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AlphaFold and similar groundbreaking, AI-based tools, have revolutionized the field of structural bioinformatics, with their remarkable accuracy in ab-initio protein structure prediction. This success has catalyzed the development of new software and pipelines aimed at incorporating AlphaFold's predictions, often focusing on addressing the algorithm's remaining challenges. Here, we present the current landscape of structural bioinformatics shaped by AlphaFold, and discuss how the field is dynamically responding to this revolution, with new software, methods, and pipelines. While the excitement around AI-based tools led to their widespread application, it is essential to acknowledge that their practical success hinges on their integration into established protocols within structural bioinformatics, often neglected in the context of AI-driven advancements. Indeed, user-driven intervention is still as pivotal in the structure prediction process as in complementing state-of-the-art algorithms with functional and biological knowledge., (© 2024 The Author(s). FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Gene therapy of dominantly inherited genetic diseases requires either the selective disruption of the mutant allele or the editing of the specific mutation. The CRISPR-Cas system holds great potential for the genetic correction of single nucleotide variants (SNVs), including dominant mutations. However, distinguishing between single-nucleotide variations in a pathogenic genomic context remains challenging. The presence of a PAM in the disease-causing allele can guide its precise targeting, preserving the functionality of the wild-type allele. The AlPaCas (Aligning Patients to Cas) webserver is an automated pipeline for sequence-based identification and structural analysis of SNV-derived PAMs that satisfy this demand. When provided with a gene/SNV input, AlPaCas can: (i) identify SNV-derived PAMs; (ii) provide a list of available Cas enzymes recognizing the SNV (s); (iii) propose mutational Cas-engineering to enhance the selectivity towards the SNV-derived PAM. With its ability to identify allele-specific genetic variants that can be targeted using already available or engineered Cas enzymes, AlPaCas is at the forefront of advancements in genome editing. AlPaCas is open to all users without a login requirement and is freely available at https://schubert.bio.uniroma1.it/alpacas., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Staphylococcus aureus is a gram-positive bacterium that may cause intestinal inflammation by secreting enterotoxins, which commonly cause food-poisoning and gastrointestinal injuries. Staphylococcal enterotoxin B (SEB) acts as a superantigen (SAg) by binding in a bivalent manner the T-cell receptor (TCR) and the costimulatory receptor CD28, thus stimulating T cells to produce large amounts of inflammatory cytokines, which may affect intestinal epithelial barrier integrity and functions. However, the role of T cell-mediated SEB inflammatory activity remains unknown. Here we show that inflammatory cytokines produced by T cells following SEB stimulation induce dysfunctions in Caco-2 intestinal epithelial cells by promoting actin cytoskeleton remodelling and epithelial cell-cell junction down-regulation. We also found that SEB-activated inflammatory T cells promote the up-regulation of epithelial-mesenchymal transition transcription factors (EMT-TFs) in a nuclear factor-κB (NF-κB)- and STAT3-dependent manner. Finally, by using a structure-based design approach, we identified a SEB mimetic peptide (pSEB 116-132 ) that, by blocking the binding of SEB to CD28, dampens inflammatory-mediated dysregulation of intestinal epithelial barrier., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision, (Copyright © 2024 Amormino, Russo, Tedeschi, Fiorillo, Paiardini, Spallotta, Rosanò, Tuosto and Kunkl.)

Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B 6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Motivation: Protein contact networks (PCNs) represent the 3D structure of a protein using network formalism. Inter-residue contacts are described as binary adjacency matrices, which are derived from the graph representation of residues (as α-carbons, β-carbons or centroids) and Euclidean distances according to defined thresholds. Functional characterization algorithms are computed on binary adjacency matrices to unveil allosteric, dynamic, and interaction mechanisms in proteins. Such strategies are usually applied in a combinatorial manner, although rarely in seamless and user-friendly implementations., Results: PyPCN is a plugin for PyMOL wrapping more than twenty PCN algorithms and metrics in an easy-to-use graphical user interface, to support PCN analysis. The plugin accepts 3D structures from the Protein Data Bank, user-provided PDBs, or precomputed adjacency matrices. The results are directly mapped to 3D protein structures and organized into interactive diagrams for their visualization. A dedicated graphical user interface combined with PyMOL visual support makes analysis more intuitive and easier, extending the applicability of PCNs., Availability and Implementation: https://github.com/pcnproject/PyPCN., (© The Author(s) 2023. Published by Oxford University Press.)

