Identification of critical residues of the serotype modifying O-acetyltransferase of Shigella flexneri

BACKGROUND Thirteen serotypes of Shigella flexneri (S. flexneri) have been recognised, all of which are capable of causing bacillary dysentery or shigellosis. With the emergence of the newer S. flexneri serotypes, the development of an effective vaccine has only become more challenging. One of the factors responsible for the generation of serotype diversity is an LPS O-antigen modifying, integral membrane protein known as O-acetyltransferase or Oac. Oac functions by adding an acetyl group to a specific O-antigen sugar, thus changing the antigenic signature of the parent S. flexneri strain. Oac is a membrane protein, consisting of hydrophobic and hydrophilic components. Oac bears homology to several known and predicted acetyltransferases with most homology existing in the N-terminal transmembrane (TM) regions. RESULTS In this study, the conserved motifs in the TM regions and in hydrophilic loops of S. flexneri Oac were targeted for mutagenesis with the aim of identifying the amino acid residues essential for the function of Oac. We previously identified three critical arginines-R73, R75 and R76 in the cytoplasmic loop 3 of Oac. Re-establishing that these arginines are critical, in this study we suggest a catalytic role for R73 and a structural role for R75 and R76 in O-acetylation. Serine-glycine motifs (SG 52-53, GS 138-139 and SYG 274-276), phenylalanine-proline motifs (FP 78-79 and FPV 282-84) and a tryptophan-threonine motif (WT141-142) found in TM segments and residues RK 110-111, GR 269-270 and D333 found in hydrophilic loops were also found to be critical to Oac function. CONCLUSIONS By studying the effect of the mutations on Oac's function and assembly, an insight into the possible roles played by the chosen amino acids in Oac was gained. The transmembrane serine-glycine motifs and hydrophilic residues (RK 110-111, GR 269-270 and D333) were shown to have an affect on Oac assembly which suggests a structural role for these motifs. The phenylalanine-proline and the tryptophan-threonine motifs affect Oac function which could suggest a catalytic role for these amino acids.This work was supported by a grant from the National Health and Medical Research Council of Australia

Triple-targeting Gram-negative selective antimicrobial peptides capable of disrupting the cell membrane and lipid A biosynthesis

Early lipid A biosynthesis is a potential antimicrobial target as this is an essential component of lipid A producing Gram-negative bacteria. Herein, we introduce a potent and highly synergistic Gram-negative selective triple-targeting antimicrobial peptide. The peptide contains a dual lipid A biosynthesis inhibiting sequence and an antimicrobial sequence

A role for human N-alpha acetyltransferase 30 (Naa30) in maintaining mitochondrial integrity

N-terminal acetylation (Nt-acetylation) by N-terminal acetyltransferases (NATs) is one of the most common protein modifications in eukaryotes. The NatC complex represents one of three major NATs of which the substrate profile remains largely unexplored. Here, we defined the in vivo human NatC Nt-acetylome on a proteome-wide scale by combining knockdown of its catalytic subunit Naa30 with positional proteomics. We identified 46 human NatC substrates, expanding our current knowledge on the substrate repertoire of NatC which now includes proteins harboring Met-Leu, Met-Ile, Met-Phe, Met-Trp, Met-Val, Met-Met, Met-His and Met-Lys N termini. Upon Naa30 depletion the expression levels of several organellar proteins were found reduced, in particular mitochondrial proteins, some of which were found to be NatC substrates. Interestingly, knockdown of Naa30 induced the loss of mitochondrial membrane potential and fragmentation of mitochondria. In conclusion, NatC N-tacetylates a large variety of proteins and is essential for mitochondrial integrity and function

Lifespan-increasing drug nordihydroguaiaretic acid inhibits p300 and activates autophagy.

Aging is characterized by the progressive loss of physiological function in all organisms. Remarkably, the aging process can be modulated by environmental modifications, including diet and small molecules. The natural compound nordihydroguaiaretic acid (NDGA) robustly increases lifespan in flies and mice, but its mechanism of action remains unclear. Here, we report that NDGA is an inhibitor of the epigenetic regulator p300. We find that NDGA inhibits p300 acetyltransferase activity in vitro and suppresses acetylation of a key p300 target in histones (i.e., H3K27) in cells. We use the cellular thermal shift assay to uniquely demonstrate NDGA binding to p300 in cells. Finally, in agreement with recent findings indicating that p300 is a potent blocker of autophagy, we show that NDGA treatment induces autophagy. These findings identify p300 as a target of NDGA and provide mechanistic insight into its role in longevity

