Uncovering the Role of the Yeast Lysine Acetyltransferase NuA4 in the Regulation of Nuclear Shape and Lipid Metabolism.

Here, we report a novel role for the yeast lysine acetyltransferase NuA4 in regulating phospholipid availability for organelle morphology. Disruption of the NuA4 complex results in 70% of cells displaying nuclear deformations and nearly 50% of cells exhibiting vacuolar fragmentation. Cells deficient in NuA4 also show severe defects in the formation of nuclear-vacuole junctions (NJV), as well as a decrease in piecemeal microautophagy of the nucleus (PMN). To determine the cause of these defects we focused on Pah1, an enzyme that converts phosphatidic acid into diacylglycerol, favoring accumulation of lipid droplets over phospholipids that are used for membrane expansion. NuA4 subunit Eaf1 was required for Pah1 localization to the inner nuclear membrane and artificially tethering of Pah1 to the nuclear membrane rescued nuclear deformation and vacuole fragmentation defects, but not defects related to the formation of NVJs. Mutation of a NuA4-dependent acetylation site on Pah1 also resulted in aberrant Pah1 localization and defects in nuclear morphology and NVJ. Our work suggests a critical role for NuA4 in organelle morphology that is partially mediated through the regulation of Pah1 subcellular localization.

Modification of histidine repeat proteins by inorganic polyphosphate.

Inorganic polyphosphate (polyP) is a linear polymer of orthophosphate that is present in nearly all organisms studied to date. A remarkable function of polyP involves its attachment to lysine residues via non-enzymatic post-translational modification (PTM), which is presumed to be covalent. Here, we show that proteins containing tracts of consecutive histidine residues exhibit a similar modification by polyP, which confers an electrophoretic mobility shift on NuPAGE gels. Our screen uncovers 30 human and yeast histidine repeat proteins that undergo histidine polyphosphate modification (HPM). This polyP modification is histidine dependent and non-covalent in nature, although remarkably it withstands harsh denaturing conditions-a hallmark of covalent PTMs. Importantly, we show that HPM disrupts phase separation and the phosphorylation activity of the human protein kinase DYRK1A, and inhibits the activity of the transcription factor MafB, highlighting HPM as a potential protein regulatory mechanism.

Ddp1 Cooperates with Ppx1 to Counter a Stress Response Initiated by Nonvacuolar Polyphosphate.

In diverse cells from bacterial to mammalian species, inorganic phosphate is stored in long chains called polyphosphate (polyP). These nearly universal polymers, ranging from three to thousands of phosphate moieties in length, are associated with molecular functions, including energy homeostasis, protein folding, and cell signaling. In many cell types, polyphosphate is concentrated in subcellular compartments or organelles. In the budding yeast Saccharomyces cerevisiae, polyP synthesis by the membrane-bound vacuolar transporter chaperone (VTC) complex is coupled to its translocation into the lumen of the vacuole, a lysosome-like organelle, where it is stored at high concentrations. In contrast, the ectopic expression of the bacterial polyphosphate kinase (PPK) results in the toxic accumulation of polyP outside the vacuole. In this study, we used label-free mass spectrometry to investigate the mechanisms underlying this toxicity. We find that PPK expression results in the activation of a stress response mediated in part by the Hog1 and Yak1 kinases and the Msn2/Msn4 transcription factors as well as by changes in protein kinase A (PKA) activity. This response is countered by the combined action of the Ddp1 and Ppx1 polyphosphatases that function together to counter polyP accumulation and downstream toxicity. In contrast, the ectopic expression of previously proposed mammalian polyphosphatases did not impact PPK-mediated toxicity in this model, suggesting either that these enzymes do not function directly as polyphosphatases in vivo or that they require cofactors unique to higher eukaryotes. Our work provides insight into why polyP accumulation outside lysosome-like organelles is toxic. Furthermore, it serves as a resource for exploring how polyP may impact conserved biological processes at a molecular level. IMPORTANCE Cells from bacteria to humans have a molecule called polyphosphate (polyP) that functions in diverse processes. In many microbes, polyP is sequestered in granules or lysosome-related organelles such as vacuoles. In this study, we use an ectopic expression system to force budding yeast to accumulate polyP outside the vacuole. We use proteomics to demonstrate that this nonvacuolar polyP initiates a stress response mediated by a signaling cascade involving the Yak1 and Hog1 kinases and the Msn2 and Msn4 transcription factors. This response is countered by a pair of polyphosphatases with different enzymatic activities that function in concert to degrade polyP. Our results provide new insights into why polyP is confined to specific cell locations in many microbial cells.

