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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A new serine-carboxyl proteinase, called kumamolisin-ac, was purified from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius. The enzyme is a monomeric protein of 45 kDa, active over a wide temperature range (5.0–70°C) and extremely acidic pHs (1.0–4.0), showing maximal proteolytic activity at pH 2.0 and 60°C. Interestingly, kumamolisin-ac displayed a significant proteolytic activity even at 5°C, thus suggesting a sort of cold-adaptation for this enzyme. The protease was remarkably stable at high temperatures (t1/2 at 80°C, 10 h, pH 2.0) and over a broad range of pH (2.0–7.0). Substrate analysis indicated that kumamolisin-ac was active on a variety of macromolecular substrates, such as haemoglobin, hide powder azure, and azocoll. In particular, a high specific activity was detected towards collagen. The corresponding gene was cloned, expressed and the recombinant protease, was found to be homologous to proteases of the ‘S53’ family. From the high identity with kumamolisin and kumamolisin-As, known as collagenolytic proteases, kumamolisin-ac can be considered as the third collagenolytic affiliate within the ‘S53’ family. Cleavage specificity investigation of kumamolisin-ac revealed a unique primary cleavage site in bovine insulin B-chain, whereas a broad specificity was detected using bovine α-globin as substrate. Thus, kumamolisin-ac could represent an attractive candidate for industrial-scale biopeptide production under thermoacidophilic conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

The archaeon Aeropyrum pernix grows optimally at 90°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 (OppAAp) 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 OppAAp-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 OppAAp was about 70% at 90°C using a 1/100 (w/w) OppAAp/substrate ratio. Sequence comparisons showed a weak but significant similarity of OppAAp 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 OppAAp as the extracellular component for the uptake of oligopeptides. [ABSTRACT FROM AUTHOR]

Abstract: The cleavage specificity of Pernisine, a subtilisin-like protease from the hyperthermophilic archaeon Aeropyrum pernix, was established by mass spectrometry, analysing the peptides generated by digestion of oxidised bovine insulin B chain. The specificity was explored by changing several factors such as substrate/enzyme ratio, temperature and reaction media. Using a S/E ratio of 1000 (w/w) and a temperature of 60°C, five primary cleavage sites in the insulin B chain were detected suggesting a broad specificity of Pernisine, which is different from that found for other bacterial subtilisin-like proteases. When the S/E ratio and/or temperature were increased, a higher selectivity of Pernisine was observed with a unique cleavage site occurring between Leu15 and Tyr16. In addition, the influence on the enzymatic hydrolysis of different organic solvent concentrations was investigated. The results demonstrated that Pernisine could specifically digest the peptide substrate even in the presence of 80% acetonitrile solution or 30% dimethyl sulfoxyde. Thereby the cleavage specificity of Pernisine can be opportunely modulated by controlling the in vitro digestion conditions, suggesting that this enzyme could be an attractive candidate to use in a variety of biotechnological applications. [Copyright &y& Elsevier]