Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS.

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells.

BACKGROUND: The C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with "cancer hallmarks" and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT). Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions. Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities. METHODS: Structured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model. RESULTS: We identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models.  CONCLUSIONS: This study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Targeting the Ubiquitin-Proteasome System and Recent Advances in Cancer Therapy.

Ubiquitination is a reversible post-translational modification based on the chemical addition of ubiquitin to proteins with regulatory effects on various signaling pathways. Ubiquitination can alter the molecular functions of tagged substrates with respect to protein turnover, biological activity, subcellular localization or protein-protein interaction. As a result, a wide variety of cellular processes are under ubiquitination-mediated control, contributing to the maintenance of cellular homeostasis. It follows that the dysregulation of ubiquitination reactions plays a relevant role in the pathogenic states of human diseases such as neurodegenerative diseases, immune-related pathologies and cancer. In recent decades, the enzymes of the ubiquitin-proteasome system (UPS), including E3 ubiquitin ligases and deubiquitinases (DUBs), have attracted attention as novel druggable targets for the development of new anticancer therapeutic approaches. This perspective article summarizes the peculiarities shared by the enzymes involved in the ubiquitination reaction which, when deregulated, can lead to tumorigenesis. Accordingly, an overview of the main pharmacological interventions based on targeting the UPS that are in clinical use or still in clinical trials is provided, also highlighting the limitations of the therapeutic efficacy of these approaches. Therefore, various attempts to circumvent drug resistance and side effects as well as UPS-related emerging technologies in anticancer therapeutics are discussed.

The DarT/DarG Toxin-Antitoxin ADP-Ribosylation System as a Novel Target for a Rational Design of Innovative Antimicrobial Strategies.

The chemical modification of cellular macromolecules by the transfer of ADP-ribose unit(s), known as ADP-ribosylation, is an ancient homeostatic and stress response control system. Highly conserved across the evolution, ADP-ribosyltransferases and ADP-ribosylhydrolases control ADP-ribosylation signalling and cellular responses. In addition to proteins, both prokaryotic and eukaryotic transferases can covalently link ADP-ribosylation to different conformations of nucleic acids, thus highlighting the evolutionary conservation of archaic stress response mechanisms. Here, we report several structural and functional aspects of DNA ADP-ribosylation modification controlled by the prototype DarT and DarG pair, which show ADP-ribosyltransferase and hydrolase activity, respectively. DarT/DarG is a toxin-antitoxin system conserved in many bacterial pathogens, for example in Mycobacterium tuberculosis, which regulates two clinically important processes for human health, namely, growth control and the anti-phage response. The chemical modulation of the DarT/DarG system by selective inhibitors may thus represent an exciting strategy to tackle resistance to current antimicrobial therapies.

ADP-Ribosylation Post-Translational Modification: An Overview with a Focus on RNA Biology and New Pharmacological Perspectives.

Cellular functions are regulated through the gene expression program by the transcription of new messenger RNAs (mRNAs), alternative RNA splicing, and protein synthesis. To this end, the post-translational modifications (PTMs) of proteins add another layer of complexity, creating a continuously fine-tuned regulatory network. ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules, regulating a multitude of key functional processes as diverse as DNA damage repair (DDR), transcriptional regulation, intracellular transport, immune and stress responses, and cell survival. Additionally, due to the emerging role of ADP-ribosylation in pathological processes, ADP-ribosyltransferases (ARTs), the enzymes involved in ADPr, are attracting growing interest as new drug targets. In this review, an overview of human ARTs and their related biological functions is provided, mainly focusing on the regulation of ADP-ribosyltransferase Diphtheria toxin-like enzymes (ARTD)-dependent RNA functions. Finally, in order to unravel novel gene functional relationships, we propose the analysis of an inventory of human gene clusters, including ARTDs, which share conserved sequences at 3' untranslated regions (UTRs).

Combinatorial Strategies to Target Molecular and Signaling Pathways to Disarm Cancer Stem Cells.