Hmgcs2-mediated ketogenesis modulates high-fat diet-induced hepatosteatosis.

OBJECTIVE: Aberrant ketogenesis is correlated with the degree of steatosis in non-alcoholic fatty liver disease (NAFLD) patients, and an inborn error of ketogenesis (mitochondrial HMG-CoA synthase deficiency) is commonly associated with the development of the fatty liver. Here we aimed to determine the impact of Hmgcs2-mediated ketogenesis and its modulations on the development and treatment of fatty liver disease. METHODS: Loss- and gain-of-ketogenic function models, achieved by Hmgcs2 knockout and overexpression, respectively, were utilized to investigate the role of ketogenesis in the hepatic lipid accumulation during postnatal development and in a high-fat diet-induced NAFLD mouse model. RESULTS: Ketogenic function was decreased in NAFLD mice with a reduction in Hmgcs2 expression. Mice lacking Hmgcs2 developed spontaneous fatty liver phenotype during postnatal development, which was rescued by a shift to a low-fat dietary composition via early weaning. Hmgcs2 heterozygous adult mice, which exhibited lower ketogenic activity, were more susceptible to diet-induced NAFLD development, whereas HMGCS2 overexpression in NAFLD mice improved hepatosteatosis and glucose homeostasis. CONCLUSIONS: Our study adds new knowledge to the field of ketone body metabolism and shows that Hmgcs2-mediated ketogenesis modulates hepatic lipid regulation under a fat-enriched nutritional environment. The regulation of hepatic ketogenesis may be a viable therapeutic strategy in the prevention and treatment of hepatosteatosis.

Model systems for studying polyphosphate biology: a focus on microorganisms.

Polyphosphates (polyP) are polymers of inorganic phosphates joined by high-energy bonds to form long chains. These chains are present in all forms of life but were once disregarded as 'molecular fossils'. PolyP has gained attention in recent years following new links to diverse biological roles ranging from energy storage to cell signaling. PolyP research in humans and other higher eukaryotes is limited by a lack of suitable tools and awaits the identification of enzymatic players that would enable more comprehensive studies. Therefore, many of the most important insights have come from single-cell model systems. Here, we review determinants of polyP metabolism, regulation, and function in major microbial systems, including bacteria, fungi, protozoa, and algae. We highlight key similarities and differences that may aid in our understanding of how polyP impacts cell physiology at a molecular level.

A Broad Response to Intracellular Long-Chain Polyphosphate in Human Cells.

Polyphosphates (polyPs) are long chains of inorganic phosphates linked by phosphoanhydride bonds. They are found in all kingdoms of life, playing roles in cell growth, infection, and blood coagulation. Unlike in bacteria and lower eukaryotes, the mammalian enzymes responsible for polyP metabolism are largely unexplored. We use RNA sequencing (RNA-seq) and mass spectrometry to define a broad impact of polyP produced inside of mammalian cells via ectopic expression of the E. coli polyP synthetase PPK. We find that multiple cellular compartments can support accumulation of polyP to high levels. Overproduction of polyP is associated with reprogramming of both the transcriptome and proteome, including activation of the ERK1/2-EGR1 signaling axis. Finally, fractionation analysis shows that polyP accumulation results in relocalization of nuclear/cytoskeleton proteins, including targets of non-enzymatic lysine polyphosphorylation. Our work demonstrates that internally produced polyP can activate diverse signaling pathways in human cells.

Evidence that Bacteria Packaging by Tetrahymena Is a Widespread Phenomenon.

Protozoa are natural predators of bacteria, but some bacteria can evade digestion once phagocytosed. Some of these resistant bacteria can be packaged in the fecal pellets produced by protozoa, protecting them from physical stresses and biocides. Depending on the bacteria and protozoa involved in the packaging process, pellets can have different morphologies. In the present descriptive study, we evaluated the packaging process with 20 bacteria that have never been tested before for packaging by ciliates. These bacteria have various characteristics (shape, size, Gram staining). All of them appear to be included in pellets produced by the ciliates Tetrahymena pyriformis and/or T. thermophila in at least one condition tested. We then focused on the packaging morphology of four of these bacteria. Our results demonstrated that, as shown previously for Mycobacterium smegmatis, the packaging of Microbacterium oxydans, Micrococcus luteus, and Cupriavidus sp. was formed of a single layer of material. The packaging of Cellulosimicrobiumfunkei was made of indistinguishable material. A different pellet morphology was obtained for each of the four bacterial strains studied. The ingestion of small bacteria resulted in rounder, denser, and more regular pellets. These results support the idea that bacteria packaging is a relatively widespread phenomenon.