Environmental nutrients control bacterial biofilm homeostasis, by regulating the intracellular levels of c-di-GMP. One component transducers can sense different classes of small molecules through a periplasmic domain; the nutrient recognition triggers the subsequent regulation of the downstream cytosolic diguanylate cyclase (GGDEF) or phosphodiesterase (EAL) domains, via transmembrane helix(ces), to finally change c-di-GMP levels.Protein studies on such transducers have been mainly carried out on isolated domains due to the presence of the transmembrane portion. Nevertheless, the cleavage of GGDEF and EAL-containing proteins could be detrimental since both tertiary and quaternary structures could be allosterically controlled; to by-pass this limitation, studies on the corresponding full-length proteins are highly desired.We have in silico selected a GGDEF-EAL transducer from Dyella thiooxydans (ann. A0A160N0B7), whose periplasmic binding domain was predicted to bind to arginine, a nutrient often associated with chronic infections and biofilm. This protein has been used as an in vitro tool for the identification of the best approach for its isolation, including (i) protein engineering to produce a water-soluble version via QTY (Glutamine, Threonine, and Tyrosine) code or (ii) nanodiscs assembly. The results on this "prototype" may represent the proof-of-concept for future isolation of other transmembrane proteins sharing the same architecture, including more complex nutrient-based transducers controlling c-di-GMP levels., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland A.)

The pathogenicity of Shigella , the intracellular pathogen responsible for human bacillary dysentery, depends on a coordinated and tightly regulated expression of its virulence determinants. This is the result of a cascade organization of its positive regulators, with VirF, a transcriptional activator belonging to the AraC-XylS family, in a pivotal position. VirF itself is submitted to several well-known regulations at the transcriptional level. In this work, we present evidence for a novel posttranslational regulatory mechanism of VirF mediated by the inhibitory interaction with specific fatty acids. By homology modeling and molecular docking analyses, we identify a jelly roll motif in the structure of ViF capable of interacting with medium-chain saturated and long-chain unsaturated fatty acids. In vitro and in vivo assays show that capric, lauric, myristoleic, palmitoleic, and sapienic acids interact effectively with the VirF protein, abolishing its transcription-promoting activity. This silences the virulence system of Shigella , leading to a drastic reduction in its ability to invade epithelial cells and proliferate in their cytoplasm. IMPORTANCE In the absence of a valid vaccine, the main therapeutic approach currently used to treat shigellosis is based on the use of antibiotics. The emergence of antibiotic resistance jeopardizes the future effectiveness of this approach. The importance of the present work resides both in the identification of a new level of posttranslational regulation of the Shigella virulence system and in the characterization of a mechanism offering new opportunities for the design of antivirulence compounds, which may change the treatment paradigm of Shigella infections by limiting the emergence of antibiotic-resistant bacteria., Competing Interests: The authors declare no conflict of interest.

KDM5B histone demethylase is overexpressed in many cancers and plays an ambivalent role in oncogenesis, depending on the specific context. This ambivalence could be explained by the expression of KDM5B protein isoforms with diverse functional roles, which could be present at different levels in various cancer cell lines. We show here that one of these isoforms, namely KDM5B-NTT, accumulates in breast cancer cell lines due to remarkable protein stability relative to the canonical PLU-1 isoform, which shows a much faster turnover. This isoform is the truncated and catalytically inactive product of an mRNA with a transcription start site downstream of the PLU-1 isoform, and the consequent usage of an alternative ATG for translation initiation. It also differs from the PLU-1 transcript in the inclusion of an additional exon (exon-6), previously attributed to other putative isoforms. Overexpression of this isoform in MCF7 cells leads to an increase in bulk H3K4 methylation and induces derepression of a gene cluster, including the tumor suppressor Cav1 and several genes involved in the interferon-alpha and -gamma response. We discuss the relevance of this finding considering the hypothesis that KDM5B may possess regulatory roles independent of its catalytic activity., (© 2023. The Author(s).)

KAT8 is a lysine acetyltransferase primarily catalyzing the acetylation of Lys16 of histone H4 (H4K16). KAT8 dysregulation is linked to the development and metastatization of many cancer types, including non-small cell lung cancer (NSCLC) and acute myeloid leukemia (AML). Few KAT8 inhibitors have been reported so far, none of which displaying selective activity. Based on the KAT3B/KDAC inhibitor C646, we developed a series of N -phenyl-5-pyrazolone derivatives and identified compounds 19 and 34 as low-micromolar KAT8 inhibitors selective over a panel of KATs and KDACs. Western blot, immunofluorescence, and CETSA experiments demonstrated that both inhibitors selectively target KAT8 in cells. Moreover, 19 and 34 exhibited mid-micromolar antiproliferative activity in different cancer cell lines, including NSCLC and AML, without impacting the viability of nontransformed cells. Overall, these compounds are valuable tools for elucidating KAT8 biology, and their simple structures make them promising candidates for future optimization studies.