TGFβ impairs HNF1α functional activity in Epithelial-to-Mesenchymal Transition interfering with the recruitment of CBP/p300 acetyltransferases

The cytokine transforming growth factor β (TGFβ) plays a crucial role in the induction of both epithelial-to-mesenchymal transition (EMT) program and fibro-cirrhotic process in the liver, where it contributes also to organ inflammation following several chronic injuries. All these pathological situations greatly increase the risk of hepatocellular carcinoma (HCC) and contribute to tumor progression. In particular, late-stage HCCs are characterized by constitutive activation of TGFβ pathway and by an EMT molecular signature leading to the acquisition of invasive and metastatic properties. In these pathological conditions, the cytokine has been shown to induce the transcriptional downregulation of HNF1α, a master regulator of the epithelial/hepatocyte differentiation and of the EMT reverse process, the mesenchymal-to-epithelial transition (MET). Therefore, the restoration of HNF1α expression/activity has been proposed as targeted therapeutic strategy for liver fibro-cirrhosis and late-stage HCCs. In this study, TGFβ is found to trigger an early functional inactivation of HNF1α during EMT process that anticipates the effects of the transcriptional downregulation of its own gene. Mechanistically, the cytokine, while not affecting the HNF1α DNA-binding capacity, impaired its ability to recruit CBP/p300 acetyltransferases on target gene promoters and, consequently, its transactivating function. The loss of HNF1α capacity to bind to CBP/p300 and HNF1α functional inactivation have been found to correlate with a change of its posttranslational modification profile. Collectively, the results obtained in this work unveil a new level of HNF1α functional inactivation by TGFβ and contribute to shed light on the early events triggering EMT in hepatocytes. Moreover, these data suggest that the use of HNF1α as anti-EMT tool in a TGFβ-containing microenvironment may require the design of new therapeutic strategies overcoming the TGFβ-induced HNF1α inactivation

Identification of a New Family of Enzymes with Potential \u3cem\u3eO\u3c/em\u3e-acetylpeptidoglycan esterase activity in both Gram-positive and Gram-negative bacteria

Background: The metabolism of the rigid bacterial cell wall heteropolymer peptidoglycan is a dynamic process requiring continuous biosynthesis and maintenance involving the coordination of both lytic and synthetic enzymes. The O-acetylation of peptidoglycan has been proposed to provide one level of control on these activities as this modification inhibits the action of the major endogenous lytic enzymes, the lytic transglycosylases. The O-acetylation of peptidoglycan also inhibits the activity of the lysozymes which serve as the first line of defense of host cells against the invasion of bacterial pathogens. Despite this central importance, there is a dearth of information regarding peptidoglycan O-acetylation and nothing has previously been reported on its de-acetylation. Results: Homology searches of the genome databases have permitted this first report on the identification of a potential family of O-Acetylpeptidoglycan esterases (Ape). These proteins encoded in the genomes of a variety of both Gram-negative and Gram-positive bacteria, including a number of important human pathogens such as species of Neisseria, Helicobacter, Campylobacter, and Bacillus anthracis, have been organized into three families based on amino acid sequence similarities with family 1 being further divided into three sub-families. The genes encoding these proteins are shown to be clustered with Peptidoglycan O-acetyltransferases (Pat) and in some cases, together with other genes involved in cell wall metabolism. Representative bacteria that encode the Ape proteins were experimentally shown to produce O-acetylated peptidoglycan. Conclusion: The hypothetical proteins encoded by the pat and ape genes have been organized into families based on sequence similarities. The Pat proteins have sequence similarity to Pseudomonas aeruginosa AlgI, an integral membrane protein known to participate in the O-acetylation of the exopolysaccaride, alginate. As none of the bacteria that harbor the pat genes produce alginate, we propose that the Pat proteins serve to O-acetylate peptidoglycan which is known to be a maturation event occurring in the periplasm. The Ape sequences have amino acid sequence similarity to the CAZy CE 3 carbohydrate esterases, a family previously known to be composed of only O-acetylxylan esterases. They are predicted to contain the α/β hydrolase fold associated with the GDSL and TesA hydrolases and they possess the signature motifs associated with the catalytic residues of the CE3 esterases. Specific signature sequence motifs were identified for the Ape proteins which led to their organization into distinct families. We propose that by expressing both Pat and Ape enzymes, bacteria would be able to obtain a high level of localized control over the degradation of peptidoglycan through the attachment and removal of O-linked acetate. This would facilitate the efficient insertion of pores and flagella, localize spore formation, and control the level of general peptidoglycan turnover