Proteins required for vacuolar function are targets of lysine polyphosphorylation in yeast.

Polyphosphates (polyP) are long chains of inorganic phosphates that can be attached to lysine residues of target proteins as a nonenzymatic post-translational modification. This modification, termed polyphosphorylation, may be particularly prevalent in bacterial and fungal species that synthesize large quantities of polyP. In this study, we evaluated the polyphosphorylation status of over 200 candidate targets in Saccharomyces cerevisiae. We report eight new polyphosphorylated proteins that interact genetically and physically with previous targets implicated in ribosome biogenesis. The expanded target network includes vacuolar proteins Prb1 and Apl5, whose modification with polyP suggests a model for feedback regulation of polyP synthesis, while raising questions regarding the location of polyphosphorylation in vivo.

A synthetic non-histone substrate to study substrate targeting by the Gcn5 HAT and sirtuin HDACs.

Gcn5 and sirtuins are highly conserved histone acetyltransferase (HAT) and histone deacetylase (HDAC) enzymes that were first characterized as regulators of gene expression. Although histone tails are important substrates of these enzymes, they also target many nonhistone proteins that function in diverse biological processes. However, the mechanisms used by these enzymes to choose their nonhistone substrates are unknown. Previously, we used SILAC-based MS to identify novel nonhistone substrates of Gcn5 and sirtuins in yeast and found a shared target consensus sequence. Here, we use a synthetic biology approach to demonstrate that this consensus sequence can direct acetylation and deacetylation targeting by these enzymes in vivo Remarkably, fusion of the sequence to a nonsubstrate confers de novo acetylation that is regulated by both Gcn5 and sirtuins. We exploit this synthetic fusion substrate as a tool to define subunits of the Gcn5-containing SAGA and ADA complexes required for nonhistone protein acetylation. In particular, we find a key role for the Ada2 and Ada3 subunits in regulating acetylations on our fusion substrate. In contrast, other subunits tested were largely dispensable, including those required for SAGA stability. In an extended analysis, defects in proteome-wide acetylation observed in ada3Δ mutants mirror those in ada2Δ mutants. Altogether, our work argues that nonhistone protein acetylation by Gcn5 is determined in part by specific amino acids surrounding target lysines but that even optimal sequences require both Ada2 and Ada3 for robust acetylation. The synthetic fusion substrate we describe can serve as a tool to further dissect the regulation of both Gcn5 and sirtuin activities in vivo.

Nonhistone targets of KAT2A and KAT2B implicated in cancer biology 1.

Lysine acetylation is a critical post-translation modification that can impact a protein's localization, stability, and function. Originally thought to only occur on histones, we now know thousands of nonhistone proteins are also acetylated. In conjunction with many other proteins, lysine acetyltransferases (KATs) are incorporated into large protein complexes that carry out these modifications. In this review we focus on the contribution of two KATs, KAT2A and KAT2B, and their potential roles in the development and progression of cancer. Systems biology demands that we take a broad look at protein function rather than focusing on individual pathways or targets. As such, in this review we examine KAT2A/2B-directed nonhistone protein acetylations in cancer in the context of the 10 "Hallmarks of Cancer", as defined by Hanahan and Weinberg. By focusing on specific examples of KAT2A/2B-directed acetylations with well-defined mechanisms or strong links to a cancer phenotype, we aim to reinforce the complex role that these enzymes play in cancer biology.

The fate of multilamellar bodies produced and secreted by Dictyostelium discoideum amoebae.

The amoeba Dictyostelium discoideum produces and secretes multilamellar bodies (MLBs) mainly composed of amoebal membranes upon digestion of bacteria. After their secretion, the fate of these MLBs remains unknown. The aim of this study was to determine if protozoa can internalize and digest secreted D. discoideum MLBs. Our results showed that MLBs were ingested by naive axenic D. discoideum cells (i. e. cells not exposed to bacteria and consequently not producing MLBs). Only a small fraction of the ingested MLBs were found in cells' post-lysosomes compared to undigestible beads suggesting that naive amoebae digest them. D. discoideum MLBs were also ingested by the ciliates Tetrahymena pyriformis and Tetrahymena thermophila. MLBs internalized by the ciliates were compacted into pellets and expelled in the extracellular medium without obvious signs of degradation. The results of this study provide new insights on the biological function of MLBs and, considering that MLBs are also involved in bacteria packaging, suggest additional layers of complexity in microbial interactions.