Cancer is an urgent public health issue with a very huge number of cases all over the world expected to increase by 2040. Despite improved diagnosis and therapeutic protocols, it remains the main leading cause of death in the world. Cancer stem cells (CSCs) constitute a tumor subpopulation defined by ability to self-renewal and to generate the heterogeneous and differentiated cell lineages that form the tumor bulk. These cells represent a major concern in cancer treatment due to resistance to conventional protocols of radiotherapy, chemotherapy and molecular targeted therapy. In fact, although partial or complete tumor regression can be achieved in patients, these responses are often followed by cancer relapse due to the expansion of CSCs population. The aberrant activation of developmental and oncogenic signaling pathways plays a relevant role in promoting CSCs therapy resistance. Although several targeted approaches relying on monotherapy have been developed to affect these pathways, they have shown limited efficacy. Therefore, an urgent need to design alternative combinatorial strategies to replace conventional regimens exists. This review summarizes the preclinical studies which provide a proof of concept of therapeutic efficacy of combinatorial approaches targeting the CSCs.

Corrigendum: Combinatorial Strategies to Target Molecularand Signaling Pathways to Disarm Cancer Stem Cells.

[This corrects the article DOI: 10.3389/fonc.2021.689131.].

WTAP and BIRC3 are involved in the posttranscriptional mechanisms that impact on the expression and activity of the human lactonase PON2.

The activity of human paraoxonase 2 (PON2) is rapidly reduced in cells incubated with the bacterial quorormone 3-Oxo-dodecanoyl Homoserine Lactone (3OC12HSL), an observation that led to hypothesize a fast PON2 post-translational modification (PTM). Recently, we detected a 3OC12HSL-induced PTM in a cell-free system in which a crude extract from 3OC12HSL-treated HeLa cells was able to inactivate and ubiquitinate at position 144 a recombinant PON2. Here we show the occurrence of this and new PTMs on PON2 in HeLa cells. PTMs were found to gather nearby the two SNPs, A148G, and S311C, that are related to type-2 diabetes and its complications. Furthermore, we detected a PTM nearby a 12 amino acids region that is deleted in PON2 Isoform 2. An in vitro mutation analysis showed that the SNPs and the deletion are involved in PON2 activity and suggested a role of PTMs on its modulation, while a SAXS analysis pointed to Isoform 2 as being largely unstructured, compared to the wild type. Besides, we discovered a control of PON2 expression via a putative mRNA operon involving the Wilms tumor 1 associated protein (WTAP) and the E3 ubiquitin ligase (E3UbL) baculoviral IAP repeat-containing 3 (BIRC3).

Targeting ADP-ribosylation as an antimicrobial strategy.

ADP-ribosylation (ADPr) is an ancient reversible modification of cellular macromolecules controlling major biological processes as diverse as DNA damage repair, transcriptional regulation, intracellular transport, immune and stress responses, cell survival and proliferation. Furthermore, enzymatic reactions of ADPr are central in the pathogenesis of many human diseases, including infectious conditions. By providing a review of ADPr signalling in bacterial systems, we highlight the relevance of this chemical modification in the pathogenesis of human diseases depending on host-pathogen interactions. The post-antibiotic era has raised the need to find alternative approaches to antibiotic administration, as major pathogens becoming resistant to antibiotics. An in-depth understanding of ADPr reactions provides the rationale for designing novel antimicrobial strategies for treatment of infectious diseases. In addition, the understanding of mechanisms of ADPr by bacterial virulence factors offers important hints to improve our knowledge on cellular processes regulated by eukaryotic homologous enzymes, which are often involved in the pathogenesis of human diseases.

PARPs and PAR as novel pharmacological targets for the treatment of stress granule-associated disorders.

Among the post-translational modifications, ADP-ribosylation has been for long time the least integrated in the scheme of the structural protein modifications affecting physiological functions. In spite of the original findings on bacterial-dependent ADP-ribosylation catalysed by toxins such as cholera and pertussis toxin, only with the discovery of the poly-ADP-ribosyl polymerase (PARP) family the field has finally expanded and the role of ADP-ribosylation has been recognised in both physiological and pathological processes, including cancer, infectious and neurodegenerative diseases. This is now a rapidly expanding field of investigation, centred on the role of the different PARPs and their substrates in various diseases, and on the potential of PARP inhibitors as novel pharmacological tools to be employed in relevant pathological context. In this review we analyse the role that members of the PARP family and poly-ADP-ribose (PAR; the product of PARP1 and PARP5a activity) play in the processes following the exposure of cells to different stresses. The cell response that arises following conditions such as heat, osmotic, oxidative stresses or viral infection relies on the formation of stress granules, which are transient cytoplasmic membrane-less structures, that include untranslated mRNA, specific proteins and PAR, this last one serving as the "collector" of all components (that bind to it in a non-covalent manner). The resulting phenotypes are cells in which translation, intracellular transport or pro-apoptotic pathways are reversibly inhibited, for the time the given stress holds. Interestingly, the formation of defective stress granules has been detected in diverse pathological conditions including neurological disorders and cancer. Analysing the molecular details of stress granule formation under these conditions offers a novel view on the pathogenesis of these diseases and, as a consequence, the possibility of identifying novel drug targets for their treatment.

