF and deacetylated MOF were much slower than the cognate substrate acetylation. Thus, autoacetylation does not seem to contribute to the intrinsic enzymatic activity in a significant manner. These data provide new insights into the mechanism and function of MYST HAT autoacetylation.     

Effects of the prosegment and pH on the activity of PCSK9: evidence for additional processing events

PCSK9, a target for the treatment of dyslipidemia, enhances the degradation of the LDL receptor (LDLR) in endosomes/lysosomes, up-regulating LDL-cholesterol levels. Whereas the targeting and degradation of the PCSK9-LDLR complex are under scrutiny, the roles of the N- and C-terminal domains of PCSK9 are unknown. Although autocatalytic zymogen processing of PCSK9 occurs at Gln(152)↓, here we show that human PCSK9 can be further cleaved in its N-terminal prosegment at Arg(46)↓ by an endogenous enzyme of insect High Five cells and by a cellular mammalian protease, yielding an ～4-fold enhanced activity. Removal of the prosegment acidic stretch resulted in ～3-fold higher binding to LDLR in vitro, in ≥4-fold increased activity on cellular LDLR, and faster cellular internalization in endosome/lysosome-like compartments. Finally, swapping the acidic stretch of PCSK9 with a similar one found in the glycosylphosphatidylinositol-anchored heparin-binding protein 1 does not impair PCSK9 autoprocessing, secretion, or activity and confirmed that the acidic stretch acts as an inhibitor of PCSK9 function. We also show that upon short exposure to pH values 6.5 to 5.5, an ～2.5-fold increase in PCSK9 activity on total and cell surface LDLR occurs, and PCSK9 undergoes a second cleavage at Arg(248), generating a two-chain PCSK9-ΔN(248). At pH values below 5.5, PCSK9 dissociates from its prosegment and loses its activity. This pH-dependent activation of PCSK9 represents a novel pathway to further activate PCSK9 in acidic endosomes. These data enhance our understanding of the functional role of the acidic prosegment and on the effect of pH in the regulation of PCSK9 activity.     

High mobility group box 1 release from hepatocytes during ischemia and reperfusion injury is mediated by decreased histone deacetylase activity

The mobilization and extracellular release of nuclear high mobility group box-1 (HMGB1) by ischemic cells activates inflammatory pathways following liver ischemia/reperfusion (I/R) injury. In immune cells such as macrophages, post-translational modification by acetylation appears to be critical for active HMGB1 release. Hyperacetylation shifts its equilibrium from a predominant nuclear location toward cytosolic accumulation and subsequent release. However, mechanisms governing its release by parenchymal cells such as hepatocytes are unknown. In this study, we found that serum HMGB1 released following liver I/R in vivo is acetylated, and that hepatocytes exposed to oxidative stress in vitro also released acetylated HMGB1. Histone deacetylases (HDACs) are a family of enzymes that remove acetyl groups and control the acetylation status of histones and various intracellular proteins. Levels of acetylated HMGB1 increased with a concomitant decrease in total nuclear HDAC activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. We identified the isoforms HDAC1 and HDAC4 as critical in regulating acetylated HMGB1 release. Activation of HDAC1 was decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promoted HMGB1 translocation and release. Furthermore, we demonstrate that HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. Together, these findings suggest that decreased nuclear HDAC1 and HDAC4 activities in hepatocytes following liver I/R is a mechanism that promotes the hyperacetylation and subsequent release of HMGB1.     

Identification of Tubulin Deglutamylase among Caenorhabditis elegans and Mammalian Cytosolic Carboxypeptidases (CCPs)