Packaging of Mycobacterium smegmatis bacteria into fecal pellets by the ciliate Tetrahymena pyriformis.

Mycobacteria are widespread microorganisms that live in various environments, including man-made water systems where they cohabit with protozoa. Environmental mycobacterial species give rise to many opportunistic human infections and can infect phagocytic protozoa. Protozoa such as amoebae and ciliates feeding on bacteria can sometimes get rid of non-digestible or pathogenic material by packaging it into secreted fecal pellets. Usually, packaged bacteria are still viable and are protected against chemical and physical stresses. We report here that mycobacteria can be packaged into pellets by ciliates. The model bacterium Mycobacterium smegmatis survived digestion in food vacuoles of the ciliate Tetrahymena pyriformis and was included in expelled fecal pellets. LIVE/DEAD® staining confirmed that packaged M. smegmatis cells preserved their viability through the process. Scanning and transmission electron microscopy revealed that bacteria are packaged in undefined filamentous and/or laminar substances and that just a thin layer of material seemed to keep the pellet contents in a spherical shape. These results imply that packaging of bacteria is more common than expected, and merits further study to understand its role in persistence and dissemination of pathogens in the environment.

Identification of Proteins Associated with Multilamellar Bodies Produced by Dictyostelium discoideum.

Dictyostelium discoideum amoebae produce and secrete multilamellar bodies (MLBs) when fed digestible bacteria. The aim of the present study was to elucidate the proteic content of MLBs. The lipid composition of MLBs is mainly amoebal in origin, suggesting that MLB formation is a protozoa-driven process that could play a significant role in amoebal physiology. We identified four major proteins on purified MLBs using mass spectrometry in order to better understand the molecular mechanisms governing MLB formation and, eventually, to elucidate the true function of MLBs. These proteins were SctA, PhoPQ, PonC and a protein containing a cytidine/deoxycytidylate deaminase (CDD) zinc-binding region. SctA is a component of pycnosomes, which are membranous materials that are continuously secreted by amoebae. The presence of SctA on MLBs was confirmed by immunofluorescence and Western blotting using a specific anti-SctA antibody. The CDD protein may be one of the proteins recognized by the H36 antibody, which was used as a MLB marker in a previous study. The function of the CDD protein is unknown. Immunofluorescence and flow cytometric analyses confirmed that the H36 antibody is a better marker of MLBs than the anti-SctA antibody. This study is an additional step to elucidate the potential role of MLBs and revealed that only a small set of proteins appeared to be present on MLBs.

Potential role of bacteria packaging by protozoa in the persistence and transmission of pathogenic bacteria.

Many pathogenic bacteria live in close association with protozoa. These unicellular eukaryotic microorganisms are ubiquitous in various environments. A number of protozoa such as amoebae and ciliates ingest pathogenic bacteria, package them usually in membrane structures, and then release them into the environment. Packaged bacteria are more resistant to various stresses and are more apt to survive than free bacteria. New evidence indicates that protozoa and not bacteria control the packaging process. It is possible that packaging is more common than suspected and may play a major role in the persistence and transmission of pathogenic bacteria. To confirm the role of packaging in the propagation of infections, it is vital that the molecular mechanisms governing the packaging of bacteria by protozoa be identified as well as elements related to the ecology of this process in order to determine whether packaging acts as a Trojan Horse.

Aeromonas salmonicida Ati2 is an effector protein of the type three secretion system.

The bacterium Aeromonas salmonicida, a fish pathogen, uses the type three secretion system (TTSS) to inject effector proteins into host cells to promote the infection. The study of the genome of A. salmonicida has revealed the existence of Ati2, a potential TTSS effector protein. In the present study, a structure-function analysis of Ati2 has been done to determine its role in the virulence of A. salmonicida. Biochemical assays revealed that Ati2 is secreted into the medium in a TTSS-dependent manner. Protein sequence analyses, molecular modelling and biochemical assays demonstrated that Ati2 is an inositol polyphosphate 5-phosphatase, which hydrolyses PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in a way similar to VPA0450, a protein from Vibrio parahaemolyticus having high sequence similarity with Ati2. Mutants of Ati2 with altered amino acids at two different locations in the catalytic site displayed no phosphatase activity. Wild-type and mutant forms of Ati2 were cloned into expression systems for Dictyostelium discoideum, a soil amoeba used as an alternative host to study A. salmonicida virulence. Expression tests allowed us to demonstrate that Ati2 is toxic for the host cell in a catalytic-dependent manner. Finally, this study demonstrated the existence of a new TTSS effector protein in A. salmonicida.