Differential display analysis of gene expression in Etrog citron leaves infected by Citrus viroid III.

Citrus are natural hosts of several viroids, which are plant pathogens composed exclusively of a non-protein-coding, small single-stranded circular RNA that is able to replicate autonomously in susceptible hosts. They are responsible for symptoms such as stunting, leaf epinasty, and chlorosis. Citrus viroid III (CVd-III) has been long regarded as a possible dwarfing agent of citrus grafted on trifoliate orange and its hybrids. To investigate molecular mechanisms involved in pathogenesis, the messenger RNA (mRNA) differential display technique was here applied to identify genes whose transcription was significantly altered in leaves of Etrog citron (Citrus medica) infected by CVd-III (variant b). Of eighteen genes identified, thirteen were up-regulated by viroid infection, while five were down-regulated. Except for two genes that encode proteins of unknown function, the remaining genes are mainly involved in plant defence/stress responses, signal transduction, amino acid transport, and cell wall structure. Among the up-regulated genes, it is noteworthy a suppressor of RNA silencing that might be involved in viroid and virus pathogenicity. The functions of these genes are discussed.

In Vitro Techniques for ADP-Ribosylated Substrate Identification.

ADP-ribosylation is a post-translational modification of proteins that has required the development of specific technical approaches for the full definition of its physiological roles and regulation. The identification of the enzymes and specific substrates of this reaction is an instrumental step toward these aims. Here we describe a method for the separation of ADP-ribosylated proteins based on the use of the ADP-ribose-binding macro domain of the thermophilic protein Af1521, coupled to mass spectrometry analysis for protein identification. This method foresees the coupling of the macro domain to resin, an affinity-based pull-down assay, coupled to a specificity step resulting from the clearing of cell lysates with a mutated macro domain unable to bind ADP-ribose. By this method both mono- and poly-ADP-ribosylated proteins have been identified.

PARP1-produced poly-ADP-ribose causes the PARP12 translocation to stress granules and impairment of Golgi complex functions.

Poly-ADP-ribose-polymerases (PARPs) 1 and 2 are nuclear enzymes that catalyze the poly-ADP-ribosylation of nuclear proteins transferring poly-ADP-ribose (PAR) polymers to specific residues. PARPs and PAR intervene in diverse functions, including DNA repair in the nucleus and stress granule assembly in the cytoplasm. Stress granules contribute to the regulation of translation by clustering and stabilizing mRNAs as well as several cytosolic PARPs and signaling proteins to modulate cell metabolism and survival. Our study is focused on one of these PARPs, PARP12, a Golgi-localized mono-ADP-ribosyltransferase that under stress challenge reversibly translocates from the Golgi complex to stress granules. PARP1 activation and release of nuclear PAR drive this translocation by direct PAR binding to the PARP12-WWE domain. Thus, PAR formation functionally links the activity of the nuclear and cytosolic PARPs during stress response, determining the release of PARP12 from the Golgi complex and the disassembly of the Golgi membranes, followed by a block in anterograde-membrane traffic. Notably, these functions can be rescued by reverting the stress condition (by drug wash-out). Altogether these data point at a novel, reversible nuclear signaling that senses stress to then act on cytosolic PARP12, which in turn converts the stress response into a reversible block in intracellular-membrane traffic.

From toxins to mammalian enzymes: the diversity of mono-ADP-ribosylation.

