In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second ...messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.
The conformational landscape of a protein is constantly expanded by genetic variations that have a minimal impact on the function(s) while causing subtle effects on protein structure. The wider the ...conformational space sampled by these variants, the higher the probabilities to adapt to changes in environmental conditions. However, the probability that a single mutation may result in a pathogenic phenotype also increases. Here we present a paradigmatic example of how protein evolution balances structural stability and dynamics to maximize protein adaptability and preserve protein fitness. We took advantage of known genetic variations of human alanine:glyoxylate aminotransferase (AGT1), which is present as a common major allelic form (AGT‐Ma) and a minor polymorphic form (AGT‐Mi) expressed in 20% of Caucasian population. By integrating crystallographic studies and molecular dynamics simulations, we show that AGT‐Ma is endowed with structurally unstable (frustrated) regions, which become disordered in AGT‐Mi. An in‐depth biochemical characterization of variants from an anticonsensus library, encompassing the frustrated regions, correlates this plasticity to a fitness window defined by AGT‐Ma and AGT‐Mi. Finally, co‐immunoprecipitation analysis suggests that structural frustration in AGT1 could favor additional functions related to protein–protein interactions. These results expand our understanding of protein structural evolution by establishing that naturally occurring genetic variations tip the balance between stability and frustration to maximize the ensemble of conformations falling within a well‐defined fitness window, thus expanding the adaptability potential of the protein.
DOPA decarboxylase, the dimeric enzyme responsible for the synthesis of neurotransmitters dopamine and serotonin, is involved in severe neurological diseases such as Parkinson disease, schizophrenia, ...and depression. Binding of the pyridoxal-5′-phosphate (PLP) cofactor to the apoenzyme is thought to represent a central mechanism for the regulation of its activity. We solved the structure of the human apoenzyme and found it exists in an unexpected open conformation: compared to the pig kidney holoenzyme, the dimer subunits move 20 Å apart and the two active sites become solvent exposed. Moreover, by tuning the PLP concentration in the crystals, we obtained two more structures with different conformations of the active site. Analysis of three-dimensional data coupled to a kinetic study allows to identify the structural determinants of the open/close conformational change occurring upon PLP binding and thereby propose a model for the preferential degradation of the apoenzymes of Group II decarboxylases.
Nutrient utilization and reshaping of metabolism in cancer cells is a well-known driver of malignant transformation. Less clear is the influence of the local microenvironment on metastasis formation ...and choice of the final organ to invade. Here we show that the level of the amino acid serine in the cytosol affects the migratory properties of lung adenocarcinoma (LUAD) cells. Inhibition of serine or glycine uptake from the extracellular milieu, as well as knockdown of the cytosolic one-carbon metabolism enzyme serine hydroxymethyltransferase (SHMT1), abolishes migration. Using rescue experiments with a brain extracellular extract, and direct measurements, we demonstrate that cytosolic serine starvation controls cell movement by increasing reactive oxygen species formation and decreasing ATP levels, thereby promoting activation of the AMP sensor kinase (AMPK) by phosphorylation. Activation of AMPK induces remodeling of the cytoskeleton and finally controls cell motility. These results highlight that cytosolic serine metabolism plays a key role in controlling motility, suggesting that cells are able to dynamically exploit the compartmentalization of this metabolism to adapt their metabolic needs to different cell functions (movement vs. proliferation). We propose a model to explain the relevance of serine/glycine metabolism in the preferential colonization of the brain by LUAD cells and suggest that the inhibition of serine/glycine uptake and/or cytosolic SHMT1 might represent a successful strategy to limit the formation of brain metastasis from primary tumors, a major cause of death in these patients.
Cancer cells reprogramme one‐carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by ...reversibly converting serine and tetrahydrofolate (THF) into 5,10‐methylene‐THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1.
Database
Structural data are available in the PDB under the accession number 6FL5
We designed a set of three serine hydroxymethyltransferase 1 (SHMT1) mutants to uncouple the oligomeric state of the enzyme from its catalytic activity. We investigated the effects of these mutations on nuclear localization, cell viability, and crosstalk between SHMT1 and SHMT2 in lung cancer cells. In these cell lines, de novo thymidylate synthesis requires SHMT1 to be active regardless of its oligomeric state.
Adaptive metabolic reprogramming gives cancer cells a proliferative advantage. Tumour cells extensively use glycolysis to sustain anabolism and produce serine, which not only refuels the one‐carbon ...units necessary for the synthesis of nucleotide precursors and for DNA methylation, but also affects the cellular redox homeostasis. Given its central role in serine metabolism, serine hydroxymethyltransferase (SHMT), a pyridoxal 5′‐phosphate (PLP)‐dependent enzyme, is an attractive target for tumour chemotherapy. In humans, the cytosolic isoform (SHMT1) and the mitochondrial isoform (SHMT2) have distinct cellular roles, but high sequence identity and comparable catalytic properties, which may complicate development of successful therapeutic strategies. Here, we investigated how binding of the cofactor PLP controls the oligomeric state of the human isoforms. The fact that eukaryotic SHMTs are tetrameric proteins while bacterial SHMTs function as dimers may suggest that the quaternary assembly in eukaryotes provides an advantage to fine‐tune SHMT function and differentially regulate intertwined metabolic fluxes, and may provide a tool to address the specificity problem. We determined the crystal structure of SHMT2, and compared it to the apo‐enzyme structure, showing that PLP binding triggers a disorder‐to‐order transition accompanied by a large rigid‐body movement of the two cofactor‐binding domains. Moreover, we demonstrated that SHMT1 exists in solution as a tetramer, both in the absence and presence of PLP, while SHMT2 undergoes a dimer‐to‐tetramer transition upon PLP binding. These findings indicate an unexpected structural difference between the two human SHMT isoforms, which opens new perspectives for understanding their differing behaviours, roles or regulation mechanisms in response to PLP availability in vivo.