Staphylococcus aureu s superantigens (SAgs) such as staphylococcal enterotoxin A (SEA) and B (SEB) are potent toxins stimulating T cells to produce high levels of inflammatory cytokines, thus causing toxic shock and sepsis. Here we used a recently released artificial intelligence-based algorithm to better elucidate the interaction between staphylococcal SAgs and their ligands on T cells, the TCR and CD28. The obtained computational models together with functional data show that SEB and SEA are able to bind to the TCR and CD28 stimulating T cells to activate inflammatory signals independently of MHC class II- and B7-expressing antigen presenting cells. These data reveal a novel mode of action of staphylococcal SAgs. By binding to the TCR and CD28 in a bivalent way, staphylococcal SAgs trigger both the early and late signalling events, which lead to massive inflammatory cytokine secretion., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Kunkl, Amormino, Spallotta, Caristi, Fiorillo, Paiardini, Kaempfer and Tuosto.)

The AurkA kinase is a well-known mitotic regulator, frequently overexpressed in tumors. The microtubule-binding protein TPX2 controls AurkA activity, localization, and stability in mitosis. Non-mitotic roles of AurkA are emerging, and increased nuclear localization in interphase has been correlated with AurkA oncogenic potential. Still, the mechanisms leading to AurkA nuclear accumulation are poorly explored. Here, we investigated these mechanisms under physiological or overexpression conditions. We observed that AurkA nuclear localization is influenced by the cell cycle phase and nuclear export, but not by its kinase activity. Importantly, AURKA overexpression is not sufficient to determine its accumulation in interphase nuclei, which is instead obtained when AURKA and TPX2 are co-overexpressed or, to a higher extent, when proteasome activity is impaired. Expression analyses show that AURKA, TPX2, and the import regulator CSE1L are co-overexpressed in tumors. Finally, using MCF10A mammospheres we show that TPX2 co-overexpression drives protumorigenic processes downstream of nuclear AurkA. We propose that AURKA/TPX2 co-overexpression in cancer represents a key determinant of AurkA nuclear oncogenic functions., (© 2023 Asteriti et al.)

Mitotic kinase Aurora A (AURKA) diverges from other kinases in its multiple active conformations that may explain its interphase roles and the limited efficacy of drugs targeting the kinase pocket. Regulation of AURKA activity by the cell is critically dependent on destruction mediated by the anaphase-promoting complex (APC/C FZR1 ) during mitotic exit and G1 phase and requires an atypical N-terminal degron in AURKA called the "A-box" in addition to a reported canonical D-box degron in the C-terminus. Here, we find that the reported C-terminal D-box of AURKA does not act as a degron and instead mediates essential structural features of the protein. In living cells, the N-terminal intrinsically disordered region of AURKA containing the A-box is sufficient to confer FZR1-dependent mitotic degradation. Both in silico and in cellulo assays predict the QRVL short linear interacting motif of the A-box to be a phospho-regulated D-box. We propose that degradation of full-length AURKA also depends on an intact C-terminal domain because of critical conformational parameters permissive for both activity and mitotic degradation of AURKA., (© 2022 Abdelbaki et al.)

Over the past few decades, the number of available structural bioinformatics pipelines, libraries, plugins, web resources and software has increased exponentially and become accessible to the broad realm of life scientists. This expansion has shaped the field as a tangled network of methods, algorithms and user interfaces. In recent years PyMOL, widely used software for biomolecules visualization and analysis, has started to play a key role in providing an open platform for the successful implementation of expert knowledge into an easy-to-use molecular graphics tool. This review outlines the plugins and features that make PyMOL an eligible environment for supporting structural bioinformatics analyses.