Dynamic chromatin: concerted nucleosome remodelling and acetylation

The flexibility of chromatin that enables translation of environmental cues into changes in genome utilisation, relies on a battery of enzymes able to modulate chromatin structure in a highly targeted and regulated manner. The most dynamic structural changes are brought about by two kinds of enzymes with different functional principles. Changes in the acetylation status of histones modulate the folding of the nucleosomal fibre. The histone-DNA interactions that define the nucleosome itself can be disrupted by ATP-dependent remodelling factors. This review focuses on recent developments that illustrate various strategies for integrating these disparate activities into complex regulatory schemes. Synergies may be brought about by consecutive or parallel action during the stepwise process of chromatin opening or closing. Tight co-ordination may be achieved by direct interaction of (de-)acetylation enzymes and remodelling ATPases or even permanent residence within the same multi-enzyme complex. The fact that remodelling ATPases can be acetylated by histone acetyltransferases themselves suggests exciting possibilities for the coordinate modulation of chromatin structure and remodelling enzymes

Epigenetics in ovarian cancer: premise, properties, and perspectives.

Malignant ovarian tumors bear the highest mortality rate among all gynecological cancers. Both late tumor diagnosis and tolerance to available chemical therapy increase patient mortality. Therefore, it is both urgent and important to identify biomarkers facilitating early identification and novel agents preventing recurrence. Accumulating evidence demonstrates that epigenetic aberrations (particularly histone modifications) are crucial in tumor initiation and development. Histone acetylation and methylation are respectively regulated by acetyltransferases-deacetylases and methyltransferases-demethylases, both of which are implicated in ovarian cancer pathogenesis. In this review, we summarize the most recent discoveries pertaining to ovarian cancer development arising from the imbalance of histone acetylation and methylation, and provide insight into novel therapeutic interventions for the treatment of ovarian carcinoma

Lysine acetyltransferase 5 in EGFR mutated non-small cell lung cancer

Histone modifications are crucial in activities such as transcriptional activation, gene silencing, and epigenetic cellular memory. In particular, lysine acetylation via lysine (K) acetyltransferases (KATs) has been implicated in cancer development. Interestingly, KAT5, also known as Tip60 (tat-interactive protein-60kDa), has been reported to possess both tumor promoting and tumor suppressing properties depending on the context of malignancy. Herein we report that KAT5 contributes to tumorigenesis in epidermal growth factor receptor (EGFR) mutated lung cancer, and Kat5-knockout mice models demonstrate significantly reduced lung tumor burden. To probe the aberrant modification of KAT5, we demonstrated that KAT5 binds to and is phosphorylated by oncogenic EGFR in co-immunoprecipitation experiments. Next, to investigate whether KAT5 is involved in cell proliferation and survival, H1975 cells harboring L858R-T790M double-activating mutations were transfected with doxycycline inducible short helical RNA (shRNA) targeting KAT5 (shKAT5). Following treatment, shKAT5 cells were observed to have suppressed proliferation rates. Pharmacological inhibition using TH1834, a known KAT5 inhibitor, also suppressed proliferation rates in shKAT5 cells; in contrast BEAS2B cells, an immortalized normal human bronchial cell line, surprisingly exhibited increased viability compared to transformed human lung H1975 cells. This finding supports KAT5’s context-dependent role in in normal and abnormal cell homeostasis. To further investigate KAT5 in lung tumorigenesis in vivo, we generated EGFR-mutant conditional Kat5 knockout mice using a tetracycline-induced Cre/loxP system. Following doxycycline treatment for 10 weeks, isolated mice lungs for EGFRTL/CCSP-rtTA/Cre/Kat5F/F possessed significantly lower tumor volume compared to EGFRTL/CCSP-rtTA/Cre/Kat5wt/F and EGFRTL/CCSP-rtTA/Cre/Kat5wt/wt mice lungs. Hemotoxylin and eosin staining showed no evident hyperproliferation in lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5F/F mice whereas lungs isolated from EGFRTL/CCSP-rtTA/Cre/Kat5wt/wt and EGFRTL/CCSP-rtTA/Cre/Kat5wt/F did, signifying that KAT5 has a potential regulatory role in cellular proliferation. RNA-Seq analysis of shKAT5 H1975 cells identified downstream targets involved in tumorigenic pathways. Subsequent quantitative polymerase chain reaction (PCR) of shKAT5 cells served to validate the reported targets. Taken together, these data offer insight into a KAT5 mediated oncogenic pathway that can provide novel therapeutic approaches in treating lung cancer