The ADP-ribosylation of proteins is a phylogenetically ancient mechanism that involves the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD⁺) to specific amino acids of target proteins post-translationally. In the first part of this review, we briefly describe ADP-ribosylation as the mechanism of action of toxins, while giving particular emphasis to a non-conventional ADP-ribosylation reaction that is mediated by the fungal toxin brefeldin A (BFA). This modification results in the loss of the membrane fission activity of the C-terminal binding protein (CtBP)1/ BFA-ADP-ribosylated substrate (BARS), thus blocking progression of cells into mitosis, with important implications for the design of new anticancer drugs. In addition, we summarize the most recent findings on mammalian, intracellular mono-ADP-ribosyl transferase enzymes, underlining the emerging functional roles in which they are involved, including immune responses, transcriptional regulation, stress responses, cell survival. The observation that several mono-ADP-ribosyl transferases, such as PARP-10, PARP-12, PARP-13, are involved in a range of physiological processes points at the multifunctional feature of these proteins.

Identification of a cell-bound extracellular protease overproduced by Sulfolobus solfataricus in peptide-rich media.

A new protease, named SsMTP was identified from the archeon Sulfolobus solfataricus. The enzyme is associated to the cell-membrane and over-produced in response to the peptide-enriched media. SsMTP has a molecular mass of 120 kDa showing optimal activity at pH 2.0 in the temperature range 70 - 90 degrees C, and a half-life of 20 days at 80 degrees C. Primary structure analysis revealed that SsMTP represents a novel type of multi-domain thermopsin-like protease containing the catalytic domain followed by two distinct domains, PKD and Y_Y_Y, which are usually involved in a range of protein-protein interactions among the extracellular proteins.

First Archaeal PEPB-Serine Protease Inhibitor from Sulfolobus solfataricus with Noncanonical Amino Acid Sequence in the Reactive-Site Loop.

The specific inhibition of serine proteinases, which are crucial switches in many important physiological processes, is of great value both for basic research and for therapeutic applications. In this study, we report the molecular cloning of the sso0767 gene from Sulfolobus solfataricus, and the functional characterization of its product, SsCEI, which represents the first archaeal phosphatidylethanolamine-binding protein (PEBP)-serine proteinase inhibitor, reported to date. SsCEI is a monomer protein with a molecular mass of 19.0 kDa and a pI of 6.7, which is able to inhibit the serine proteases alpha-chymotrypsin and elastase with K(i) values of 0.08 and 0.1 microM, respectively. Moreover SsCEI is extremely resistant to both thermal inactivation and proteolytic attack suggesting compact folding of the protein. Within the I51 family, the archaeal inhibitor shows strong similarity to the human and murine members. The three-dimensional model of SsCEI revealed a general beta-fold and the presence of an anion-binding pocket, the hallmark of the PEBP family. Moreover SsCEI binds the cognate proteases according to a common, substrate-like standard mechanism. Point mutation experiments supported the prediction of the protease-binding site located on the surface at the C- terminal region of the protein. Interestingly, searches based on preidentified structural reactive loop motifs revealed the occurrence of a sequence (T123-N130) that is not represented in all serine-protease inhibitor families. This unique motif may provide new insights into both the inhibitor/protease binding mode and the specific biological functions of SsCEI within the PEBP family.

Aspartic proteinases in Antarctic fish.

The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases "Nothepsins" and classified them relative to aspartic proteinases from other species.

Identification of the first archaeal oligopeptide-binding protein from the hyperthermophile Aeropyrum pernix.

The archaeon Aeropyrum pernix grows optimally at 90 degrees C and derives energy primarily from aerobic degradation of complex proteinaceous substrates. The ability of these nutrients to sustain growth is generally associated with the presence of oligopeptide transport systems, such as the well-known protein-dependent ATP-binding cassette (ABC) transporters. This study is concerned with the isolation and characterisation of the first archaeal oligopeptide-binding protein (OppA(Ap)) from the extracellular medium of A. pernix. The protein shows a pI of 3.9 and a molecular mass of about 90 kDa under native conditions. By using a proteomic approach, the OppA(Ap)-encoding gene was identified (APE1583) and about 55% of the protein amino-acid sequence was validated. The extracellular purified protein was able to efficiently bind oligopeptide substrates such as Xenopsin. The amount of a liganded peptide to OppA(Ap) was about 70% at 90 degrees C using a 1/100 (w/w) OppA(Ap)/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppA(Ap) with bacterial oligopeptide binding proteins. Furthermore, APE1583 neighbouring genes encode for the cognate components of an ABC transport system, suggesting that these ORFs are organised in an operon-like structure, with OppA(Ap )as the extracellular component for the uptake of oligopeptides.