Unexpected Histone H3 Tail-clipping Activity of Glutamate Dehydrogenase

Clipping of histone tails has been reported in several organisms. However, the significance and regulation of histone tail clipping largely remains unclear. According to recent discoveries H3 clipping has been found to be involved in regulation of gene expression and chromatin dynamics. Earlier we had provided evidence of tissue-specific proteolytic processing of histone H3 in White Leghorn chicken liver nuclei. In this study we identify a novel activity of glutamate dehydrogenase (GDH) as a histone H3-specific protease in chicken liver tissue. This protease activity is regulated by divalent ions and thiol-disulfide conversion in vitro. GDH specifically clips H3 in its free as well as chromatin-bound form. Furthermore, we have found an inhibitor that inhibits the H3-clipping activity of GDH. Like previously reported proteases, GDH too may have the potential to regulate/modulate post-translational modifications of histone H3 by removing the N-terminal residues of the histone. In short, our findings identify an unexpected proteolytic activity of GDH specific to histone H3 that is regulated by redox state, ionic concentrations, and a cellular inhibitor in vitro.     

JMJD5 cleaves monomethylated histone H3 N‐tail under DNA damaging stress

The histone H3 N‐terminal protein domain (N‐tail) is regulated by multiple posttranslational modifications, including methylation, acetylation, phosphorylation, and by proteolytic cleavage. However, the mechanism underlying H3 N‐tail proteolytic cleavage is largely elusive. Here, we report that JMJD5, a Jumonji C (JmjC) domain‐containing protein, is a Cathepsin L‐type protease that mediates histone H3 N‐tail proteolytic cleavage under stress conditions that cause a DNA damage response. JMJD5 clips the H3 N‐tail at the carboxyl side of monomethyl‐lysine (Kme1) residues. In vitro H3 peptide digestion reveals that JMJD5 exclusively cleaves Kme1 H3 peptides, while little or no cleavage effect of JMJD5 on dimethyl‐lysine (Kme2), trimethyl‐lysine (Kme3), or unmethyl‐lysine (Kme0) H3 peptides is observed. Although H3 Kme1 peptides of K4, K9, K27, and K36 can all be cleaved by JMJD5 in vitro, K9 of H3 is the major cleavage site in vivo, and H3.3 is the major H3 target of JMJD5 cleavage. Cleavage is enhanced at gene promoters bound and repressed by JMJD5 suggesting a role for H3 N‐tail cleavage in gene expression regulation.     

Peptidomimetic ethyl propenoate covalent inhibitors of the enterovirus 71 3C protease: a P2–P4 study

Enterovirus 71 (EV71) is a highly infectious pathogen primarily responsible for Hand, Foot, and Mouth Disease, particularly among children. Currently, no approved antiviral drug has been developed against this disease. The EV71 3C protease is deemed an attractive drug target due to its crucial role in viral polyprotein processing. Rupintrivir, a peptide-based inhibitor originally developed to target the human rhinovirus 3C protease, was found to inhibit the EV71 3C protease. In this communication, we report the inhibitory activities of 30 Rupintrivir analogs against the EV71 3C protease. The most potent inhibitor, containing a P2 ring-constrained phenylalanine analog (compound 9), was found to be two-fold more potent than Rupintrivir (IC₅₀ value 3.4?±?0.4 versus 7.3?±?0.8?μM). Our findings suggest that employing geometrically constrained residues in peptide-based protease inhibitors can potentially enhance their inhibitory activities.     

UHRF1 double tudor domain and the adjacent PHD finger act together to recognize K9me3-containing histone H3 tail

Human multi-domain-containing protein UHRF1 has recently been extensively characterized as a key epigenetic regulator for maintaining DNA methylation patterns. UHRF1 SRA domain preferentially binds to hemimethylated CpG sites, and double Tudor domain has been implicated in recognizing H3K9me3 mark, but the role of the adjacent PHD finger remains unclear. Here, we report the high-resolution crystal structure of UHRF1 PHD finger in complex with N-terminal tail of histone H3. We found that the preceding zinc-Cys4 knuckle is indispensable for the PHD finger of UHRF1 to recognize the first four unmodified residues of histone H3 N-terminal tail. Quantitative binding studies indicated that UHRF1 PHD finger (including the preceding zinc-Cys4 knuckle) acts together with the adjacent double Tudor domain to specifically recognize the H3K9me3 mark. Combinatorial recognition of H3K9me3-containing histone H3 tail by UHRF1 PHD finger and double Tudor domain may play a role in establishing and maintaining histone H3K9 methylation patterns during the cell cycle.     

