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Members of the KCTD protein family play key roles in fundamental physio-pathological processes including cancer, neurodevelopmental/neuropsychiatric, and genetic diseases. Here, we report the crystal structure of the KCTD1 P20S mutant, which causes the scalp-ear-nipple syndrome, and molecular dynamics (MD) data on the wild-type protein. Surprisingly, the structure unravels that the N-terminal region, which precedes the BTB domain (preBTB) and bears the disease-associated mutation, adopts a folded polyproline II (PPII) state. The KCTD1 pentamer is characterized by an intricate architecture in which the different subunits mutually exchange domains to generate a closed domain swapping motif. Indeed, the BTB of each chain makes peculiar contacts with the preBTB and the C-terminal domain (CTD) of an adjacent chain. The BTB-preBTB interaction consists of a PPII-PPII recognition motif whereas the BTB-CTD contacts are mediated by an unusual (+/-) helix discontinuous association. The inspection of the protein structure, along with the data emerged from the MD simulations, provides an explanation of the pathogenicity of the P20S mutation and unravels the role of the BTB-preBTB interaction in the insurgence of the disease. Finally, the presence of potassium bound to the central cavity of the CTD pentameric assembly provides insights into the role of KCTD1 in metal homeostasis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Allostery is an important property of biological macromolecules which regulates diverse biological functions such as catalysis, signal transduction, transport, and molecular recognition. However, the concept was expressed using two different definitions by J. Monod and, over time, more have been added by different authors, making it fuzzy. Here, we reviewed the different meanings of allostery in the current literature and found that it has been used to indicate that the function of a protein is regulated by heterotropic ligands, and/or that the binding of ligands and substrates presents homotropic positive or negative cooperativity, whatever the hypothesized or demonstrated reaction mechanism might be. Thus, proteins defined to be allosteric include not only those that obey the two-state concerted model, but also those that obey different reaction mechanisms such as ligand-induced fit, possibly coupled to sequential structure changes, and ligand-linked dissociation-association. Since each reaction mechanism requires its own mathematical description and is defined by it, there are many possible 'allosteries'. This lack of clarity is made even fuzzier by the fact that the reaction mechanism is often assigned imprecisely and/or implicitly in the absence of the necessary experimental evidence. In this review, we examine a list of proteins that have been defined to be allosteric and attempt to assign a reaction mechanism to as many as possible., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Organochlorine pesticides (OCPs) are a class of environmentally persistent and bioaccumulative pollutants. Among these, β-hexachlorocyclohexane (β-HCH) is a byproduct of lindane synthesis, one of the most worldwide widespread pesticides. β-HCH cellular mechanisms inducing chemical carcinogenesis correspond to many of those inducing chemoresistance, in particular, by the activation of signal transducer and activator of transcription 3 (STAT3) signaling pathways. For this purpose, four cell lines, representative of breast, lung, prostate, and hepatocellular cancers, were treated with β-HCH, specific tyrosine kinase inhibitors (TKIs), and a STAT3 inhibitor. All cell samples were analyzed by a viability assay, immunoblotting analysis, a wound-healing assay, and a colony formation assay. The results show that β-HCH reduces the efficacy of TKIs. The STAT3 protein, in this context, plays a central role. In fact, by inhibiting its activity, the efficacy of the anticancer drug is restored. Furthermore, this manuscript aimed to draw the attention of the scientific and socio-healthcare community to the issue of prolonged exposure to contaminants and their impact on drug efficacy.

The surveillance of COVID-19 pandemic has led to the determination of millions of genome sequences of the SARS-CoV-2 virus, with the accumulation of a wealth of information never collected before for an infectious disease. Exploring the information retrieved from the GISAID database reporting at that time >13 million genome sequences, we classified the 141,639 unique missense mutations detected in the first two-and-a-half years (up to October 2022) of the pandemic. Notably, our analysis indicates that 98.2 % of all possible conservative amino acid replacements occurred. Even non-conservative mutations were highly represented (73.9 %). For a significant number of residues (3 %), all possible replacements with the other nineteen amino acids have been observed. These observations strongly indicate that, in this time interval, the virus explored all possible alternatives in terms of missense mutations for all sites of its polypeptide chain and that those that are not observed severely affect SARS-CoV-2 integrity. The implications of the present findings go well beyond the structural biology of SARS-CoV-2 as the huge amount of information here collected and classified may be valuable for the elucidation of the sequence-structure-function relationships in proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The acetylation of α-tubulin on lysine 40 is a well-studied post-translational modification which has been associated with the presence of long-lived stable microtubules that are more resistant to mechanical breakdown. The discovery of α-tubulin acetyltransferase 1 (ATAT1), the enzyme responsible for lysine 40 acetylation on α-tubulin in a wide range of species, including protists, nematodes, and mammals, dates to about a decade ago. However, the role of ATAT1 in different cellular activities and molecular pathways has been only recently disclosed. This review comprehensively summarizes the most recent knowledge on ATAT1 structure and substrate binding and analyses the involvement of ATAT1 in a variety of cellular processes such as cell motility, mitosis, cytoskeletal organization, and intracellular trafficking. Finally, the review highlights ATAT1 emerging roles in human diseases and discusses ATAT1 potential enzymatic and non-enzymatic roles and the current efforts in developing ATAT1 inhibitors., (© 2024. The Author(s).)

E2-conjugating enzymes (E2s) play a central role in the enzymatic cascade that leads to the attachment of ubiquitin to a substrate. This process, termed ubiquitylation, is required to maintain cellular homeostasis and affects almost all cellular process. By interacting with multiple E3 ligases, E2s dictate the ubiquitylation landscape within the cell. Since its discovery, ubiquitylation has been regarded as a posttranslational modification that specifically targets lysine side chains (canonical ubiquitylation). We used Matrix-Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry to identify and characterize a family of E2s that are instead able to conjugate ubiquitin to serine and/or threonine. We used structural modeling and prediction tools to identify the key activity determinants that these E2s use to interact with ubiquitin as well as their substrates. Our results unveil the missing E2s necessary for noncanonical ubiquitylation, underscoring the adaptability and versatility of ubiquitin modifications.

