A conserved scaffold with heterogeneous metal ion binding site: the multifaceted example of HD-GYP proteins

The control of the intracellular level of cyclic dinucleotides is a major strategy to transduce external signals into a cellular response, particularly in bacteria. The HD-GYP metalloproteins, a subgroup of the larger family of histidine-aspartate (HD) hydrolases, can catalyze the cleavage of the phosphodiester bond(s) of cyclic dinucleotides. The HD signature is involved in metal binding in the active site, whereas the GYP motif is likely involved in recognition and interaction with other partners. The most representative substrate of HD-GYPs is the second messenger cyclic-di-GMP (c-di-GMP), a global regulator of bacterial biofilm, motility, and virulence. Hydrolysis of c-di-GMP into the linear form pGpG or into the monomeric guanosine derivative (GMP) reprograms the cellular phenotype, usually promoting biofilm dispersion and virulence. Recent data indicate that members of HD-GYP group can also hydrolyze the bacterial cyclic diGMP-AMP (cGAMP) dinucleotide or act as possible sensors of pGpG. The HD-GYP diversity is not limited to substrate recognition: the most striking trait is the extraordinary heterogeneity of the active sites characterized so far, showing different metals ions assisting catalysis, both in terms of their nature and number. In this review, we provide a critical overview of the structural, functional, and kinetic properties of the bacterial HD-GYP metalloproteins

The emerging role of amino acids of the brain microenvironment in the process of metastasis formation

Brain metastases are the most severe clinical manifestation of aggressive tumors. Melanoma, breast, and lung cancers are the types that prefer the brain as a site of metastasis formation, even if the reasons for this phenomenon still remain to be clarified. One of the main characteristics that makes a cancer cell able to form metastases in the brain is the ability to interact with the endothelial cells of the microvasculature, cross the blood–brain barrier, and metabolically adapt to the nutrients available in the new microenvironment. In this review, we analyzed what makes the brain a suitable site for the development of metastases and how this microenvironment, through the continuous release of neurotransmitters and amino acids in the extracellular milieu, is able to support the metabolic needs of metastasizing cells. We also suggested a possible role for amino acids released by the brain through the endothelial cells of the blood–brain barrier into the bloodstream in triggering the process of extravasation/invasion of the brain parenchyma

Crystal structure of Aspergillus fumigatus AroH, an aromatic amino acid aminotransferase

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis (IPA). To survive in the lungs the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5'-phosphate (PLP)-dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L-kynurenine and α-aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus related diseases. We have solved the crystal structure of Af-AroH at 2.4 å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH

Crystal structure of Aspergillus fumigatus AroH, an aromatic amino acid aminotransferase

Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 50-phosphate-dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L-kynurenine and \u3b1-aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus-related diseases. We have solved the crystal structure of Af-AroH at 2.4 \uc5 resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

Cancer cells reprogramme one­carbon metabolism (OCM) to support enhanced growth and proliferation, in this contest Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme. SHMT mainly exists in three isoforms; two localized in the cytosol (SHMT1/SHMT2 α) and one (SHMT2) in the mitochondria. SHMT1 undergoes to a nuclear localization during the S­phase of the cell cycle to sustain de novo dTMP synthesis1. The de novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), the latter two are targets of widely used chemotherapeutics such as antifolates and 5­fluorouracil. It had been suggested that these three proteins assemble in the nucleus into the thymidylate synthesis complex (dTMP­SC)1. We have recently understood the intracellular dynamics of dTMP synthesis complex in lung cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result strongly indicates that the role of the dTMP­SC assembly may go beyond dTMP synthesis. We have also successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR by using a different array of techniques. Moreover, we have demonstrated that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionary selected in eukaryotes to optimize protein­protein interactions. Lastly, we have set­up an activity assay of the complete thymidylate cycle in vitro, which may provide a useful tool to develop drugs targeting the entire complex instead of the individual components. [1] Anderson, D. and Stover, P. (2009). SHMT1 and SHMT2 Are Functionally Redundant in Nuclear De novo Thymidylate Biosynthesis. PLoS ONE, 4(6), p.e5839

Best oral young crystallographer presentation award

I have presented my latest results about the Structural clues of Serine hidroxymethyltransferase (SHMT) riboregulation

Design, Synthesis and Discovery of N,N'-Carbazoyl-aryl-urea Inhibitors of Zika NS5 Methyltransferase and Virus Replication

The recent outbreaks of Zika virus (ZIKV) infection worldwide make the discovery of novel antivirals against flaviviruses a research priority. This work describes the identification of novel inhibitors of ZIKV through a structure-based virtual screening approach using the ZIKV NS5-MTase. A novel series of molecules with a carbazoyl-aryl-urea structure has been discovered and a library of analogues has been synthesized. The new compounds inhibit ZIKV MTase with IC50 between 23-48 μM. In addition, carbazoyl-aryl-ureas also proved to inhibit ZIKV replication activity at micromolar concentration.status: publishe

Cytosolic localization and in vitro assembly of human de novo thymidylate synthesis complex

De novo thymidylate synthesis is a crucial pathway for normal and cancer cells. Deoxythymidine monophosphate (dTMP) is synthesized by the combined action of three enzymes: serine hydroxymethyltransferase (SHMT), dihydrofolate reductase (DHFR) and thymidylate synthase (TYMS), with the latter two being targets of widely used chemotherapeutics such as antifolates and 5-fluorouracil. These proteins translocate to the nucleus after SUMOylation and are suggested to assemble in this compartment into the thymidylate synthesis complex (dTMP-SC). We report the intracellular dynamics of the complex in cancer cells by in situ proximity ligation assay, showing that it is also detected in the cytoplasm. This result indicates that the role of the dTMP-SC assembly may go beyond dTMP synthesis. We have successfully assembled the dTMP synthesis complex in vitro, employing tetrameric SHMT1 and a bifunctional chimeric enzyme comprising human TYMS and DHFR. We show that the SHMT1 tetrameric state is required for efficient complex assembly, indicating that this aggregation state is evolutionarily selected in eukaryotes to optimize protein-protein interactions. Lastly, our results on the activity of the complete thymidylate cycle in vitro, may provide a useful tool to develop drugs targeting the entire complex instead of the individual components