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CDC25 phosphatases play a critical role in the regulation of the cell cycle and thus represent attractive cancer therapeutic targets. We previously discovered the 4-(2-carboxybenzoyl)phthalic acid (NSC28620) as a new CDC25 inhibitor endowed with promising anticancer activity in breast, prostate and leukemia cells. Herein, we report a structure-based optimization of NSC28620, leading to the identification of a series of novel naphthylphenylketone (NPK) and naphthylphenylamine (NPA) derivatives as CDC25B inhibitors. Compounds 7j, 7i, 6e, 7f and 3 showed higher inhibitory activity than the initial lead, with Ki values in the low micromolar range. Kinetic analysis, intrinsic fluorescence studies and induced fit docking simulations provided a mechanistic understanding of the activity of these derivatives. All compounds were tested in the highly aggressive human melanoma cell lines A2058 and A375. Compound 4a potently inhibited cell proliferation and colony formation, causing an increase of the G2/M phase and a reduction of G0/G1 phase of the cell cycle in both cell lines.

In this study, we explored the ability of Annurca apple flesh polyphenol extract (AFPE) to affect the activity of key enzymes involved in neurodegenerative disorders&mdash, in particular, Acetyl- and Butirryl-cholinesterases, and type A and B monoamine oxidase. The effect of AFPE on enzyme activity was analyzed by in vitro enzyme assays, and the results showed concentration-dependent enzyme inhibition, with IC50 values corresponding to 859 &plusmn, 18 &micro, M and 966 &plusmn, 72 &micro, M for AChE and BuChE respectively, and IC50 corresponding to 145 &plusmn, 3 &micro, M and 199 &plusmn, 7 &micro, M for MAO-A and MAO-B, respectively, with a preference for MAO-A. Moreover, in this concentration range, AFPE did not affect the viability of human neuroblastoma SH-SY5Y and fibroblast BJ-5ta cell lines, as determined by an MTT assay. In conclusion, our results demonstrate that AFPE shows the new biological properties of inhibiting the activity of enzymes that are involved in brain functions, neurodegenerative disorders, and aging.

Among matrix metalloproteinases (MMPs), MMP-9/2 are key enzymes involved in the proteolysis of extracellular matrices in the inflammatory process and in cancer. Since MMP-9/2 expression levels, activity, and secretion is up-regulated during inflammation in response to pro-inflammatory cytokines, such as interleukin-6 (IL-6), many efforts have been devoted to identifying factors that could inhibit the IL-6-induced MMP-9/2 expression. Up to now, several reports indicated that polyphenols from fruits and vegetables are among the major components of health promotion for their antioxidant properties and also for their anti-inflammatory and anti-cancer agents. Among plant derived polyphenols, lemon (Citrus limon) peel extract (LPE) shows anti-cancer properties in various cancer types. In our previous work, we demonstrated that LPE can reduce IL-6-induced migration/invasiveness and MMP-9/2 up-regulation in some gastric cancer cell lines. This study aims to exploit the anti-cancer properties of LPE using an in vitro system model of inflammation, consisting of IL-6-exposed human primary colon cancer cells. We first analyzed the effect of LPE on IL-6-induced cell migration and invasiveness by wound healing and Boyden chamber assay, respectively. The MMP-2 mRNA expression levels and gelatinolytic activity in the cell culture media were determined by q-PCR analysis and gelatin zymography, respectively, and finally, the effects of LPE on IL-6-induced JAK2/STAT3 signaling pathways have been investigated by Western blotting analysis. Our results show that LPE is able to inhibit the IL-6-dependent cell migration and invasiveness associated with the up-regulation of MMP-2 expression levels and that these effects are correlated to the STAT3 phosphorylation in human primary T88 and T93 colon cancer cells.

Lin28 is an evolutionary conserved RNA-binding protein that plays important roles during embryonic development and tumorigenesis. It regulates gene expression through two different post-transcriptional mechanisms. The first one is based on the regulation of miRNA biogenesis, in particular that of the let-7 family, whose expression is suppressed by Lin28. Thus, loss of Lin28 leads to the upregulation of mRNAs that are targets of let-7 species. The second mechanism is based on the direct interaction of Lin28 with a large number of mRNAs, which results in the regulation of their translation. This second mechanism remains poorly understood. To address this issue, we purified high molecular weight complexes containing Lin28a in mouse embryonic stem cells (ESCs). Numerous proteins, co-purified with Lin28a, were identified by proteomic procedures and tested for their possible role in Lin28a-dependent regulation of the mRNA encoding DNA methyltransferase 3a (Dnmt3a). The results show that Lin28a activity is dependent on many proteins, including three helicases and four RNA-binding proteins. The suppression of four of these proteins, namely Ddx3x, Hnrnph1, Hnrnpu or Syncrip, interferes with the binding of Lin28a to the Dnmt3a mRNA, thus suggesting that they are part of an oligomeric ribonucleoprotein complex that is necessary for Lin28a activity.