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.)

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IK Ca ) channel. IK Ca channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IK Ca current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IK Ca channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IK Ca channel., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Ada Maria Tata reports article publishing charges was provided by University of Rome La Sapienza. Ada Maria Tata reports a relationship with University of Rome La Sapienza that includes: board membership and funding grants. Ada Maria Tata has No patent pending to Not assigned. No conflict of interest to declare., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

KCTD ((K)potassium Channel Tetramerization Domain-containing) proteins constitute an emerging class of proteins involved in fundamental physio-pathological processes. In these proteins, the BTB domain, which represents the defining element of the family, may have the dual role of promoting oligomerization and favoring functionally important partnerships with different interactors. Here, by exploiting the potential of recently developed methodologies for protein structure prediction, we report a comprehensive analysis of the interactions of all KCTD proteins with their most common partner Cullin 3 (Cul3). The data here presented demonstrate the impressive ability of this approach to discriminate between KCTDs that interact with Cul3 and those that do not. Indeed, reliable and stable models of the complexes were only obtained for the 15 members of the family that are known to interact with Cul3. The generation of three-dimensional models for all KCTD-Cul3 complexes provides interesting clues on the determinants of the structural basis of this partnership as clear structural differences emerged between KCTDs that bind or do not bind Cul3. Finally, the availability of accurate three-dimensional models for KCTD-Cul3 interactions may be valuable for the ad hoc design and development of compounds targeting specific KCTDs that are involved in several common diseases.

Background: Peritoneal metastasis, which accounts for 85% of all epithelial ovarian carcinoma (EOC) metastases, is a multistep process that requires the establishment of adhesive interactions between cancer cells and the peritoneal membrane. Interrelations between EOC and the mesothelial stroma are critical to facilitate the metastatic process. No data is available so far on the impact of histone acetylation/deacetylation, a potentially relevant mechanism governing EOC metastasis, on mesothelial cells (MCs)-mediated adhesion., Methods: Static adhesion and peritoneal clearance experiments were performed pretreating mesenchymal-like MCs and platinum-sensitive/resistant EOC cell lines with MS-275-a Histone deacetylase (HDAC)1-3 pharmacological inhibitor currently used in combination trials. Results were acquired by confocal microscopy and were analyzed with an automated Opera software. The role of HDAC1/2 was validated by genetic silencing. The role of α4-, α5-α1 Integrins and Fibronectin-1 was validated using specific monoclonal antibodies. Quantitative proteomic analysis was performed on primary MCs pretreated with MS-275. Decellularized matrices were generated from either MS-275-exposed or untreated cells to study Fibronectin-1 extracellular secretion. The effect of MS-275 on β1 integrin activity was assessed using specific monoclonal antibodies. The role of Talin-1 in MCs/EOC adhesion was analyzed by genetic silencing. Talin-1 ectopic expression was validated as a rescue tool from MS-275-induced phenotype. The in vivo effect of MS-275-induced MC remodeling was validated in a mouse model of peritoneal EOC dissemination., Results: Treatment of MCs with non-cytotoxic concentrations of MS-275 caused a consistent reduction of EOC adhesion. Proteomic analysis revealed several pathways altered upon MC treatment with MS-275, including ECM deposition/remodeling, adhesion receptors and actin cytoskeleton regulators. HDAC1/2 inhibition hampered actin cytoskeleton polymerization by downregulating actin regulators including Talin-1, impairing β1 integrin activation, and leading to abnormal extracellular secretion and distribution of Fibronectin-1. Talin-1 ectopic expression rescued EOC adhesion to MS-275-treated MCs. In an experimental mouse model of metastatic EOC, MS-275 limited tumor invasion, Fibronectin-1 secretion and the sub-mesothelial accumulation of MC-derived carcinoma-associated fibroblasts., Conclusion: Our study unveils a direct impact of HDAC-1/2 in the regulation of MC/EOC adhesion and highlights the regulation of MC plasticity by epigenetic inhibition as a potential target for therapeutic intervention in EOC peritoneal metastasis., (© 2024. The Author(s).)

The extent of the devastation of the Black Death pandemic (1346-1353) on European populations is known from documentary sources and its bacterial source illuminated by studies of ancient pathogen DNA. What has remained less understood is the effect of the pandemic on human mobility and genetic diversity at the local scale. Here, we report 275 ancient genomes, including 109 with coverage >0.1×, from later medieval and postmedieval Cambridgeshire of individuals buried before and after the Black Death. Consistent with the function of the institutions, we found a lack of close relatives among the friars and the inmates of the hospital in contrast to their abundance in general urban and rural parish communities. While we detect long-term shifts in local genetic ancestry in Cambridgeshire, we find no evidence of major changes in genetic ancestry nor higher differentiation of immune loci between cohorts living before and after the Black Death.