Motivation: The primary strategy for predicting the binding mode of small molecules to their receptors and for performing receptor-based virtual screening studies is protein-ligand docking, which is undoubtedly the most popular and successful approach in computer-aided drug discovery. The increased popularity of docking has resulted in the development of different docking algorithms and scoring functions. Nonetheless, it is unlikely that a single approach outperforms the others in terms of reproducibility and precision. In this ground, consensus docking techniques are taking hold., Results: We have developed DockingPie, an open source PyMOL plugin for individual, as well as consensus docking analyses. Smina, AutoDock Vina, ADFR and RxDock are the four docking engines that DockingPie currently supports in an easy and extremely intuitive way, thanks to its integrated docking environment and its GUI, fully integrated within PyMOL., Availability and Implementation: https://github.com/paiardin/DockingPie., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The Aurora-A kinase regulates cell division, by controlling centrosome biology and spindle assembly. Cancer cells often display elevated levels of the kinase, due to amplification of the gene locus, increased transcription or post-translational modifications. Several inhibitors of Aurora-A activity have been developed as anti-cancer agents and are under evaluation in clinical trials. Although the well-known mitotic roles of Aurora-A point at chromosomal instability, a hallmark of cancer, as a major link between Aurora-A overexpression and disease, recent evidence highlights the existence of non-mitotic functions of potential relevance. Here we focus on a nuclear-localised fraction of Aurora-A with oncogenic roles. Interestingly, this pool would identify not only non-mitotic, but also kinase-independent functions of the kinase. We review existing data in the literature and databases, examining potential links between Aurora-A stabilisation and localisation, and discuss them in the perspective of a more effective targeting of Aurora-A in cancer therapy.

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal ( Sulfolobus solfataricus ) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lo Gullo, De Santis, Paiardini, Rosignoli, Romagnoli, La Teana, Londei and Benelli.)

Chromatin architect of muscle expression (Charme) is a muscle-restricted long noncoding RNA (lncRNA) that plays an important role in myogenesis. Earlier evidence indicates that the nuclear Charme isoform, named pCharme, acts on the chromatin by assisting the formation of chromatin domains where myogenic transcription occurs. By combining RNA antisense purification (RAP) with mass spectrometry and loss-of-function analyses, we have now identified the proteins that assist these chromatin activities. These proteins-which include a sub-set of splicing regulators, principally PTBP1 and the multifunctional RNA/DNA binding protein MATR3-bind to sequences located within the alternatively spliced intron-1 to form nuclear aggregates. Consistent with the functional importance of pCharme interactome in vivo, a targeted deletion of the intron-1 by a CRISPR-Cas9 approach in mouse causes the release of pCharme from the chromatin and results in cardiac defects similar to what was observed upon knockout of the full-length transcript., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

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.

Pancreatic cancer-melanoma syndrome (PCMS) is an inherited condition in which mutation carriers have an increased risk of malignant melanoma and/or pancreatic cancer. About 30% of PCMS cases carry mutations in CDKN2A. This gene encodes several protein isoforms, one of which, known as p16, regulates the cell-cycle by interacting with CDK4/CDK6 kinases and with several non-CDK proteins. Herein, we report on a novel CDKN2A germline in-frame deletion (c.52_57delACGGCC) found in an Italian family with PCMS. By segregation analysis, the c.52_57delACGGCC was proven to segregate in kindred with cutaneous melanoma (CM), in kindred with CM and pancreatic cancer, and in a single case presenting only with pancreatic cancer. In the literature, duplication mapping in the same genic region has been already reported at the germline level in several unrelated CM cases as a variant of unknown clinical significance. A computational approach for studying the effect of mutational changes over p16 protein structure showed that both the deletion and the duplication of the c.52_57 nucleotides result in protein misfolding and loss of interactors' binding. In conclusion, the present results argue that the quantitative alteration of nucleotides c.52_57 has a pathogenic role in p16 function and that the c.52_57delACGGCC is associated with PCMS.

Guanine-quadruplexes (G4) included in RNA molecules exert several functions in controlling gene expression at post-transcriptional level; however, the molecular mechanisms of G4-mediated regulation are still poorly understood. Here, we describe a regulatory circuitry operating in the early phases of murine muscle differentiation in which a long non-coding RNA (SMaRT) base pairs with a G4-containing mRNA (Mlx-γ) and represses its translation by counteracting the activity of the DHX36 RNA helicase. The time-restricted, specific effect of lnc-SMaRT on the translation of Mlx-γ isoform modulates the general subcellular localization of total MLX proteins, impacting on their transcriptional output and promoting proper myogenesis and mature myotube formation. Therefore, the circuitry made of lnc-SMaRT, Mlx-γ, and DHX36 not only plays an important role in the control of myogenesis but also unravels a molecular mechanism where G4 structures and G4 unwinding activities are regulated in living cells., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

PPARγ represents a key target for the treatment of type 2 diabetes and metabolic syndrome. Synthetic antidiabetic drugs activating PPARγ are accompanied by serious undesirable side effects related to their agonism. In the search for new PPARγ regulators, inhibitors of PPARγ phosphorylation on S245 mediated by CDK5 represent an opportunity for the development of an improved generation of antidiabetic drugs acting through this nuclear receptor. We have employed a multidisciplinary approach, including protein-protein docking, X-ray crystallography, NMR, HDX, MD simulations, and site-directed mutagenesis to investigate conformational changes in PPARγ that impair the ability of CDK5 to interact with PPARγ and hence inhibit PPARγ phosphorylation. Finally, we describe an alternative inhibition mechanism adopted by a ligand bound far from the phosphorylation site.

