Mesh1

The stringent response, originally identified in Escherichia coli as a signal that leads to reprogramming of gene expression under starvation or nutrient deprivation, is now recognized as ubiquitous in all bacteria, and also as part of a broader survival strategy in diverse, other stress conditions. Much of our insight into this phenomenon derives from the role of hyperphosphorylated guanosine derivatives (pppGpp, ppGpp, pGpp; guanosine penta-, tetra- and tri-phosphate, respectively) that are synthesized on starvation cues and act as messengers or alarmones...

MESH1 is the metazoan homolog of bacterial SpoT, the main phosphatase that dephosphorylates and degrades (p)ppGpp, the alarmone involved in the bacterial stringent response. The functional role of MESH1 in human cells is unknown. To define the global transcriptional response to MESH1 knockdown, we employed microarrays to perform transcriptome analysis of H1975 when the MESH1 was knocked down using three independent siRNAs targeting MESH1. The changes of each gene were derived by zero-transformation, followed by filtering to derive the genes affected by MESH1 knockdown...

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Small alarmone hydrolases (SAHs) are alarmone metabolizing enzymes found in both metazoans and bacteria. In metazoans, the SAH homolog Mesh1 is reported to function in cofactor metabolism by hydrolyzing NADPH to NADH. In bacteria, SAHs are often identified in genomes with toxic alarmone synthetases for self-resistance. Here, we characterized a bacterial orphan SAH, i.e., without a toxic alarmone synthetase, in the phytopathogen Xanthomonas campestris pv. campestris ( Xcc SAH) and found that it metabolizes both cellular alarmones and cofactors...

All organisms are constantly exposed to various stresses, necessitating adaptive strategies for survival. In bacteria, the main metabolic stress-coping mechanism is the stringent response, which is triggered by the accumulation of "alarmone" (p)ppGpp to arrest proliferation and reprogram the transcriptome. The level of (p)ppGpp is regulated by its synthetase RelA and its hydrolase SpoT. MESH1 is the metazoan homolog of bacterial SpoT that regulates the bacterial stringent response by degrading the alarmone (p)ppGpp...

Growth and division are central to cell size. Bacteria achieve size homeostasis by dividing when growth has added a constant size since birth, termed the adder principle, by unknown mechanisms.1 , 2 Growth is well known to be regulated by guanosine tetraphosphate (ppGpp), which controls diverse processes from ribosome production to metabolic enzyme activity and replication initiation and whose absence or excess can induce stress, filamentation, and small growth-arrested cells.3-6 These observations raise unresolved questions about the relation between ppGpp and size homeostasis mechanisms during normal exponential growth...

While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments...

All organisms exposed to metabolic and environmental stresses have developed various stress adaptive strategies to maintain homeostasis. The main bacterial stress survival mechanism is the stringent response triggered by the accumulation "alarmone" (p)ppGpp, whose level is regulated by RelA and SpoT. While metazoan genomes encode MESH1 (Metazoan SpoT Homolog 1) with ppGpp hydrolase activity, neither ppGpp nor the stringent response is found in metazoa. The deletion of Mesh1 in Drosophila triggers a transcriptional response reminiscent of the bacterial stringent response...

A biomembrane's role is to be a barrier for interior cytosol from an exterior environment to execute the cell's normal biological functions. However, a water-soluble peptide called cell-penetrating peptide (CPP) has been known for its ability to directly penetrate through the biomembranes into cells (cytolysis) without perturbating cell viability and expected to be a promising drug delivery vector. Examples of CPP include peptides with multiple arginine units with strong cationic properties, which is the key to cytolysis...

The Mesh1 class of hydrolases found in bacteria, metazoans and humans was discovered as able to cleave an intact pyrophosphate residue esterified on the 3'hydroxyl of (p)ppGpp in a Mn2+ dependent reaction. Here, thin layer chromatography (TLC) qualitative evidence is presented indicating the substrate specificity of Mesh1 from Drosophila melanogaster and human MESH1 also extends to the (p)ppApp purine analogs. More importantly, we developed real time enzymatic assays, coupling ppNpp hydrolysis to NADH oxidation and pppNpp hydrolysis to NADP+ reduction, which facilitate estimation of kinetic constants...

Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila...

Cell-penetrating peptide (CPP) can directly penetrate the cytosol (cytolysis) and is expected to be a potent vector for a drug delivery system (DDS). Although there is general agreement that CPP cytolysis is related to dynamic membrane deformation, a distinctive process has yet to be established. Here, we report the key process and factors controlling CPP cytolysis. To elucidate the task, we have introduced trypsin digestion of adsorbed CPP onto giant unilamellar vesicle (GUV) to quantify the adsorption and internalization (cytolysis) separately...

Critical to the bacterial stringent response is the rapid relocation of resources from proliferation toward stress survival through the respective accumulation and degradation of (p)ppGpp by RelA and SpoT homologues. While mammalian genomes encode MESH1, a homologue of the bacterial (p)ppGpp hydrolase SpoT, neither (p)ppGpp nor its synthetase has been identified in mammalian cells. Here, we show that human MESH1 is an efficient cytosolic NADPH phosphatase that facilitates ferroptosis. Visualization of the MESH1-NADPH crystal structure revealed a bona fide affinity for the NADPH substrate...

Understanding how bacteria coordinate gene expression with biomass growth to adapt to various stress conditions remains a grand challenge in biology. Stress response is often associated with dramatic accumulation of cellular guanosine tetra- or penta-phosphate (p)ppGpp (also known as 'magic spot'), which is a key second messenger participating in regulating various biochemical and physiological processes of bacteria. Despite of the extensive studies on the mechanism of gene regulation by (p)ppGpp during stringent response, the connection between (p)ppGpp and bacterial steady-state exponential growth remains elusive...

Bacteria living in marine environment encounter various challenges and limitations, thus in order to survive, they need to employ efficient stress-response mechanisms. One of these mechanisms is the stringent response, where unusual nucleotides, guanosine tetra- and pentaphosphates, herald starvation and physico-chemical stresses. All so far sequenced free-living bacteria contain the gene(s) responsible for (p)ppGpp synthesis - rsh (named after Escherichia coli genes, relA and spoT). Two similar genes were identified mostly in β- and γ-proteobacteria while other bacteria have only one gene coding the dual function of (p)ppGpp synthesis and degradation...

In nutrient-starved bacteria, RelA and SpoT proteins have key roles in reducing cell growth and overcoming stresses. Here we identify functional SpoT orthologs in metazoa (named Mesh1, encoded by HDDC3 in human and Q9VAM9 in Drosophila melanogaster) and reveal their structures and functions. Like the bacterial enzyme, Mesh1 proteins contain an active site for ppGpp hydrolysis and a conserved His-Asp-box motif for Mn(2+) binding. Consistent with these structural data, Mesh1 efficiently catalyzes hydrolysis of guanosine 3',5'-diphosphate (ppGpp) both in vitro and in vivo...