Peptides are able to self-organize in structural elements including cross-β structures. Taking advantage of this tendency, in the last decades, peptides have been scrutinized as molecular elements for the development of multivalent supramolecular architectures. In this context, different classes of peptides, also with completely aromatic sequences, were proposed. Our previous studies highlighted that the (FY)3 peptide, which alternates hydrophobic phenylalanine and more hydrophilic tyrosine residues, is able to self-assemble, thanks to the formation of both polar and apolar interfaces. It was observed that the replacement of Phe and Tyr residues with other noncoded aromatic amino acids like 2-naphthylalanine (Nal) and Dopa affects the interactions among peptides with consequences on the supramolecular organization. Herein, we have investigated the self-assembling behavior of two novel (FY)3 analogues with Trp and Dopa residues in place of the Phe and Tyr ones, respectively. Additionally, PEGylation of the N-terminus was analyzed too. The supramolecular organization, morphology, and capability to gel were evaluated using complementary techniques, including fluorescence, Fourier transform infrared spectroscopy, and scanning electron microscopy. Structural periodicities along and perpendicular to the fiber axis were detected by grazing incidence wide-angle X-ray scattering. Finally, molecular dynamics studies provided interesting insights into the atomic structure of the cross-β that constitutes the basic motif of the assemblies formed by these novel peptide systems.

Aptamers are synthetic nucleic acids that are developed to target with high affinity and specificity chemical entities ranging from single ions to macromolecules and present a wide range of chemical and physical properties. Their ability to selectively bind proteins has made these compounds very attractive and versatile tools, in both basic and applied sciences, to such an extent that they are considered an appealing alternative to antibodies. Here, by exhaustively surveying the content of the Protein Data Bank (PDB), we review the structural aspects of the protein-aptamer recognition process. As a result of three decades of structural studies, we identified 144 PDB entries containing atomic-level information on protein-aptamer complexes. Interestingly, we found a remarkable increase in the number of determined structures in the last two years as a consequence of the effective application of the cryo-electron microscopy technique to these systems. In the present paper, particular attention is devoted to the articulated architectures that protein-aptamer complexes may exhibit. Moreover, the molecular mechanism of the binding process was analyzed by collecting all available information on the structural transitions that aptamers undergo, from their protein-unbound to the protein-bound state. The contribution of computational approaches in this area is also highlighted.

AMBRA1 is a crucial factor for nervous system development, and its function has been mainly associated with autophagy. It has been also linked to cell proliferation control, through its ability to regulate c-Myc and D-type cyclins protein levels, thus regulating G1-S transition. However, it remains still unknown whether AMBRA1 is differentially regulated during the cell cycle, and if this pro-autophagy protein exerts a direct role in controlling mitosis too. Here we show that AMBRA1 is phosphorylated during mitosis on multiple sites by CDK1 and PLK1, two mitotic kinases. Moreover, we demonstrate that AMBRA1 phosphorylation at mitosis is required for a proper spindle function and orientation, driven by NUMA1 protein. Indeed, we show that the localization and/or dynamics of NUMA1 are strictly dependent on AMBRA1 presence, phosphorylation and binding ability. Since spindle orientation is critical for tissue morphogenesis and differentiation, our findings could account for an additional role of AMBRA1 in development and cancer ontogenesis., (© 2023. The Author(s).)

The coagulation process relies on an intricate network of three-dimensional structural interactions and subtle biological regulations. In the present review, we illustrate the state of the art of the structural biology of the coagulation cascade by surveying the Protein Data Bank and the EBI AlphaFold databases. Investigations performed in the last decade have provided structural information on essentially all players involved in the process. Indeed, the initial characterization of specific and rather canonical domains has been progressively extended to complicated multidomain proteins. Recently, the application of cryogenic electron microscopy techniques has unraveled the structural features of highly complex coagulation factors, which has led to enhanced understanding. This review initially focuses on the structure of the individual factors as a function of their involvement in intrinsic, extrinsic, and common pathways. A specific emphasis is given to what is known or unknown on the structural basis of each step of the cascade. Available data providing clues on the structural recognition of the factors involved in the functional partnerships of the pathways are illustrated. Recent structures of important complexes formed by these proteins with regulators are described, focusing on the drugs used as anticoagulants and on their reversal agents. Finally, we highlight the different roles that innovative biomolecules such as aptamers may have in the regulation of the cascade., (© 2023 The Author(s).)

Macroautophagy/autophagy has been shown to exert a dual role in cancer i.e., promoting cell survival or cell death depending on the cellular context and the cancer stage. Therefore, development of potent autophagy modulators, with a clear mechanistic understanding of their target action, has paramount importance in both mechanistic and clinical studies. In the process of exploring the mechanism of action of a previously identified cytotoxic small molecule (SM15) designed to target microtubules and the interaction domain of microtubules and the kinetochore component NDC80/HEC1, we discovered that the molecule acts as a potent autophagy inhibitor. By using several biochemical and cell biology assays we demonstrated that SM15 blocks basal autophagic flux by inhibiting the fusion of correctly formed autophagosomes with lysosomes. SM15-induced autophagic flux blockage promoted apoptosis-mediated cell death associated with ROS production. Interestingly, autophagic flux blockage, apoptosis induction and ROS production were rescued by genetic or pharmacological inhibition of OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) or by expressing an O-GlcNAcylation-defective mutant of the SNARE fusion complex component SNAP29, pointing to SNAP29 as the molecular target of SM15 in autophagy. Accordingly, SM15 was found to enhance SNAP29 O-GlcNAcylation and, thereby, inhibit the formation of the SNARE fusion complex. In conclusion, these findings identify a new pathway in autophagy connecting O-GlcNAcylated SNAP29 to autophagic flux blockage and autophagosome accumulation, that, in turn, drives ROS production and apoptotic cell death. Consequently, modulation of SNAP29 activity may represent a new opportunity for therapeutic intervention in cancer and other autophagy-associated diseases.

