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.
.
The synthesis of superheavy elements stimulates the effort to study the peculiarities of the complete fusion with massive nuclei and to improve theoretical models in order to extract knowledge ...about reaction mechanism in heavy-ion collisions at low energies. We compare the theoretical results of the compound nucleus (CN) formation and evaporation residue (ER) cross sections obtained for the
48
Ca +
248
Cm and
58
Fe +
232
Th reactions leading to formation of CN with
A
= 296 and
A
= 290 of the superheavy element Lv (
Z
= 116 , respectively. The ER cross sections, which can be measured directly, are determined by the complete fusion and survival probabilities of the heated and rotating CN. Those probabilities cannot be measured unambiguously but the knowledge about them is important to study the formation mechanism of the observed products and to estimate the ER cross sections of the expected isotopes of elements. To this aim, the
48
Ca +
249
Cf and
64
Ni +
232
Th reactions are considered too. The use of the mass values of superheavy nuclei calculated in the framework of the macroscopic-microscopic model by the Warsaw group leads to smaller ER cross section for all of the reactions in comparison with the case of using the masses calculated by P. Möller
et al.
.
Pseudomonas aeruginosa is responsible for a plethora of biofilm mediated chronic infections among which cystic fibrosis pneumonia is the most frightening. The long-term survival strategy of P. ...aeruginosa in the patients lungs is based on a fine balance of virulence vs dormant states and on genetic adaptation, in order to select persistent phenotypes as the small colony variants (SCVs), which strongly correlate with antibiotic resistance and poor lung function. Recent studies have coupled SCV with increased levels of the signaling molecule cyclic di-GMP, and demonstrated the central role of the diguanylate cyclase YfiN, part of the tripartite signaling module YifBNR, in c-di-GMP dependent SCV regulation. YfiN, also called TpbB, is a multi-domain membrane enzyme connecting periplasmic stimuli to cytosolic c-di-GMP production by an allosteric inside-out signaling mechanism that, due to the lack of structural data, is still largely hypothetical. We have solved the crystal structure of the catalytic domain (GGDEF), and measured the enzymatic activity of the cytosolic portion in real-time by means of a newly developed method. Based on these results we demonstrate that, unlike other diguanylate cyclase, YfiN does not undergo product feedback inhibition, and that the presence of the HAMP domain is required for dimerization and catalysis. Coupling our structural and kinetic data with an in silico study we are now able to propose a model for the allosteric regulation of YfiN.
Dopa decarboxylase (DDC), a pyridoxal 5'-phosphate (PLP) enzyme responsible for the biosynthesis of dopamine and serotonin, is involved in Parkinson's disease (PD). PD is a neurodegenerative disease ...mainly due to a progressive loss of dopamine-producing cells in the midbrain. Co-administration of L-Dopa with peripheral DDC inhibitors (carbidopa or benserazide) is the most effective symptomatic treatment for PD. Although carbidopa and trihydroxybenzylhydrazine (the in vivo hydrolysis product of benserazide) are both powerful irreversible DDC inhibitors, they are not selective because they irreversibly bind to free PLP and PLP-enzymes, thus inducing diverse side effects. Therefore, the main goals of this study were (a) to use virtual screening to identify potential human DDC inhibitors and (b) to evaluate the reliability of our virtual-screening (VS) protocol by experimentally testing the "in vitro" activity of selected molecules. Starting from the crystal structure of the DDC-carbidopa complex, a new VS protocol, integrating pharmacophore searches and molecular docking, was developed. Analysis of 15 selected compounds, obtained by filtering the public ZINC database, yielded two molecules that bind to the active site of human DDC and behave as competitive inhibitors with K(i) values ≥10 µM. By performing in silico similarity search on the latter compounds followed by a substructure search using the core of the most active compound we identified several competitive inhibitors of human DDC with K(i) values in the low micromolar range, unable to bind free PLP, and predicted to not cross the blood-brain barrier. The most potent inhibitor with a K(i) value of 500 nM represents a new lead compound, targeting human DDC, that may be the basis for lead optimization in the development of new DDC inhibitors. To our knowledge, a similar approach has not been reported yet in the field of DDC inhibitors discovery.
Serine hydroxymethyltransferase (SHMT) is a central enzyme in the metabolic reprogramming of cancer cells, providing activated one-carbon units in the serine-glycine one-carbon metabolism. Previous ...studies demonstrated that the cytoplasmic isoform of SHMT (SHMT1) plays a relevant role in lung cancer. SHMT1 is overexpressed in lung cancer patients and NSCLC cell lines. Moreover, SHMT1 is required to maintain DNA integrity. Depletion in lung cancer cell lines causes cell cycle arrest and uracil accumulation and ultimately leads to apoptosis. We found that a pyrazolopyran compound, namely 2.12, preferentially inhibits SHMT1 compared to the mitochondrial counterpart SHMT2. Computational and crystallographic approaches suggest binding at the active site of SHMT1 and a competitive inhibition mechanism. A radio isotopic activity assay shows that inhibition of SHMT by 2.12 also occurs in living cells. Moreover, administration of 2.12 in A549 and H1299 lung cancer cell lines causes apoptosis at LD50 34 μM and rescue experiments underlined selectivity towards SHMT1. These data not only further highlight the relevance of the cytoplasmic isoform SHMT1 in lung cancer but, more importantly, demonstrate that, at least in vitro, it is possible to find selective inhibitors against one specific isoform of SHMT, a key target in metabolic reprogramming of many cancer types.