Artesunic acid and artemisinin are natural substances with promiscuous anticancer activity against different types of cancer cell lines. The mechanism of action of these compounds is associated with the formation of reactive radical species by cleavage of the sesquiterpene pharmacophore endoperoxide bridge. Here we suggested topoisomerase 1 as a possible molecular target for the improvement of the anticancer activity of these compounds. In this context, we report that novel hybrid and dimer derivatives of artesunic acid and artemisinin, bearing camptothecin and SN38 as side-chain biological effectors, can inhibit growth of yeast cells overexpressing human topoisomerase 1 and its enzymatic activity in vitro . These derivatives showed also anticancer activity in melanoma cell lines higher than camptothecin and paclitaxel. In silico molecular docking calculations highlighted a common binding mode for the novel derivatives, with the sesquiterpene lactone scaffold being located near the traditional recognition site for camptothecin, while the bioactive side-chain effector laid in the camptothecin cleft., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Tankyrases (TNKSs) have recently gained great consideration as potential targets in Wnt/β-catenin pathway-dependent solid tumors. Previously, we reported the 2-mercaptoquinazolin-4-one MC2050 as a micromolar PARP1 inhibitor. Here we show how the resolution of the X-ray structure of PARP1 in complex with MC2050, combined with the computational investigation of the structural differences between TNKSs and PARP1/2 active sites, provided the rationale for a structure-based drug design campaign that with a limited synthetic effort led to the discovery of the bis-quinazolinone 5 as a picomolar and selective TNKS2 inhibitor, endowed with antiproliferative effects in a colorectal cancer cell line (DLD-1) where the Wnt pathway is constitutively activated., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Recent studies have uncovered an important role for RNA modifications in gene expression regulation, which led to the birth of the epitranscriptomics field. It is now acknowledged that RNA modifiers play a crucial role in the control of differentiation of stem and progenitor cells and that changes in their levels are a relevant feature of different types of cancer. To date, among more than 160 different RNA chemical modifications, the more relevant in cancer biology is the reversible and dynamic N 6 -methylation of adenosine, yielding N 6 -methyladenosine (m 6 A). m 6 A is the more abundant internal modification in mRNA, regulating the expression of the latter at different levels, from maturation to translation. Here, we will describe the emerging role of m 6 A modification in acute myeloid leukemia (AML), which, among first, has demonstrated how mis-regulation of the m6A modifying system can contribute to the development and progression of cancer. Moreover, we will discuss how AML is paving the way to the development of new therapeutic options based on the inhibition of m 6 A deposition.

Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme in one-carbon metabolism that catalyses the reversible conversion of serine and tetrahydrofolate into glycine and methylenetetrahydrofolate. It exists in cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms. Research on one-carbon metabolism in cancer cell lines has shown that SHMT1 preferentially catalyses serine synthesis, whereas in mitochondria SHMT2 is involved in serine breakdown. Recent research has focused on the identification of inhibitors that bind at the folate pocket. We have previously found that a representative derivative of the pyrazolopyran scaffold, namely 2.12, inhibits both SHMT isoforms, with a preference for SHMT1, causing apoptosis in lung cancer cell lines. Here we show that the affinity of 2.12 for SHMT depends on the identity of the amino acid substrate bound to the enzyme. The dissociation constant of 2.12 is 50-fold lower when it binds to SHMT1 enzyme-serine complex, as compared to the enzyme-glycine complex. Evidence is presented for a similar behaviour of compound 2.12 in the cellular environment. These findings suggest that the presence and identity of the amino acid substrate should be considered when designing SHMT inhibitors. Moreover, our data provide the proof-of-concept that SHMT inhibitors selectively targeting the directionality of one-carbon metabolism flux could be designed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We report here a clinical case of a patient with a novel mutation (Arg347→Gly) in the gene encoding aromatic amino acid decarboxylase (AADC) that is associated with AADC deficiency. The variant R347G in the purified recombinant form exhibits, similarly to the pathogenic mutation R347Q previously studied, a 475-fold drop of kcat compared to the wild-type enzyme. In attempting to unravel the reason(s) for this catalytic defect, we have carried out bioinformatics analyses of the crystal structure of AADC-carbidopa complex with the modelled catalytic loop (residues 328-339). Arg347 appears to interact with Phe103, as well as with both Leu333 and Asp345. We have then prepared and characterized the artificial F103L, R347K and D345A mutants. F103L, D345A and R347K exhibit about 13-, 97-, and 345-fold kcat decrease compared to the wild-type AADC, respectively. However, unlike F103L, the R347G, R347K and R347Q mutants as well as the D345A variant appear to be more defective in catalysis than in protein folding. Moreover, the latter mutants, unlike the wild-type protein and the F103L variant, share a peculiar binding mode of dopa methyl ester consisting of formation of a quinonoid intermediate. This finding strongly suggests that their catalytic defects are mainly due to a misplacement of the substrate at the active site. Taken together, our results highlight the importance of the Arg347-Leu333-Asp345 hydrogen-bonds network in the catalysis of AADC and reveal the molecular basis for the pathogenicity of the variants R347. Following the above results, a therapeutic treatment for patients bearing the mutation R347G is proposed., (Copyright © 2016 Elsevier B.V. All rights reserved.)