Actinic keratosis (AK) is a carcinoma in situ precursor of cutaneous squamous cell carcinoma (cSCC), the second most common cancer affecting the Caucasian population. AK is frequently present in the sun-exposed skin of the elderly population, UV radiation being the main cause of this cancer, and other risk factors contributing to AK incidence. The dysregulation of microRNAs (miRNAs) observed in different cancers leads to an improper expression of miRNA targets involved in several cellular pathways. The TaqMan Array Human MicroRNA Card assay for miRNA expression profiling was performed in pooled AK compared to healthy skin scraping samples from the same patients. Forty-three miRNAs were modulated in the AK samples. The expression of miR-19b ( p < 0.05), -31, -34a ( p < 0.001), -126, -146a ( p < 0.01), -193b, and -222 ( p < 0.05) was validated by RT-qPCR. The MirPath tool was used for MiRNA target prediction and enriched pathways. The top DIANA-mirPath pathways regulated by the targets of the 43 miRNAs are TGF-beta signaling, Proteoglycans in cancer, Pathways in cancer, and Adherens junction (7.30 × 10 -10 < p < 1.84 × 10 -8 ). Selected genes regulating the KEGG pathways, i.e., TP53 , MDM2 , CDKN1A , CDK6 , and CCND1 , were analyzed. MiRNAs modulated in AK regulate different pathways involved in tumorigenesis, indicating miRNA regulation as a critical step in keratinocyte cancer.

Dissemination of high-grade serous ovarian cancer (HG-SOC) in the omentum and intercalation into a mesothelial cell (MC) monolayer depends on functional α5β1 integrin (Intα5β1) activity. Although the binding of Intα5β1 to fibronectin drives these processes, other molecular mechanisms linked to integrin inside-out signaling might support metastatic dissemination. Here, we report a novel interactive signaling that contributes to Intα5β1 activation and accelerates tumor cells toward invasive disease, involving the protein β-arrestin1 (β-arr1) and the activation of the endothelin A receptor (ET A R) by endothelin-1 (ET-1). As demonstrated in primary HG-SOC cells and SOC cell lines, ET-1 increased Intβ1 and downstream FAK/paxillin activation. Mechanistically, β-arr1 directly interacts with talin1 and Intβ1, promoting talin1 phosphorylation and its recruitment to Intβ1, thus fueling integrin inside-out activation. In 3D spheroids and organotypic models mimicking the omentum, ET A R/β-arr1-driven Intα5β1 signaling promotes the survival of cell clusters, with mesothelium-intercalation capacity and invasive behavior. The treatment with the antagonist of ET A R, Ambrisentan (AMB), and of Intα5β1, ATN161, inhibits ET-1-driven Intα5β1 activity in vitro, and tumor cell adhesion and spreading to intraperitoneal organs and Intβ1 activity in vivo. As a prognostic factor, high EDNRA/ITGB1 expression correlates with poor HG-SOC clinical outcomes. These findings highlight a new role of ET A R/β-arr1 operating an inside-out integrin activation to modulate the metastatic process and suggest that in the new integrin-targeting programs might be considered that ET A R/β-arr1 regulates Intα5β1 functional pathway., (© 2023. The Author(s).)

Cytochrome P450 OleP catalytic activity is strongly influenced by its structural dynamic conformational behavior. Here, we combine equilibrium-binding experiments with all-atom molecular dynamics simulations to clarify how different environments affect OleP conformational equilibrium between the open and the closed-catalytic competent-forms. Our data clearly show that at high-ionic strength conditions, the closed form is favored, and, very interestingly, different mechanisms, depending on the chemistry of the cations, can be used to rationalize such an effect.

Here, we describe a conserved motor neuron-specific long non-coding RNA, Lhx1os, whose knockout in mice produces motor impairment and postnatal reduction of mature motor neurons (MNs). The ER stress-response pathway result specifically altered with the downregulation of factors involved in the unfolded protein response (UPR). Lhx1os was found to bind the ER-associated PDIA3 disulfide isomerase and to affect the expression of the same set of genes controlled by this protein, indicating that the two factors act in conjunction to modulate the UPR. Altogether, the observed phenotype and function of Lhx1os indicate its important role in the control of MN homeostasis and function., Competing Interests: Authors declare that they have no competing interests., (© 2023 The Authors.)

Background: 1,3-β-glucan is a polysaccharide widely distributed in the cell wall of several phylogenetically distant organisms, such as bacteria, fungi, plants and microalgae. The presence of highly active 1,3-β-glucanases in fungi evokes the biological question on how these organisms can efficiently metabolize exogenous sources of 1,3-β-glucan without incurring in autolysis., Results: To elucidate the molecular mechanisms at the basis of 1,3-β-glucan metabolism in fungal saprotrophs, the putative exo-1,3-β-glucanase G9376 and a truncated form of the putative glucan endo-1,3-β-glucosidase (ΔG7048) from Penicillium sumatraense AQ67100 were heterologously expressed in Pichia pastoris and characterized both in terms of activity and structure. G9376 efficiently converted laminarin and 1,3-β-glucan oligomers into glucose by acting as an exo-glycosidase, whereas G7048 displayed a 1,3-β-transglucanase/branching activity toward 1,3-β-glucan oligomers with a degree of polymerization higher than 5, making these oligomers more recalcitrant to the hydrolysis acted by exo-1,3-β-glucanase G9376. The X-ray crystallographic structure of the catalytic domain of G7048, solved at 1.9 Å of resolution, consists of a (β/α) 8 TIM-barrel fold characteristic of all the GH17 family members. The catalytic site is in a V-shaped cleft containing the two conserved catalytic glutamic residues. Molecular features compatible with the activity of G7048 as 1,3-β-transglucanase are discussed., Conclusions: The antagonizing activity between ΔG7048 and G9376 indicates how opportunistic fungi belonging to Penicillium genus can feed on substrates similar for composition and structure to their own cell wall without incurring in a self-deleterious autohydrolysis., (© 2022. The Author(s).)

An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate.