Fragment-wise design of inhibitors to 3C proteinase from enterovirus 71

BackgroundEnterovirus 71 (EV71) is a causative agent of hand, foot and mouth disease (HFMD), which can spread its infection to central nervous and other systems with severe consequence. A key factor in the replication of EV71 is its 3C proteinase (3C^pro), a significant drug target. Peptidomimetics were employed as inhibitors of this enzyme for developing antivirals. However, the peptide bonds in these peptidomimetics are a source of low bioavailability due to their susceptibility to protease digestion. To produce non-peptidomimetic inhibitors by replacing these peptide bonds, it would be important to gain better understanding on the contribution of each component to the interaction and potency.MethodsA series of compounds of different lengths targeting 3C^pro and having an α,β-unsaturated ester as the warhead were synthesized and their interactions with the enzyme were evaluated by complex structure analyses and potency assays for a better understanding on the relationship between potency and evolution of interaction.ResultsThe P2 moiety of the compound would need to be oriented to interact in the S2 site in the substrate binding cleft and the P3–P4 moieties were required to generate sufficient potency. A hydrophobic terminal group will benefit the cellular uptake and improve the activity in vivo.Conclusions and general significanceThe data presented here provide a basis for designing a new generation of non-peptidomimetics to target EV71 3C^pro.     

Meiosis-specific loading of the centromere-specific histone CENH3 in Arabidopsis thaliana

Centromere behavior is specialized in meiosis I, so that sister chromatids of homologous chromosomes are pulled toward the same side of the spindle (through kinetochore mono-orientation) and chromosome number is reduced. Factors required for mono-orientation have been identified in yeast. However, comparatively little is known about how meiotic centromere behavior is specialized in animals and plants that typically have large tandem repeat centromeres. Kinetochores are nucleated by the centromere-specific histone CENH3. Unlike conventional histone H3s, CENH3 is rapidly evolving, particularly in its N-terminal tail domain. Here we describe chimeric variants of CENH3 with alterations in the N-terminal tail that are specifically defective in meiosis. Arabidopsis thaliana cenh3 mutants expressing a GFP-tagged chimeric protein containing the H3 N-terminal tail and the CENH3 C-terminus (termed GFP-tailswap) are sterile because of random meiotic chromosome segregation. These defects result from the specific depletion of GFP-tailswap protein from meiotic kinetochores, which contrasts with its normal localization in mitotic cells. Loss of the GFP-tailswap CENH3 variant in meiosis affects recruitment of the essential kinetochore protein MIS12. Our findings suggest that CENH3 loading dynamics might be regulated differently in mitosis and meiosis. As further support for our hypothesis, we show that GFP-tailswap protein is recruited back to centromeres in a subset of pollen grains in GFP-tailswap once they resume haploid mitosis. Meiotic recruitment of the GFP-tailswap CENH3 variant is not restored by removal of the meiosis-specific cohesin subunit REC8. Our results reveal the existence of a specialized loading pathway for CENH3 during meiosis that is likely to involve the hypervariable N-terminal tail. Meiosis-specific CENH3 dynamics may play a role in modulating meiotic centromere behavior.     

Differential miRNA expression profiling of highly pathogenic avian influenza virus H5N1 infected chicken lungs reveals critical microRNAs,biological pathways and genes involved in the molecular pathogenesis

Highlights
· Highthrouput sequencing of small RNA of H5N1 infected and mock infected chicken lungs.
· 297 miRNAs identified in mock-infected and 201 miRNAs identified in AIV infected chicken lungs.
· 36 miRNAs were upregulated and 90 were downregulated during H5N1 infection.
· Functional analysis and gene ontology of predicted target genes of expressed miRNAs.
· MAPK pathway, NF-κB, IGF and gga-let-7b might play important role during H5N1 pathogenesis.     