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.
The intracellular level of the bacterial secondary messenger cyclic di-3′,5′-GMP (c-di-GMP) is determined by a balance between its biosynthesis and degradation, the latter achieved via dedicated ...phosphodiesterases (PDEs) bearing a characteristic EAL or HD-GYP domain. We here report the crystal structure of PA4781, one of the three Pseudomonas aeruginosa HD-GYP proteins, which we have previously characterized in vitro . The structure shows a bimetallic active site whose metal binding mode is different from those of both HD-GYP PDEs characterized so far. Purified PA4781 does not contain iron in the active site as for other HD-GYPs, and we show that it binds to a wide range of transition metals with similar affinities. Moreover, the structural features of PA4781 indicate that this is preferentially a pGpG binding protein, as we previously suggested. Our results point out that the structural features of HD-GYPs are more complex than predicted so far and identify the HD-GYP domain as a conserved scaffold which has evolved to preferentially interact with a partner GGDEF but which harbors different functions obtained through diversification of the active site. IMPORTANCE In bacteria, the capability to form biofilms, responsible for increased pathogenicity and antibiotic resistance, is almost universally stimulated by the second messenger cyclic di-GMP (c-di-GMP). To design successful strategies for targeting biofilm formation, a detailed characterization of the enzymes involved in c-di-GMP metabolism is crucial. We solved the structure of the HD-GYP domain of PA4781 from Pseudomonas aeruginosa , involved in c-di-GMP degradation. This is the third structure of this class of phosphodiesterases to be solved, and with respect to its homologues, it shows significant differences both in the nature and in the binding mode of the coordinated metals, indicating that HD-GYP proteins are able to fine-tune their function, thereby increasing the chances of the microorganism to adapt to different environmental needs.
The anticancer actions of the biguanide metformin involve the functioning of the serine/glycine one-carbon metabolic network. We report that metformin directly and specifically targets the enzymatic ...activity of mitochondrial serine hydroxymethyltransferase (SHMT2). In vitro competitive binding assays with human recombinant SHMT1 and SHMT2 isoforms revealed that metformin preferentially inhibits SHMT2 activity by a non-catalytic mechanism. Computational docking coupled with molecular dynamics simulation predicted that metformin could occupy the cofactor pyridoxal-5′-phosphate (PLP) cavity and destabilize the formation of catalytically active SHMT2 oligomers. Differential scanning fluorimetry-based biophysical screening confirmed that metformin diminishes the capacity of PLP to promote the conversion of SHMT2 from an inactive, open state to a highly ordered, catalytically competent closed state. CRISPR/Cas9-based disruption of SHMT2, but not of SHMT1, prevented metformin from inhibiting total SHMT activity in cancer cell lines. Isotope tracing studies in SHMT1 knock-out cells confirmed that metformin decreased the SHMT2-channeled serine-to-formate flux and restricted the formate utilization in thymidylate synthesis upon overexpression of the metformin-unresponsive yeast equivalent of mitochondrial complex I (mCI). While maintaining its capacity to inhibit mitochondrial oxidative phosphorylation, metformin lost its cytotoxic and antiproliferative activity in SHMT2-null cancer cells unable to produce energy-rich NADH or FADH2 molecules from tricarboxylic acid cycle (TCA) metabolites. As currently available SHMT2 inhibitors have not yet reached the clinic, our current data establishing the structural and mechanistic bases of metformin as a small-molecule, PLP-competitive inhibitor of the SHMT2 activating oligomerization should benefit future discovery of biguanide skeleton-based novel SHMT2 inhibitors in cancer prevention and treatment.
1 Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, Rome, Italy
2 Consorzio INBB, 00136 Rome, Italy
Pseudomonas aeruginosa is a well-known pathogen in chronic ...respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli -based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro . Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro , suggesting that DNR responds specifically to NO.
Correspondence Francesca Cutruzzolà francesca.cutruzzola{at}uniroma1.it
Abbreviations: ALA, 5-aminolaevulinic acid; CRP, cAMP receptor protein; DNR, dissimilatory nitrate respiration regulator; FNR, fumarate and nitrate reductase regulator; HTH, helix–turn–helix; SNP, sodium nitroprusside; β -gal, β -galactosidase
The interactions between biomolecules often require flexible surfaces to perform many different tasks such as recognition, regulation and signaling. These interactions rely on sophisticated, dynamic ...molecular mechanisms that must be tightly controlled within very crowded environments, such as cellular compartments, to allow tailored and tuned complexes, e.g., receptor- and enzyme-ligand or chaperone-client. This Research Topic pointed out special emphasis on the characterization of the interactions between molecular partners forming complexes, be it fully biological of hybrid.
PDF