Oligomerization endows proteins with some key properties such as extra-stabilization, long-range allosteric regulation(s), and partnerships not accessible to their monomeric counterparts. How oligomerization is achieved and preserved during evolution is a subject of remarkable scientific relevance. By exploiting the abilities of the machine-learning algorithms implemented in AlphaFold (AF) in predicting protein structures, herein, we report a comprehensive analysis of the structural states of functional oligomers of all members of the KCTD protein family. Interestingly, our approach led to the identification of reliable three-dimensional models for the pentameric states of KCNRG, KCTD6, KCTD4, KCTD7, KCTD9, and KCTD14 and possibly for KCTD11 and KCTD21 that are involved in key biological processes and that were previously uncharacterized from a structural point of view. Although for most of these proteins, the CTD domains lack any sequence similarity, they share some important structural features, such as a propeller-like structure with a central cavity delimited by five exposed and regular β-strands. Moreover, the structure of the related proteins KCTD7 and KCTD14, although pentameric, appears to be characterized by a different organization of the CTD region, with the five chains forming a circle-like structure with a large cavity. Our predictions also suggest that other members of the family, such as KCTD10, KCTD13, and TNFAIP1, present a strong propensity to assume dimeric states. Although the structures of the functional oligomers reported herein represent models that require additional validations, they provide a consistent and global view of KCTD protein oligomerization.

The definition of the structural basis of the conformational preferences of the genetically encoded amino acid residues is an important yet unresolved issue of structural biology. In order to gain insights into this intricate topic, we here determined and compared the amino acid propensity scales for different (φ, ψ) regions of the Ramachandran plot and for different secondary structure elements. These propensities were calculated using the Chou-Fasman approach on a database of non-redundant protein chains retrieved from the Protein Data Bank. Similarities between propensity scales were evaluated by linear regression analyses. One of the most striking and unexpected findings is that distant regions of the Ramachandran plot may exhibit significantly similar propensity scales. On the other hand, contiguous regions of the Ramachandran plot may present anticorrelated propensities. In order to provide an interpretative background to these results, we evaluated the role that the local variability of protein backbone geometry plays in this context. Our analysis indicates that (dis)similarities of propensity scales between different regions of the Ramachandran plot are coupled with (dis)similarities in the local geometry. The concept that similarities of the propensity scales are dictated by the similarity of the NC α C angle and not necessarily by the similarity of the (φ, ψ) conformation may have far-reaching implications in the field.

Tetrameric hemoglobins (Hbs) are prototypal systems for studies aimed at unveiling basic structure-function relationships as well as investigating the molecular/structural basis of adaptation of living organisms to extreme conditions. However, a chronological analysis of decade-long studies conducted on Hbs is illuminating on the difficulties associated with the attempts of gaining functional insights from static structures. Here, we applied molecular dynamics (MD) simulations to explore the functional transition from the T to the R state of the hemoglobin of the Antarctic fish Trematomus bernacchii (HbTb). Our study clearly demonstrates the ability of the MD technique to accurately describe the transition of HbTb from the T to R-like states, as shown by a number of global and local structural indicators. A comparative analysis of the structural states that HbTb assumes in the simulations with those detected in previous MD analyses conducted on HbA (human Hb) highlights interesting analogies (similarity of the transition pathway) and differences (distinct population of intermediate states). In particular, the ability of HbTb to significantly populate intermediate states along the functional pathway explains the observed propensity of this protein to assume these structures in the crystalline state. It also explains some functional data reported on the protein that indicate the occurrence of other functional states in addition to the canonical R and T ones. These findings are in line with the emerging idea that the classical two-state view underlying tetrameric Hb functionality is probably an oversimplification and that other structural states play important roles in these proteins. The ability of MD simulations to accurately describe the functional pathway in tetrameric Hbs suggests that this approach may be effectively applied to unravel the molecular and structural basis of Hbs exhibiting peculiar functional properties as a consequence of the environmental adaptation of the host organism.

Euphorbia resinifera latex has been extensively utilized in traditional medicine due to its range of bioactivities. Chromatographic separations on silica gel of ethanol extract of E. resinifera latex led to the development of a new procedure for isolating resiniferatoxin ( 4 ) via dried E. resinifera latex and the identification of nine compounds. Among these, catechol ( 7 ), protocatechuic acid ( 8 ) and 3,4-dihydroxyphenylacetic acid ( 9 ), known phenolic compounds, were identified for the first time in E. resinifera latex. Herein we investigated the effects of major compounds of the latex of E. resinifera on the yeast Saccharomyces cerevisiae , on the growth of Aspergillus carbonarius , a widespread fungal contaminant, and on the breast cancer cell line MCF7 as well as on MCF10A normal breast cells. 12-deoxyphorbol-13-isobutyrate-20-acetate ( 2 ) had an inhibiting effect on the growth of A. carbonarius , and 7- p -metoxyphenylacetate-3,8,12-triacetate ingol ( 3 ) showed a negative effect on yeast cell growth and also a cytotoxic effect on breast cancer cell line MCF7, but not on MCF10A cells. Deglucosyl euphorbioside A ( 5 ) and euphorbioside A ( 6 ) showed a discoloration effect that was possibly related to mitochondrial functionality in yeast, and also cytotoxicity only on the cancer cell line that was tested. Interestingly, treatment of MCF7 cells with 7- p -metoxyphenylacetate-3,8,12-triacetate ingol ( 3 ) and deglucosyl euphorbioside A ( 5 ) not only led to a specific cytotoxic effect but also to the increase in the level of intracellular ROS., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results

Human herpes simplex virus 1 (HSV-1), a life-long infection spread by oral contact, infects a majority of adults globally. Phylogeographic clustering of sampled diversity into European, pan-Eurasian, and African groups has suggested the virus codiverged with human migrations out of Africa, although a much younger origin has also been proposed. We present three full ancient European HSV-1 genomes and one partial genome, dating from the 3rd to 17th century CE, sequenced to up to 9.5× with paired human genomes up to 10.16×. Considering a dataset of modern and ancient genomes, we apply phylogenetic methods to estimate the age of sampled modern Eurasian HSV-1 diversity to 4.68 (3.87 to 5.65) ka. Extrapolation of estimated rates to a global dataset points to the age of extant sampled HSV-1 as 5.29 (4.60 to 6.12) ka, suggesting HSV-1 lineage replacement coinciding with the late Neolithic period and following Bronze Age migrations.