The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation

Plant homeodomain (PHD) fingers are often present in chromatin-binding proteins and have been shown to bind histone H3 N-terminal tails. Mutations in the autoimmune regulator (AIRE) protein, which harbours two PHD fingers, cause a rare monogenic disease, autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). AIRE activates the expression of tissue-specific antigens by directly binding through its first PHD finger (AIRE-PHD1) to histone H3 tails non-methylated at K4 (H3K4me0). Here, we present the solution structure of AIRE-PHD1 in complex with H3K4me0 peptide and show that AIRE-PHD1 is a highly specialized non-modified histone H3 tail reader, as post-translational modifications of the first 10 histone H3 residues reduce binding affinity. In particular, H3R2 dimethylation abrogates AIRE-PHD1 binding in vitro and reduces the in vivo activation of AIRE target genes in HEK293 cells. The observed antagonism by R2 methylation on AIRE-PHD1 binding is unique among the H3K4me0 histone readers and represents the first case of epigenetic negative cross-talk between non-methylated H3K4 and methylated H3R2. Collectively, our results point to a very specific histone code responsible for non-modified H3 tail recognition by AIRE-PHD1 and describe at atomic level one crucial step in the molecular mechanism responsible for antigen expression in the thymus.     

Chicken liver glutamate dehydrogenase (GDH) demonstrates a histone H3 specific protease (H3ase) activity in vitro

Site-specific proteolysis of the N or C-terminus of histone tails has emerged as a novel form of irreversible post-translational modifications assigned to histones. Though there are many reports describing histone specific proteolysis, there are very few studies on purification of a histone specific protease. Here, we demonstrate a histone H3 specific protease (H3ase) activity in chicken liver nuclear extract. H3ase was purified to homogeneity and identified as glutamate dehydrogenase (GDH) by sequencing. A series of biochemical experiments further confirmed that the H3ase activity was due to GDH. The H3ase clipped histone H3 products were sequenced by N-terminal sequencing and the precise clipping sites of H3ase were mapped. H3ase activity was only specific to chicken liver as it was not demonstrated in other tissues like heart, muscle and brain of chicken. We assign a novel serine like protease activity to GDH which is specific to histone H3.     

Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3C^pro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C^pro. CstF-64 was cleaved in vitro by 3C^pro but neither by mutant 3C^pro (in which the catalytic site was inactivated) nor by another EV71 protease 2A^pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C^pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3C^pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C^pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.     

Transgenerational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

Centromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes, CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is reestablished on chromatin during diplotene of meiosis I. Here, we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but then its presence becomes dispensable for centromere maintenance during development. Worms homozygous for a CENP-A tail deletion maintain functional centromeres during development but give rise to inviable offspring because they fail to reestablish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2 and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

This study of the nematode Caenorhabditis elegans shows that centromere identity is set in the maternal germ line and passed on to the progeny via an epigenetic mechanism that requires the N-terminal tail of the centromeric histone H3 variant CENP-A.     

EV71 3C protease cleaves host anti-viral factor OAS3 and enhances virus replication

The global spread of enteroviruses (EVs) has become more frequent, severe and life-threatening. Intereron (IFN) I has been proved to control EVs by regulating IFN-stimulated genes (ISG) expression. 20-50-oligoadenylate synthetases 3 (OAS3) is an important ISG in the OAS/RNase L antiviral system. The relationship between OAS3 and EVs is still unclear. Here, we reveal that OAS3, superior to OAS1 and OAS2, significantly inhibited EV71 replication in vitro. However, EV71 utilized autologous 3C protease (3C^pro) to cleave intracellular OAS3 and enhance viral replication. Rupintrivir, a human rhinovirus 3C protease inhibitor, completely abolished the cleavage of EV71 3C^pro on OAS3. And the proteolytically deficient mutants H40G, E71A, and C147G of EV71 3C^pro also lost the ability of OAS3 cleavage. Mechanistically, the Q982-G983 motif in C-terminal of OAS3 was identified as a crucial 3C^pro cutting site. Further investigation indicated that OAS3 inhibited not only EV71 but also Coxsackievirus B3 (CVB3), Coxsackievirus A16 (CA16), Enterovirus D68 (EVD68), and Coxsackievirus A6 (CA6) subtypes. Notably, unlike other four subtypes, CA16 3C^pro could not cleave OAS3. Two key amino acids variation Ile36 and Val86 in CA16 3C^pro might result in weak and delayed virus replication of CA16 because of failure of OAS and 3AB cleavage. Our works elucidate the broad anti-EVs function of OAS3, and illuminate a novel mechanism by which EV71 use 3C^pro to escape the antiviral effect of OAS3. These findings can be an important entry point for developing novel therapeutic strategies for multiple EVs infection.