Grass Carp (Ctenopharyngodon idella) KAT8 Inhibits IFN 1 Response Through Acetylating IRF3/IRF7

Post-translational modifications (PTMs), such as phosphorylation and ubiquitination, etc., have been reported to modulate the activities of IRF3 and IRF7. In this study, we found an acetyltransferase KAT8 in grass carp (CiKAT8, MW286472) that acetylated IRF3/IRF7 and then resulted in inhibition of IFN 1 response. CiKAT8 expression was up-regulated in the cells under poly I:C, B-DNA or Z-DNA stimulation as well as GCRV(strain 873) or SVCV infection. The acetyltransferase domain (MYST domain) of KAT8 promoted the acetylation of IRF3 and IRF7 through the direct interaction with them. So, the domain is essential for KAT8 function. Expectedly, KAT8 without MYST domain (KAT8-△264-487) was granularly aggregated in the nucleus and failed to down-regulate IFN 1 expression. Subcellular localization analysis showed that KAT8 protein was evenly distributed in the nucleus. In addition, we found that KAT8 inhibited the recruitment of IRF3 and IRF7 to ISRE response element. Taken together, our findings revealed that grass carp KAT8 blocked the activities of IRF3 and IRF7 by acetylating them, resulting in a low affinity interaction of ISRE response element with IRF3 and IRF7, and then inhibiting nucleic acids-induced innate immune response.

Mitigation of Injury from Myocardial Infarction by TH1834, an Inhibitor of the Acetyltransferase Tip60

It is estimated that up to one billion cardiomyocytes (CMs) can be lost during myocardial infarction (MI), which results in contractile dysfunction, adverse ventricular remodeling, and systolic heart failure. Pharmacologic strategies that target factors having both pro-apoptotic and anti-proliferative functions in CMs may be useful for the treatment of ischemic heart disease. One such multifunctional candidate for drug targeting is the acetyltransferase Tip60, which is a member of the MYST family of acetyltransferases known to acetylate both histone and non-histone protein targets that have been shown in cultured cancer cells to promote apoptosis and to initiate the DNA damage response (DDR) thereby limiting cellular expansion. Using a murine model, we recently published findings demonstrating that CM-specific disruption of the Kat5 gene encoding Tip60 markedly protected against the damaging effects of MI. In the experiments described here, in lieu of genetic targeting, we administered TH1834, an experimental drug designed to specifically inhibit the acetyltransferase domain of Tip60. We report that, similar to the effect of disrupting the Kat5 gene, daily systemic administration of TH1834 beginning 3 days after induction of MI and continuing for two weeks of a 4-week timeline resulted in improved systolic function assessed by echocardiography, reduced apoptosis and scarring, reduced expression of markers of the DDR, and increased activation of the CM cell-cycle. Our results support that idea that drugs that inhibit the acetyltransferase activity of Tip60 may be useful agents for the treatment of ischemic heart disease.

Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

In bacteria, the nucleotide-based second messenger bis-(3’-5’)-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP effectors. We confirm that the stand-alone PilZ-domain protein PlpA is important for regulation of motility independently of the Frz chemosensory system, and that Pkn1, which is composed of a Ser/Thr kinase domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility in a Frz-dependent manner as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility independently of the Frz system likely by setting up the polarity of the two motility systems. Our results support a model whereby PlpA, PixA and PixB act in independent pathways and have distinct functions in regulation of motility. Importance c-di-GMP signaling controls bacterial motility in many bacterial species by binding to downstream effector proteins. Here, we identify two PilZ domain-containing proteins in Myxococcus xanthus that bind c-di-GMP. We show that PixB, which contains two PilZ domains and an acetyltransferase domain, acts in a manner that depends on the Frz chemosensory system to regulate motility via the acetyltransferase domain while the intact protein and c-di-GMP binding are essential for PixB to support development. By contrast, PixA acts acts in Frz-independent mannerto regulate motility. Together with previous observations, we conclude that PilZ-domain proteins and c-di-GMP act in multiple independent pathways to regulate motility and development in M. xanthus.