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.

Unlabelled: Biofilm formation is responsible for increased antibiotic tolerance in pathogenic bacteria. Cyclic di-GMP (c-di-GMP) is a widely used second-messenger signal that plays a key role in bacterial biofilm formation. c-di-GMP is synthesized by diguanylate cyclases (DGCs), a conserved class of enzymes absent in mammals and hence considered attractive molecular targets for the development of antibiofilm agents. Here, the results of a virtual screening approach aimed at identifying small-molecule inhibitors of the DGC PleD from Caulobacter crescentus are described. A three-dimensional (3D) pharmacophore model, derived from the mode of binding of GTP to the active site of PleD, was exploited to screen the ZINC database of compounds. Seven virtual hits were tested in vitro for their ability to inhibit the activity of purified PleD by using circular dichroism spectroscopy. Two drug-like molecules with a catechol moiety and a sulfonohydrazide scaffold were shown to competitively inhibit PleD at the low-micromolar range (50% inhibitory concentration [IC50] of ∼11 μM). Their predicted binding mode highlighted key structural features presumably responsible for the efficient inhibition of PleD by both hits. These molecules represent the most potent in vitro inhibitors of PleD identified so far and could therefore result in useful leads for the development of novel classes of antimicrobials able to hamper biofilm formation., Importance: Biofilm-mediated infections are difficult to eradicate, posing a threatening health issue worldwide. The capability of bacteria to form biofilms is almost universally stimulated by the second messenger c-di-GMP. This evidence has boosted research in the last decade for the development of new antibiofilm strategies interfering with c-di-GMP metabolism. Here, two potent inhibitors of c-di-GMP synthesis have been identified in silico and characterized in vitro by using the well-characterized DGC enzyme PleD from C. crescentus as a structural template and molecular target. Given that the protein residues implied as crucial for enzyme inhibition are found to be highly conserved among DGCs, the outcome of this study could pave the way for the future development of broad-spectrum antibiofilm compounds., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

New anti-malarial treatments are desperately required to face the spread of drug resistant parasites. Inhibition of metalloaminopeptidases, PfA-M1 and PfA-M17, is a validated therapeutic strategy for treatment of Plasmodium falciparum malaria. Here, we describe the crystal structures of PfA-M1 and PfA-M17 bound to chemotherapeutic agent Tosedostat. The inhibitor occupies the enzymes' putative product egress channels in addition to the substrate binding pockets; however, adopts different binding poses when bound to PfA-M1 and PfA-M17. These findings will be valuable for the continued development of selective inhibitors of PfA-M1 and PfA-M17., (© 2015 Wiley Periodicals, Inc.)

L-threonine aldolases (L-TAs) represent a family of homologous pyridoxal 5'-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several L-3-hydroxy-α-amino acids. L-TAs have great biotechnological potential, as they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for the bioorganic synthesis of L-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many L-TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, L-TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low-specificity L-TA from Escherichia coli at 2.2-Å resolution, in the unliganded form and cocrystallized with L-serine and L-threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro-aldol cleavage reaction. Interestingly, the very large active site opening of E. coli L-TA suggests that much larger molecules than L-threonine isomers may be easily accommodated, and L-TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues., (© 2013 FEBS.)