The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma., Graphical Abstract

Mannose-binding lectin (MBL)-Associated Serine Proteases (MASP)-1 and 3, key enzymes in the lectin complement pathway of innate immune response, are also expressed in glioma cell lines. We investigated MASP-1 and MASP-3 expression during dibutyryl cyclic AMP (dbcAMP)- or Interleukin-6 (rIL-6)-induced astrocytic differentiation of C6 glioma cells. Our results demonstrate that C6 cells express basal levels of MASP-1 and MASP-3 and following exposure to dbcAMP or IL-6, a consistent MASP-1 and MASP-3 mRNA up-regulation was found, with a behavior similar to that showed by the fibrillary acidic protein (GFAP). Furthermore, in cell conditioned media, rIL-6 stimulated MASP-3 secretion which reached levels similar to those obtained by dbcAMP treatment. Moreover, the detection of a 46-kDa MASP-3 suggested its processing to the mature form in the extracellular cell medium. Interestingly, the H89 PKA inhibitor, mostly affected dbcAMP-induced MASP-1 and MASP-3 mRNA levels, compared to that of rIL-6, suggesting that cAMP/PKA pathway contributes to MASP-1 and MASP-3 up-regulation. MASP-1 and MASP-3 expression increase was concomitant with dbcAMP- or rIL-6-induced phosphorylation of STAT3. Our findings suggest that the increase in intracellular cAMP concentration or rIL-6 stimulation can play a role in innate immunity enhancing MASP-1 and MASP-3 expression level in C6 glioma cells.

Skeletal muscle is an extremely plastic tissue for its ability to respond to different stimuli such as physiological variation and external stress. Muscle hypertrophy involves an increase in muscle mass, changes in myofibril composition and adaptation of metabolic pathways. Plasticity of skeletal muscle in response to exercise training is also caused by proliferation and differentiation of the satellite muscle stem cells in response to various growth and differentiation factors. This process is mainly mediated by myokines secreted during skeletal muscle contraction. Myokines are proteins that act as hormones both locally in the muscle and/or in an endocrine manner in other organs, mainly liver, brain and adipose tissue. Myostatin, known as growth differentiation factor-8, a myokine member of transforming growth factor-b (TGF-b) superfamily, can act on muscle cells in an autocrine manner leading to inhibition of muscle myogenesis. Muscle myostatin expression and its plasma concentration are downregulated after acute and long-term physical exercise thus allowing muscle hypertrophy. In addition, myostatin is correlated to obesity and insulin resistance for its ability to affect energy metabolism and insulin-sensitivity in muscle cells, respectively. These findings reveal that myostatin may have potential therapeutic applications to treat muscle atrophy diseases in humans. Even in sports, drugs able to inhibit myostatin expression can lead athletes to increase their sport performance. Here, we present a brief overview of myostatin and its role in biological mechanisms involved in exercise-induced plasticity of skeletal muscle.

Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.

Human health implies complex mechanisms and involves diseases prevention aimed at ensuring a psycho-physical wellbeing homoeostasis. The search for longevity can be related to the slowdown in aging, and psycho-physical wellbeing is often related to the lifestyle, mainly to the human physical activity and nutrition. Circadian rhythms are processes that affect the behaviour, physiology and metabolism of mammals across cyclic periods of 24 hours. These rhythms are regulated by multiple physiological systems, whose key elements are the alternation between light and dark and between food consumption and periods of fasting. Therefore, during evolution, a constant adaptation to natural rhythms by humans has been established towards the surrounding environment determining periods of food consumption and periods of fasting coinciding with rest. Recent sociological surveys have shown that there is a slight increase in the number of people who devote themselves to a constant physical/sport activity, but this is an ephemeral percentage compared to the dramatic increase in the incidence of diseases related to aging such as obesity and diabetes, easily preventable with healthy lifestyles. Therefore, the connection between circadian rhythms, physical activity and lifestyles represents an important feature involved in human longevity. Here, a survive of the biological mechanisms, implied in this behaviour, is presented.

Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of &alpha, smooth muscle actin (&alpha, SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.

gamma-Glutamyl transpeptidase (GGT) is an enzyme located on the surface of cellular membranes and involved in GSH metabolism and maintenance of redox homeostasis. High GGT expression on tumor cells is associated with increased cell proliferation and resistance against chemotherapy. GGT inhibitors evaluated so far in clinical trials are too toxic for human use. In this study, using enzyme kinetics analyses, we demonstrate that ovothiols, 5(N pi)-methyl thiohistidines of marine origin, act as noncompetitive inhibitors of GGT, with an apparent K-i of 21 mu m, when we fixed the concentrations of the donor substrate. We found that these compounds are more potent than the known GGT inhibitor 6-diazo-5-oxo-L-norleucine and are not toxic toward human embryonic cells. In particular, cellular process-specific fluorescence-based assays revealed that ovothiols induce a mixed cell-death phenotype of apoptosis and autophagy in GGT-overexpressing cell lines, including human liver cancer and chronic B leukemic cells. The findings of our study provide the basis for further development of 5-thiohistidines as therapeutics for GGT-positive tumors and highlight that GGT inhibition is involved in autophagy.

The dual phosphatases cell division cycle 25 (CDC25) are crucial regulators of cell cycle progression, also involved in cell response to DNA damage. Their overexpression was detected in many tumors, including melanoma [1]. The characterization of molecules with an inhibitory potency on CDC25 activity, such as compound 19 (NSC 28620) [2], contributes to the research of novel anti-cancer drugs. The structure of this lead compound was engineered to produce more potent inhibitors. The most active compounds synthesized had Ki values in the 0.79 – 10.0 μM interval. Kinetic studies identified two different mechanisms of inhibition, related to specific functional groups possessed by the inhibitors. Information on positioning/interaction of the inhibitors was also obtained by intrinsic fluorescence measurements of CDC25B. The possible cytotoxicity of the most active compounds on melanoma cell lines was analysed. Data from MTT assay indicated that at a low concentration, i.e. 10 μM, essentially one compound (cpd 21) significantly reduced the cell growth of melanoma cells in a time dependent manner. The effect of cpd 21 on cell viability was further confirmed by the colony-forming assay, that showed a similar trend in the ability to inhibit the clonogenicity of melanoma cells. Moreover, cpd 21 provoked an early increase of pCdk1 protein level and an arrest of melanoma cells in G2/M of cell cycle, followed by the activation of a caspase-mediated apoptotic program. Conversely, cpd 21 didn’t show cytotoxic effect on BJ-5ta, a nonmalignant fibroblast cell line. 85 Finally, a reduction of pAkt and an increase of p53 protein level could mediate the cytotoxic effect of cpd 21 on melanoma cells. References 1) Boutros et al. Nat Rev Cancer (2007),7,495-507. 2) Lavecchia et al. J. Med. Chem. (2012), 55, 4142-4158.

Among plant polyphenols, lemon peels extract (LPE) from the residues of the industrial processing of lemon (Citrus limon) shows anti-proliferative properties in cancer cells and anticholinesterase activity. In this study, we analyze the anti-cancer properties of LPE on migration and invasiveness in MKN-28 and AGS human gastric cancer cell lines either in the absence or presence of the pro-inflammatory cytokine IL-6. We find that the pretreatment with non-cytotoxic concentrations (0.5&ndash, 1 &mu, g/ml of gallic acid equivalent) of LPE inhibits interleukin-6 (IL-6)-induced cell migration and invasiveness in MKN-28 and AGS cells, as analyzed by wound and matrigel assays. Pretreatment with LPE is able to prevent either IL-6-induced matrix metalloproteinases (MMP)-9/2 activity, as assessed by gel zymography, or mRNA and protein MMP-9/2 expression, as evaluated by qPCR and Western blotting analysis, respectively. These LPE effects are associated with an IL-6-dependent STAT3 signaling pathway in MKN-28 and AGS cells. Furthermore, LPE shows acetylcholinesterase inhibitory activity when assayed by the Ellman method. In conclusion, our results demonstrate that LPE reduces the invasiveness of gastric MKN-28 and AGS cancer cells through the reduction of IL-6-induced MMP-9/2 up-regulation. Therefore, these data suggest that LPE exerts a protective role against the metastatic process in gastric cancer.