Three PilZ domain proteins, PlpA, PixA and PixB, have distinct functions in regulation of motility and development in Myxococcus xanthus

In bacteria, the nucleotide-based second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) binds to effectors to generate outputs in response to changes in the environment. In Myxococcus xanthus, c-di-GMP regulates type IV pili-dependent motility and the starvation-induced developmental program that results in the formation of spore-filled fruiting bodies; however, little is known about the effectors that bind c-di-GMP. Here, we systematically inactivated all 24 genes encoding PilZ domain-containing proteins, which are among the most common c-di-GMP receptors. We confirm that PlpA, a stand-alone PilZ-domain protein, is specifically important for motility and that Pkn1, which is composed of a Ser/Thr domain and a PilZ domain, is specifically important for development. Moreover, we identify two PilZ-domain proteins that have distinct functions in regulating motility and development. PixB, which is composed of two PilZ domains and an acetyltransferase domain, binds c-di-GMP in vitro and regulates type IV pili-dependent and gliding motility upstream of the Frz chemosensory system as well as development. The acetyltransferase domain is required and sufficient for function during growth while all three domains and c-di-GMP binding are essential for PixB function during development. PixA is a response regulator composed of a PilZ domain and a receiver domain, binds c-di-GMP in vitro, and regulates motility downstream of the Frz chemosensory system by setting up the polarity of the two motility systems. Our results support a model whereby the three proteins PlpA, PixA and PixB act in parallel pathways and have distinct functions to regulation of motility.

Global Lysine Crotonylation Alterations of Host Cell Proteins Caused by Brucella Effector BspF

In Brucella spp., the type IV secretion system (T4SS) is essential for bacterial intracellular survival and inhibition of the host innate immune response. The Brucella T4SS secretes 15 different effectors to escape host immunity and promote intracellular replication. Among them, BspF has a GNAT-family acetyltransferase domain, implying its acetyltransferase activity. We confirmed that BspF has acetyltransferase activity (data not shown) and de-crotonyltransferase activity. However, BspF overexpressed in HEK-293T cells can also enhance octamer crotonylation in vitro. Then we enriched crotonylated proteins and conducted LC-MS to study the crotonylation changes of proteins in HEK-293T cells caused by BspF overexpression. A total of 5,559 crotonylation sites were identified on 1,525 different proteins, of which 331 sites on 265 proteins were significantly changed. We found that Rab9A and RAP1B in proteomics data have a great impact on Brucella survival, so we speculate that BspF may influence the function of host proteins by altering crotonylation, thereby promoting the intracellular propagation of Brucella.

Crystal structure of GCN5 PCAF N-terminal domain reveals atypical ubiquitin ligase structure

General control nonderepressible 5 (GCN5, also known as Kat2a) and p300/CBP-associated factor (PCAF, also known as Kat2b) are two homologous acetyltransferases. Both proteins share similar domain architecture consisting of a PCAF N-terminal (PCAF_N) domain, acetyltransferase domain, and a bromodomain. PCAF also acts as a ubiquitin E3 ligase whose activity is attributable to the PCAF_N domain, but its structural aspects are largely unknown. Here, we demonstrated that GCN5 exhibited ubiquitination activity in a similar manner to PCAF and its activity was supported by the ubiquitin-conjugating enzyme UbcH5. Moreover, we determined the crystal structure of the PCAF_N domain at 1.8 Å resolution and found that PCAF_N domain folds into a helical structure with a characteristic binuclear zinc region, which was not predicted from sequence analyses. The zinc region is distinct from known E3 ligase structures, suggesting this region may form a new class of E3 ligase. Our biochemical and structural study provides new insight into not only the functional significance of GCN5 but also into ubiquitin biology.