Tubulin polyglutamylation is a reversible post-translational modification, serving important roles in microtubule (MT)-related processes. Polyglutamylases of the tubulin tyrosine ligase-like (TTLL) family add glutamate moieties to specific tubulin glutamate residues, whereas as yet unknown deglutamylases shorten polyglutamate chains. First we investigated regulatory machinery of tubulin glutamylation in MT-based sensory cilia of the roundworm Caenorhabditis elegans. We found that ciliary MTs were polyglutamylated by a process requiring ttll-4. Conversely, loss of ccpp-6 gene function, which encodes one of two cytosolic carboxypeptidases (CCPs), resulted in elevated levels of ciliary MT polyglutamylation. Consistent with a deglutamylase function for ccpp-6, overexpression of this gene in ciliated cells decreased polyglutamylation signals. Similarly, we confirmed that overexpression of murine CCP5, one of two sequence orthologs of nematode ccpp-6, caused a dramatic loss of MT polyglutamylation in cultured mammalian cells. Finally, using an in vitro assay for tubulin glutamylation, we found that recombinantly expressed Myc-tagged CCP5 exhibited deglutamylase biochemical activities. Together, these data from two evolutionarily divergent systems identify C. elegans CCPP-6 and its mammalian ortholog CCP5 as a tubulin deglutamylase.     

Histone Variant H2A.Z Regulates Centromere Silencing and Chromosome Segregation in Fission Yeast

The incorporation of histone variant H2A.Z into nucleosomes plays essential roles in regulating chromatin structure and gene expression. A multisubunit complex containing chromatin remodeling protein Swr1 is responsible for the deposition of H2A.Z in budding yeast and mammals. Here, we show that the JmjC domain protein Msc1 is a novel component of the fission yeast Swr1 complex and is required for Swr1-mediated incorporation of H2A.Z into nucleosomes at gene promoters. Loss of Msc1, Swr1, or H2A.Z results in loss of silencing at centromeres and defective chromosome segregation, although centromeric levels of CENP-A, a centromere-specific histone H3 variant that is required for setting up the chromatin structure at centromeres, remain unchanged. Intriguingly, H2A.Z is required for the expression of another centromere protein, CENP-C, and overexpression of CENP-C rescues centromere silencing defects associated with H2A.Z loss. These results demonstrate the importance of H2A.Z and CENP-C in maintaining a silenced chromatin state at centromeres.     

Ubiquitin Ligase Components Cullin4 and DDB1 Are Essential for DNA Methylation in Neurospora crassa

DNA methylation and H3K9 trimethylation are involved in gene silencing and heterochromatin assembly in mammals and fungi. In the filamentous fungus Neurospora crassa, it has been demonstrated that H3K9 trimethylation catalyzed by histone methyltransferase DIM-5 is essential for DNA methylation. Trimethylated H3K9 is recognized by HP1, which then recruits the DNA methyltransferase DIM-2 to methylate the DNA. Here, we show that in Neurospora, ubiquitin ligase components Cullin4 and DDB1 are essential for DNA methylation. These proteins regulate DNA methylation through their effects on the trimethylation of histone H3K9. In addition, we showed that the E3 ligase activity of the Cul4-based ubiquitin ligase is required for its function in histone H3K9 trimethylation in Neurospora. Furthermore, we demonstrated that Cul4 and DDB1 are associated with the histone methyltransferase DIM-5 protein in vivo. Together, these results suggest a mechanism for DNA methylation control that may be applicable in other eukaryotic organisms.     

Activity of a C-terminal Plant Homeodomain (PHD) of Msc1 Is Essential for Function

Msc1, a member of the Jarid1 family of putative histone demethylases, is required for chromosome stability in fission yeast. Msc1 associates with the Swr1 complex that facilitates deposition of histone H2A.Z into chromatin. To assess the function of Msc1 in the Swr1 complex, domains of Msc1 necessary for interaction with Swr1 were identified. The C-terminal plant homeodomain (PHD) 2 and PHD3 of Msc1 are sufficient to confer association with Swr1 and allow Msc1 to function in the context of kinetochore mutants. On the other hand, a mutant with a single amino acid substitution in PHD2 within the full-length Msc1 protein retains the ability to bind to Swr1 but eliminates the function of Msc1 in combination with kinetochore mutants. Thus, Swr1 association is critical but not sufficient for Msc1 function. An activity of Msc1 that depends on the cysteine residue within PHD2 of Msc1 is likewise critical for function. On the basis of our observation that the PHDs of Msc1 act as E3 ubiquitin ligases and that mutations of cysteine residues within those domains abolish ligase activity, we speculate that the ability of Msc1 to facilitate ubiquitin transfer is critical for the function it mediates through its association with Swr1.