KAT Gcn5 and DUB Ubp8 are required for respiration and mitochondria functions in budding yeast, and in this study we show that loss of respiratory activity is acquired over time. Interestingly, we show that absence of Ubp8 allows cells to grow in hypoxic conditions with altered mitophagy. Comparatively, the aggressive glioblastoma (GBM) multiforme tumor shows survival mechanisms able to overcome hypoxia in the brain. Starting from yeast and our findings on the role of Ubp8 in hypoxia, we extended our analysis to the human ortholog and signature cancer gene Usp22 in glioblastoma tumor specimens. Here we demonstrate that Usp22 is localized and overexpressed in the pseudo-palisade tissue around the necrotic area of the tumor. In addition, Usp22 colocalizes with the mitophagy marker Parkin, indicating a link with mitochondria function in GBM. Collectively, this evidence suggests that altered expression of Usp22 might provide a way for tumor cells to survive in hypoxic conditions, allowing the escape of cells from the necrotic area toward vascularized tissues. Collectively, our experimental data suggest a model for a possible mechanism of uncontrolled proliferation and invasion in glioblastoma.

The cross-talk between axon and glial cells during development and in adulthood is mediated by several molecules. Among them are neurotransmitters and their receptors, which are involved in the control of myelinating and non-myelinating glial cell development and physiology. Our previous studies largely demonstrate the functional expression of cholinergic muscarinic receptors in Schwann cells. In particular, the M2 muscarinic receptor subtype, the most abundant cholinergic receptor expressed in Schwann cells, inhibits cell proliferation downregulating proteins expressed in the immature phenotype and triggers promyelinating differentiation genes. In this study, we analysed the in vitro modulation of the Neuregulin-1 (NRG1)/erbB pathway, mediated by the M2 receptor activation, through the selective agonist arecaidine propargyl ester (APE). M2 agonist treatment significantly downregulates NRG1 and erbB receptors expression, both at transcriptional and protein level, and causes the internalization and intracellular accumulation of the erbB2 receptor. Additionally, starting from our previous results concerning the negative modulation of Notch-active fragment NICD by M2 receptor activation, in this work, we clearly demonstrate that the M2 receptor subtype inhibits erbB2 receptors by Notch-1/NICD downregulation. Our data, together with our previous results, demonstrate the existence of a cross-interaction between the M2 receptor and NRG1/erbB pathway-Notch1 mediated, and that it is responsible for the modulation of Schwann cell proliferation/differentiation.

Circular RNAs (circRNAs) are widely expressed in eukaryotes and are regulated in many biological processes. Although several studies indicate their activity as microRNA (miRNA) and protein sponges, little is known about their ability to directly control mRNA homeostasis. We show that the widely expressed circZNF609 directly interacts with several mRNAs and increases their stability and/or translation by favoring the recruitment of the RNA-binding protein ELAVL1. Particularly, the interaction with CKAP5 mRNA, which interestingly overlaps the back-splicing junction, enhances CKAP5 translation, regulating microtubule function in cancer cells and sustaining cell-cycle progression. Finally, we show that circZNF609 downregulation increases the sensitivity of several cancer cell lines to different microtubule-targeting chemotherapeutic drugs and that locked nucleic acid (LNA) protectors against the pairing region on circZNF609 phenocopy such effects. These data set an example of how the small effects tuned by circZNF609/CKAP5 mRNA interaction might have a potent output in tumor growth and drug response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Substrate binding to the cytochrome P450 OleP is coupled to a large open-to-closed transition that remodels the active site, minimizing its exposure to the external solvent. When the aglycone substrate binds, a small empty cavity is formed between the I and G helices, the BC loop, and the substrate itself, where solvent molecules accumulate mediating substrate-enzyme interactions. Herein, we analyzed the role of this cavity in substrate binding to OleP by producing three mutants (E89Y, G92W, and S240Y) to decrease its volume. The crystal structures of the OleP mutants in the closed state bound to the aglycone 6DEB showed that G92W and S240Y occupied the cavity, providing additional contact points with the substrate. Conversely, mutation E89Y induces a flipped-out conformation of this amino acid side chain, that points towards the bulk, increasing the empty volume. Equilibrium titrations and molecular dynamic simulations indicate that the presence of a bulky residue within the cavity impacts the binding properties of the enzyme, perturbing the conformational space explored by the complexes. Our data highlight the relevance of this region in OleP substrate binding and suggest that it represents a key substrate-protein contact site to consider in the perspective of redirecting its activity towards alternative compounds.

Microtubules are key components of the cytoskeleton of eukaryotic cells. Microtubule dynamic instability together with the "tubulin code" generated by the choice of different α- and β- tubulin isoforms and tubulin post-translational modifications have essential roles in the control of a variety of cellular processes, such as cell shape, cell motility, and intracellular trafficking, that are deregulated in cancer. In this review, we will discuss available evidence that highlights the crucial role of the tubulin code in determining different cancer phenotypes, including metastatic cell migration, drug resistance, and tumor vascularization, and the influence of modulating tubulin-modifying enzymes on cancer cell survival and aggressiveness. We will also discuss the role of post-translationally modified microtubules in autophagy-the lysosomal-mediated cellular degradation pathway-that exerts a dual role in many cancer types, either promoting or suppressing cancer growth. We will give particular emphasis to the role of tubulin post-translational modifications and their regulating enzymes in controlling the different stages of the autophagic process in cancer cells, and consider how the experimental modulation of tubulin-modifying enzymes influences the autophagic process in cancer cells and impacts on cancer cell survival and thereby represents a new and fruitful avenue in cancer therapy.