Enhancer RNAs predict enhancer–gene regulatory links and are critical for enhancer function in neuronal systems

Genomic enhancer elements regulate gene expression programs important for neuronal fate and function and are implicated in brain disease states. Enhancers undergo bidirectional transcription to generate non-coding enhancer RNAs (eRNAs). However, eRNA function remains controversial. Here, we combined Assay for Transposase-Accessible Chromatin using Sequencing (ATAC-Seq) and RNA-Seq datasets from three distinct neuronal culture systems in two activity states, enabling genome-wide enhancer identification and prediction of putative enhancer–gene pairs based on correlation of transcriptional output. Notably, stimulus-dependent enhancer transcription preceded mRNA induction, and CRISPR-based activation of eRNA synthesis increased mRNA at paired genes, functionally validating enhancer–gene predictions. Focusing on enhancers surrounding the Fos gene, we report that targeted eRNA manipulation bidirectionally modulates Fos mRNA, and that Fos eRNAs directly interact with the histone acetyltransferase domain of the enhancer-linked transcriptional co-activator CREB-binding protein (CBP). Together, these results highlight the unique role of eRNAs in neuronal gene regulation and demonstrate that eRNAs can be used to identify putative target genes.

Identification of four unconventional kinetoplastid kinetochore proteins KKT22–25 in Trypanosoma brucei

The kinetochore is a multi-protein complex that drives chromosome segregation in eukaryotes. It assembles onto centromere DNA and interacts with spindle microtubules during mitosis and meiosis. Although most eukaryotes have canonical kinetochore proteins, kinetochores of evolutionarily divergent kinetoplastid species consist of at least 20 unconventional kinetochore proteins (KKT1–20). In addition, 12 proteins (KKT-interacting proteins 1–12, KKIP1–12) are known to localize at kinetochore regions during mitosis. It remains unclear whether KKIP proteins interact with KKT proteins. Here, we report the identification of four additional kinetochore proteins, KKT22–25, in Trypanosoma brucei . KKT22 and KKT23 constitutively localize at kinetochores, while KKT24 and KKT25 localize from S phase to anaphase. KKT23 has a Gcn5-related N -acetyltransferase domain, which is not found in any kinetochore protein known to date. We also show that KKIP1 co-purifies with KKT proteins, but not with KKIP proteins. Finally, our affinity purification of KKIP2/3/4/6 identifies a number of proteins as their potential interaction partners, many of which are implicated in RNA binding or processing. These findings further support the idea that kinetoplastid kinetochores are unconventional.

Development and maintenance of synaptic structure is mediated by the alpha-tubulin acetyltransferase MEC-17/αTAT1

Microtubules are fundamental elements of neuronal structure and function. They are dynamic structures formed from protofilament chains of α- and β-tubulin heterodimers. Acetylation of the lysine 40 (K40) residue of α-tubulin protects microtubules from mechanical stresses by imparting structural elasticity. The enzyme responsible for this acetylation event is MEC-17/αTAT1. However, despite its functional importance, the consequences of MEC-17/αTAT1 misregulation on neuronal structure and function are incompletely defined. Using overexpression and loss of function approaches, we have analysed the effects of MEC-17 misregulation on the development and maintenance of synaptic branches in the mechanosensory neurons of Caenorhabditis elegans. We find that synaptic branch extension is delayed, and that synaptogenesis is defective in these animals. Strikingly, by adulthood the synaptic branches specifically and spontaneously degenerate. This phenotype is dependent on the acetyltransferase domain on MEC-17, revealing that correct levels of K40 acetylation are essential for the maintenance of neuronal structure. Finally, we investigate the genetic pathways in which mec-17 functions, uncovering novel interactions with dual leucine-zipper kinase dlk-1 and the focal adhesion gene zyx-1/Zyxin. These interactions link MEC-17 together with factors involved in neuronal and actin remodelling to protect synaptic branches. Together, our results reveal that appropriate levels of α-tubulin K40 acetylation by MEC-17 are crucial for the development and maintenance of neuronal architecture.