The substitution of Ser187, a residue located far from the active site of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), by Phe gives rise to a variant associated with primary hyperoxaluria type I. Unexpectedly, previous studies revealed that the recombinant form of S187F exhibits a remarkable loss of catalytic activity, an increased pyridoxal 5'-phosphate (PLP) binding affinity and a different coenzyme binding mode compared with normal AGT. To shed light on the structural elements responsible for these defects, we solved the crystal structure of the variant to a resolution of 2.9 Å. Although the overall conformation of the variant is similar to that of normal AGT, we noticed: (i) a displacement of the PLP-binding Lys209 and Val185, located on the re and si side of PLP, respectively, and (ii) slight conformational changes of other active site residues, in particular Trp108, the base stacking residue with the pyridine cofactor moiety. This active site perturbation results in a mispositioning of the AGT-pyridoxamine 5'-phosphate (PMP) complex and of the external aldimine, as predicted by molecular modeling studies. Taken together, both predicted and observed movements caused by the S187F mutation are consistent with the following functional properties of the variant: (i) a 300- to 500-fold decrease in both the rate constant of L-alanine half-transamination and the kcat of the overall transamination, (ii) a different PMP binding mode and affinity, and (iii) a different microenvironment of the external aldimine. Proposals for the treatment of patients bearing S187F mutation are discussed on the basis of these results., (Copyright © 2013 Wiley Periodicals, Inc.)

It was recently discovered that glycine consumption is strongly related to the rate of proliferation across cancer cells. This is very intriguing and raises the question of what is the actual role of this amino acid in cancer metabolism. Cancer cells are greedy for glycine. In particular, the mitochondrial production of glycine seems to be utterly important. Overexpression of mitochondrial serine hydroxymethyltransferase, the enzyme converting l-serine to glycine, assures an adequate supply of glycine to rapidly proliferating cancer cells. In fact, silencing of mitochondrial serine hydroxymethyltransferase was shown to halt cancer cell proliferation. Direct incorporation of glycine carbon atoms into the purine ring has been proposed to be one main reason for the importance of glycine in cancer cell metabolism. We believe that, as far as the importance of glycine in cancer is concerned, a central role of this amino acid, namely its participation to heme biosynthesis, has been neglected. In mitochondria, glycine condenses with succinyl-CoA to form 5-aminolevulinate, the universal precursor of the different forms of heme contained in cytochromes and oxidative phosphorylation complexes. Our hypothesis is that mitochondrial serine hydroxymethyltransferase is fundamental to sustain cancer metabolism since production of glycine fuels heme biosynthesis and therefore oxidative phosphorylation. Respiration of cancer cells may then ultimately rely on endogenous glycine synthesis by mitochondrial serine hydroxymethyltransferase. The link between mitochondrial serine hydroxymethyltransferase activity and heme biosynthesis represents an important and still unexplored aspect of the whole picture of cancer cell metabolism. Our hypothesis might be tested using a combination of metabolic tracing and gene silencing on different cancer cell lines. The experiments should be devised so as to assess the importance of mitochondrial serine hydroxymethyltransferase and the glycine deriving from its reaction as a precursor of heme. If the observed increase of glycine consumption in rapidly proliferating cancer cells has its basis in the need for heme biosynthesis, then mitochondrial serine hydroxymethyltransferase should be considered as a key target for the development of new chemotherapeutic agents., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder of glyoxylate metabolism caused by the deficiency of liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme. The PH1 pathogenesis is mostly due to single point mutations (more than 150 so far identified) on the AGXT gene, and is characterized by a marked heterogeneity in terms of genotype, enzymatic and clinical phenotypes. This article presents an up to date review of selected aspects of the biochemical properties of the two allelic forms of AGT and of some PH1-causing variants. These recent discoveries highlight the effects at the protein level of the pathogenic mutations, and, together with previous cell biology and clinical data, (i) improve the understanding of the molecular basis of PH1 pathogenesis, and (ii) help to delineate perspectives for predicting the response to pyridoxine treatment or for suggesting new strategies for PH1 patients bearing the analyzed mutations.