Transcoelomic spread of serous ovarian cancer (SOC) results from the cooperative interactions between cancer and host components. Tumor-derived factors might allow the conversion of mesothelial cells (MCs) into tumor-associated MCs, providing a favorable environment for SOC cell dissemination. However, factors and molecular mechanisms involved in this process are largely unexplored. Here we investigated the tumor-related endothelin-1 (ET-1) as an inducer of changes in MCs supporting SOC progression. Here, we report a significant production of ET-1 from MCs associated with the expression of its cognate receptors, ET A and ET B , along with the protein β-arrestin1. ET-1 triggers MC proliferation via β-arrestin1-dependent MAPK and NF-kB pathways and increases the release of cancer-related factors. The ET A /ET B receptor activation supports the genetic reprogramming of mesothelial-to-mesenchymal transition (MMT), with upregulation of mesenchymal markers, as fibronectin, α-SMA, N-cadherin and vimentin, NF-kB-dependent Snail transcriptional activity and downregulation of E-cadherin and ZO-1, allowing to enhanced MC migration and invasion, and SOC transmesothelial migration. These effects are impaired by either blockade of ET A R and ET B R or by β-arrestin1 silencing. Notably, in peritoneal metastases both ET A R and ET B R are co-expressed with MMT markers compared to normal control peritoneum. Collectively, our report shows that the ET-1 axis may contribute to the early stage of SOC progression by modulating MC pro-metastatic behaviour via MMT., 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 Del Rio, Masi, Caprara, Spadaro, Ottavi, Strippoli, Sandoval, López-Cabrera, Sainz de la Cuesta, Bagnato and Rosanò.)

Pancreatic cancer (PC) represents an intriguing topic for researchers. To date, the prognosis of metastasized PC is poor with just 7% of patients exceeding a five-year survival period. Thus, molecular modifications of existing drugs should be developed to change the course of the disease. Our previously generated nanocages of Mitoxantrone (MIT) encapsulated in human H-chain Ferritin (HFt), designated as HFt-MP-PASE-MIT, has shown excellent tumor distribution and extended serum half-life meriting further investigation for PC treatment. Thus, in this study, we used the same nano-formulation to test its cytotoxicity using both in vitro and in vivo assays. Interestingly, both encapsulated and free-MIT drugs demonstrated similar killing capabilities on PaCa44 cell line. Conversely, in vivo assessment in a subcutaneous PaCa44 tumor model of PC demonstrated a remarkable capability for encapsulated MIT to control tumor growth and improve mouse survival with a median survival rate of 65 vs. 33 days for loaded and free-MIT, respectively. Interestingly, throughout the course of mice treatment, MIT encapsulation did not present any adverse side effects as confirmed by histological analysis of various murine tissue organs and body mass weights. Our results are promising and pave the way to effective PC targeted chemotherapy using our HFt nanodelivery platforms.

Template-free nonenzymatic polymerization of 3',5' cyclic nucleotides is an emerging topic of the origin of life research. In the last ten years, a number of papers have been published addressing various aspects of this process. These works evoked a vivid discussion among scientists working in the field of prebiotic chemistry. The aim of the current review is to answer the most frequently raised questions related to the detection and characterization of oligomeric products as well as to the geological context of this chemistry.

The impact of protein-coding genes on cancer onset and progression is a well-established paradigm in molecular oncology. Nevertheless, unveiling the contribution of the noncoding genes-including long noncoding RNAs (lncRNAs)-to tumorigenesis represents a great challenge for personalized medicine, since they (i) constitute the majority of the human genome, (ii) are essential and flexible regulators of gene expression and (iii) present all types of genomic alterations described for protein-coding genes. LncRNAs have been increasingly associated with cancer, their highly tissue- and cancer type-specific expression making them attractive candidates as both biomarkers and therapeutic targets. Medulloblastoma is one of the most common malignant pediatric brain tumors. Group 3 is the most aggressive subgroup, showing the highest rate of metastasis at diagnosis. Transcriptomics and reverse genetics approaches were combined to identify lncRNAs implicated in Group 3 Medulloblastoma biology. Here we present the first collection of lncRNAs dependent on the activity of the MYC oncogene, the major driver gene of Group 3 Medulloblastoma. We assessed the expression profile of selected lncRNAs in Group 3 primary tumors and functionally characterized these species. Overall, our data demonstrate the direct involvement of three lncRNAs in Medulloblastoma cancer cell phenotypes.

Centrins are conserved calcium (Ca2+)-binding proteins typically associated with centrosomes that have been implicated in several biological processes. In Toxoplasma gondii, a parasite that causes toxoplasmosis, three centrin isoforms have been recognized. We have recently characterized the metal binding and structural features of isoform 1 (TgCEN1), demonstrating that it possesses properties consistent with a role as a Ca2+ sensor and displays a Ca2+-dependent tendency to self-assemble. Herein, we expanded our studies, focusing on the self-association and target binding properties of TgCEN1 by combining biophysical techniques including dynamic light scattering, isothermal titration calorimetry, nuclear magnetic resonance, circular dichroism, and fluorescence spectroscopy. We found that the self-assembly process of TgCEN1 depends on different physicochemical factors, including Ca2+ concentration, temperature, and protein concentration, and is mediated by both electrostatic and hydrophobic interactions. The process is completely abolished upon removal of the first 21-residues of the protein and is significantly reduced in the presence of a binding target peptide derived from the human XPC protein (P17-XPC). Titration of P17-XPC to the intact protein and isolated domains showed that TgCEN1 possesses two binding sites with distinct affinities and Ca2+ sensitivity; a high-affinity site in the C-lobe which may be constitutively bound to the peptide and a low-affinity site in the N-lobe which is active only upon Ca2+ stimulus. Overall, our results suggest a specific mechanism of TgCEN1 for Ca2+-modulated target binding and support a N-to-C self-assembly mode, in which the first 21-residues of one molecule likely interact with the C-lobe of the other., (© 2021 The Author(s).)