Cystic fibrosis (CF) is an autosomal recessive disorder characterized by respiratory failure due to a vicious cycle of defective Cystic Fibrosis Transmembrane conductance Regulator (CFTR) function, ...chronic inflammation and recurrent bacterial and fungal infections. Although the recent introduction of CFTR correctors/potentiators has revolutionized the clinical management of CF patients, resurgence of inflammation and persistence of pathogens still posit a major concern and should be targeted contextually. On the background of a network-based selectivity that allows to target the same enzyme in the host and microbes with different outcomes, we focused on sphingosine-1-phosphate (S1P) lyase (SPL) of the sphingolipid metabolism as a potential candidate to uniquely induce anti-inflammatory and antifungal activities in CF. As a feasibility study, herein we show that interfering with S1P metabolism improved the immune response in a murine model of CF with aspergillosis while preventing germination of Aspergillus fumigatus conidia. In addition, in an early drug discovery process, we purified human and A. fumigatus SPL, characterized their biochemical and structural properties, and performed an in silico screening to identify potential dual species SPL inhibitors. We identified two hits behaving as competitive inhibitors of pathogen and host SPL, thus paving the way for hit-to-lead and translational studies for the development of drug candidates capable of restraining fungal growth and increasing antifungal resistance.
Aromatic amino acid, cysteine sulfinic acid, glutamate and histidine decarboxylases, belonging to group II of pyridoxal 5'-phosphate-dependent enzymes, catalyze the synthesis of dopamine/serotonin, ...hypotaurine, γ-aminobutyric acid and histamine, respectively. Considering that these reaction products are all essential bioactive molecules, group II decarboxylases have been long studied from an evolutionary, biochemical and pharmacological standpoint. Despite the fact that they all belong to a common fold-type, during evolution each decarboxylase has evolved unique structural elements responsible for its substrate specificity. Combining a literature update with bioinformatic analyses, this review focuses on some structural determinants shared by these enzymes revealing their intrinsic substrate specificity and highlighting the importance of some residues/regions for catalytic competence. In particular, two key structural features emerge: 1) a mobile catalytic loop, and 2) an open-to-close conformation accompanying the apo-holo transition. Drawing attention on these elements is crucial in correlating subtle structural modifications to functional properties for the understanding, at a molecular level of a pathological condition. This is corroborated by the increasingly important role played by these decarboxylases in several different pathological states (autoimmune diseases, type I diabetes, Parkinson's disease, aromatic amino acid decarboxylase deficiency, Tourette's syndrome and cholangiocarcinoma).
The K-homology (KH) domains are small, structurally conserved domains found in proteins of different origins characterized by a central conserved βααβ “core” and a GxxG motif in the loop between the ...two helices of the KH core. In the eukaryotic KHI type, additional αβ elements decorate the “core” at the C-terminus. Proteins containing KH domains perform different functions and several diseases have been associated with mutations in these domains, including those in the fragile X mental retardation protein (FMRP). FMRP is an RNA-binding protein crucial for the control of RNA metabolism whose lack or mutations lead to fragile X syndrome (FXS). Among missense mutations, the R138Q substitution is in the KH0 degenerated domain lacking the classical GxxG motif. By combining equilibrium and kinetic experiments, we present a characterization of the folding mechanism of the KH0 domain from the FMRP wild-type and of the R138Q variant showing that in both cases the folding mechanism implies the accumulation of an on-pathway transient intermediate. Moreover, by exploiting a battery of biophysical techniques, we show that the KH0 domain has the propensity to form amyloid-like aggregates in mild conditions in vitro and that the R138Q mutation leads to a general destabilization of the protein and to an increased fibrillogenesis propensity.
Extravasation is a fundamental step in the metastatic journey, where cancer cells exit the bloodstream and breach the endothelial cell barrier to infiltrate target tissues. The tactics cancer cells ...employ are sophisticated, closely reflecting those used by the immune system for tissue surveillance. Remarkably, tumor cells have been observed to form distinct associations or clusters with immune cells where neutrophils stand out as particularly crucial partners. These interactions are not accidental; they are critical for cancer cells to exploit the immune functions of neutrophils and successfully extravasate. In another strategy, tumor cells mimic the behavior and characteristics of immune cells. They release a suite of inflammatory mediators, which under normal circumstances, guide the processes of endothelium reshaping and facilitate the entry and movement of immune cells within tissues. In this review, we offer a new perspective on the tactics employed by cancer cells to extravasate and infiltrate target tissues. We delve into the myriad mechanisms that tumor cells borrow, adapt, and refine from the immune playbook. Video Abstract.
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