G41 is an interfacial residue located within the alpha-helix 34-42 of alanine:glyoxylate aminotransferase (AGT). Its mutations on the major (AGT-Ma) or the minor (AGT-Mi) allele give rise to the variants G41R-Ma, G41R-Mi, and G41V-Ma causing hyperoxaluria type 1. Impairment of dimerization in these variants has been suggested to be responsible for immunoreactivity deficiency, intraperoxisomal aggregation, and sensitivity to proteasomal degradation. However, no experimental evidence supports this view. Here we report that G41 mutations, besides increasing the dimer-monomer equilibrium dissociation constant, affect the protein conformation and stability, and perturb its active site. As compared to AGT-Ma or AGT-Mi, G41 variants display different near-UV CD and intrinsic emission fluorescence spectra, larger exposure of hydrophobic surfaces, sensitivity to Met53-Tyr54 peptide bond cleavage by proteinase K, decreased thermostability, reduced coenzyme binding affinity, and catalytic efficiency. Additionally, unlike AGT-Ma and AGT-Mi, G41 variants under physiological conditions form insoluble inactive high-order aggregates (approximately 5,000 nm) through intermolecular electrostatic interactions. A comparative molecular dynamics study of the putative structures of AGT-Mi and G41R-Mi predicts that G41 --> R mutation causes a partial unwinding of the 34-42 alpha-helix and a displacement of the first 44 N-terminal residues including the active site loop 24-32. These simulations help us to envisage the possible structural basis of AGT dysfunction associated with G41 mutations. The detailed insight into how G41 mutations act on the structure-function of AGT may contribute to achieve the ultimate goal of correcting the effects of these mutations.

Human liver peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate into glycine. AGT deficiency causes primary hyperoxaluria type 1 (PH1), a rare autosomal recessive disorder, due to a marked increase in hepatic oxalate production. Normal human AGT exists as two polymorphic variants: the major (AGT-Ma) and the minor (AGT-Mi) allele. AGT-Mi causes the PH1 disease only when combined with some mutations. In this study, the molecular basis of the synergism between AGT-Mi and F152I mutation has been investigated through a detailed biochemical characterization of AGT-Mi and the Phe(152) variants combined either with the major (F152I-Ma, F152A-Ma) or the minor allele (F152I-Mi). Although these species show spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma, the Phe(152) variants exhibit the following differences with respect to AGT-Ma and AGT-Mi: (i) pyridoxamine 5'-phosphate (PMP) is released during the overall transamination leading to the conversion into apoenzymes, and (ii) the PMP binding affinity is at least 200-1400-fold lower. Thus, Phe(152) is not an essential residue for transaminase activity, but plays a role in selectively stabilizing the AGT-PMP complex, by a proper orientation of Trp(108), as suggested by bioinformatic analysis. These data, together with the finding that apoF152I-Mi is the only species that at physiological temperature undergoes a time-dependent inactivation and concomitant aggregation, shed light on the molecular defects resulting from the association of the F152I mutation with AGT-Mi, and allow to speculate on the responsiveness to pyridoxine therapy of PH1 patients carrying this mutation.

SyrC, a component of the multienzyme system of syringomycin biosynthesis, has been shown to shuttle Thr/4-Cl-Thr between the thiolation domains SyrB1-T1 and SyrE-T8,9 by transiently linking it to Cys224 in the enzyme active site. We present data on the structure-function relationship in vivo of this protein and an in silico model of its three-dimensional structure. The biosynthetic activity of SyrC was not influenced when either Asp348 or His376 that together with Cys224 form a putative catalytic triad, were replaced with Ala, but it was abolished by the exchange Cys224 with Ser. The presence of the FLAG peptide on either the N- or C-terminus of the protein did not affect activity, whereas the deletion of the first 16 amino acids at the N-terminus or the insertion of Maltose Binding Protein abolished the production of syringomycin. We present the model of the three-dimensional structure of SyrC suggesting a homodimeric structure for the protein and biochemical data that are supportive of this model.

Reelin is a large molecule of the extracellular matrix (ECM) which regulates neuronal positioning during the early stages of cortical development in vertebrate species. The Reelin molecule can be subdivided into a smaller N-terminal domain, showing homology with F-spondin, and a larger C-terminal region containing 8 EGF-like repeats. The localization of Reelin in the ECM, its large dimensions and the modular organization of its primary structure led us to suppose a structure of its modules similar to domains commonly found in ECM proteins such as Agrin, laminins and thrombospondins. We therefore performed a sequence alignment and molecular modeling analysis to study the three-dimensional fold of the Reelin subrepeat regions. Our analysis produces a tentative model of the core region of the Reelin subrepeat sequences and suggests the presence in this 3D model of structural features common to polysaccharide-binding modules which are often found on proteoglycans of the ECM. These findings provide a conceptual framework for further experiments aimed at testing the functions of the EGF-like repeat regions of Reelin.