Background: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest., Methods: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression., Results: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines., Conclusions: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

Across Europe, the genetics of the Chalcolithic/Bronze Age transition is increasingly characterized in terms of an influx of Steppe-related ancestry. The effect of this major shift on the genetic structure of populations in the Italian Peninsula remains underexplored. Here, genome-wide shotgun data for 22 individuals from commingled cave and single burials in Northeastern and Central Italy dated between 3200 and 1500 BCE provide the first genomic characterization of Bronze Age individuals (n = 8; 0.001-1.2× coverage) from the central Italian Peninsula, filling a gap in the literature between 1950 and 1500 BCE. Our study confirms a diversity of ancestry components during the Chalcolithic and the arrival of Steppe-related ancestry in the central Italian Peninsula as early as 1600 BCE, with this ancestry component increasing through time. We detect close patrilineal kinship in the burial patterns of Chalcolithic commingled cave burials and a shift away from this in the Bronze Age (2200-900 BCE) along with lowered runs of homozygosity, which may reflect larger changes in population structure. Finally, we find no evidence that the arrival of Steppe-related ancestry in Central Italy directly led to changes in frequency of 115 phenotypes present in the dataset, rather that the post-Roman Imperial period had a stronger influence, particularly on the frequency of variants associated with protection against Hansen's disease (leprosy). Our study provides a closer look at local dynamics of demography and phenotypic shifts as they occurred as part of a broader phenomenon of widespread admixture during the Chalcolithic/Bronze Age transition., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

GGDEF-containing proteins respond to different environmental cues to finely modulate cyclic diguanylate (c-di-GMP) levels in time and space, making the allosteric control a distinctive trait of the corresponding proteins. The diguanylate cyclase mechanism is emblematic of this control: two GGDEF domains, each binding one GTP molecule, must dimerize to enter catalysis and yield c-di-GMP. The need for dimerization makes the GGDEF domain an ideal conformational switch in multidomain proteins. A re-evaluation of the kinetic profile of previously characterized GGDEF domains indicated that they are also able to convert GTP to GMP: this unexpected reactivity occurs when conformational issues hamper the cyclase activity. These results create new questions regarding the characterization and engineering of these proteins for in solution or structural studies.

Some constituents of the Mediterranean diet, such as extra-virgin olive oil (EVOO) contain substances such as hydroxytyrosol (HT) and its metabolite homovanillic alcohol (HA). HT has aroused much interest due to its antioxidant activity as a radical scavenger, whereas only a few studies have been made on the HA molecule. Both chemical synthesis and extraction techniques have been developed to obtain these molecules, with each method having its advantages and drawbacks. In this study, we report the use of tyrosol from olive mill wastewaters as a starting molecule to synthesize HT and HA, using a sustainable procedure characterized by high efficiency and low cost. The effects of HT and HA were evaluated on two cell lines, THP-1 human leukemic monocytes and L-6 myoblasts from rat skeletal muscle, after treating the cells with a radical generator. Both HT and HA efficiently inhibited ROS production. In particular, HT inhibited the proliferation of the THP-1 leukemic monocytes, while HA protected L-6 myoblasts from cytotoxicity.

The 3-hydroxy-3-methylglutaryl-CoA reductase, a key enzyme of the mevalonate pathway for the synthesis of cholesterol in mammals (ergosterol in fungi), is inhibited by statins, a class of cholesterol lowering drugs. Indeed, statins are in a wide medical use, yet statins treatment could induce side effects as hepatotoxicity and myopathy in patients. We used Saccharomyces cerevisiae as a model to investigate the effects of statins on mitochondria. We demonstrate that statins are active in S.cerevisiae by lowering the ergosterol content in cells and interfering with the attachment of mitochondrial DNA to the inner mitochondrial membrane. Experiments on murine myoblasts confirmed these results in mammals. We propose that the instability of mitochondrial DNA is an early indirect target of statins.

One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.

The majority of ovarian cancer (OC) patients recur with a platinum-resistant disease. OC cells activate adaptive resistance mechanisms that are only partially described. Here we show that OC cells can adapt to chemotherapy through a positive-feedback loop that favors chemoresistance. In platinum-resistant OC cells we document that the endothelin-1 (ET-1)/endothelin A receptor axis intercepts the YAP pathway. This cross-talk occurs through the LATS/RhoA/actin-dependent pathway and contributes to prevent the chemotherapy-induced apoptosis. Mechanistically, β-arrestin1 (β-arr1) and YAP form a complex shaping TEAD-dependent transcriptional activity on the promoters of YAP target genes, including EDN1, which fuels a feed-forward signaling circuit that sustains a platinum-tolerant state. The FDA approved dual ET-1 receptor antagonist macitentan in co-therapy with cisplatin sensitizes resistant cells to the platinum-based therapy, reducing their metastatic potential. Furthermore, high ET A R/YAP gene expression signature is associated with a poor platinum-response in OC patients. Collectively, our findings identify in the networking between ET-1 and YAP pathways an escape strategy from chemotherapy. ET-1 receptor blockade interferes with such adaptive network and enhances platinum-induced apoptosis, representing a promising therapeutic opportunity to restore drug sensitivity in OC patients., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)