Breast cancer is the most common cancer in women, which incidence has increased in recent years. It is constituted by very heterogeneous tissue characterized by an abnormal microenvironment regulating tumor progression and providing evasion from cancer therapies. Breast cancer-associated fibroblasts (BCAFs) are the main cell type of breast cancer microenvironment and can represent up to 80% of the tumor mass. In particular, BCAFs induce cancer initiation, proliferation, invasion and metastasis by undergoing an activation process associated with the secretion of growth factors, cytokines, and paracrine interactions. Therapy resistance is the main cause of poor therapeutic results or even failure in breast cancer patients. Despite recent advances in breast cancer management, there is a need for new prognostic markers and novel agents for targeting key signalling pathways to either improve the efficacy of the current therapies, or reduce toxicity. In this view, BCAFs represent markers useful to clinical diagnosis, therapy, and prognosis of breast cancer. This review focuses on the role of BCAFs in cancer, and describes the processes of endocrine/chemotherapy resistance linked to BCAFs action. Moreover, it points to molecules and pathways regulating therapy resistance induced by BCAFs. Finally, potential therapeutic strategies targeting BCAFs and offering new tools in breast cancer therapy are highlighted.

Multitargeting or polypharmacological approaches, looking for single chemical entities retaining the ability to bind two or more molecular targets, are a potentially powerful strategy to fight complex, multifactorial pathologies. Unfortunately, the search for multiligand agents is challenging because only a small subset of molecules contained in molecular databases are bioactive and even fewer are active on a preselected set of multiple targets. However, collections of natural compounds feature a significantly higher fraction of bioactive molecules than synthetic ones. In this view, we searched our library of 1175 natural compounds from marine sources for molecules including a 2-aminoimidazole+aromatic group motif, found in known compounds active on single relevant targets for Alzheimer's disease (AD). This identified two molecules, a pseudozoanthoxanthin (1) and a bromo-pyrrole alkaloid (2), which were predicted by a computational approach to possess interesting multitarget profiles on AD target proteins. Biochemical assays experimentally confirmed their biological activities. The two compounds inhibit acetylcholinesterase, butyrylcholinesterase, and beta-secretase enzymes in high-to submicromolar range. They are also able to prevent and revert beta-amyloid (A beta) aggregation of both A beta(1-40) and A beta(1-42) peptides, with 1 being more active than 2. Preliminary in vivo studies suggest that compound 1 is able to restore cholinergic corticohippocampal functional connectivity.

Pseudoalteromonas haloplanktis, a psychrotrophilic marine bacterium of biotechnological interest, shows anti-biofilm properties and is particularly relevant for cold storage of vacuum packed seafood. We focused our interest on the activation of cholesterol metabolism in this bacterium as the presence in its genome of a putative 3-ketosteroid-Δ(1) -dehydrogenase. This study reports GC-MS and LC-MS/MS profiles of sterols/steroids and their derivatives found in cell extracts of P. haloplanktis grown in a medium with a low content of cholesterol. Here, for the first time, we suggest that P. haloplanktis produces some intermediates of cholesterol catabolism, putatively identified as 24-hydroxycholest-1,4-dien-3-one-26-oic acid, chol-1,4-dien-3-one-24-oic acid, 26-hydroxycholest-4-en-3-one, and pregn-4-en-3-one-20-carboxylic acid, a finding already reported in other microorganisms. The presence of these compounds, also considered steroid precursors, produced by P. haloplanktis in vacuum packed seafood could be of interest for healthy of consumers, as well as, for biotechnological applications in pharmaceutical industry.

Gamma-glutamyl transpeptidase (GGT) is a cell surface enzyme involved in glutathione metabolism and maintenance of redox homeostasis. High expression of GGT on tumor cells is associated with an increase of cell proliferation and resistance against chemotherapy. GGT inhibitors that have been evaluated in clinical trials are too toxic for human use. We have previously identified ovothiols, 5(N&pi, )-methyl-thiohistidines of marine origin, as non-competitive-like inhibitors of GGT that are more potent than the known GGT inhibitor, 6-diazo-5-oxo-l-norleucine (DON), and are not toxic for human embryonic cells. We extended these studies to the desmethylated form of ovothiol, 5-thiohistidine, and confirmed that this ovothiol derivative also acts as a non-competitive-like GGT inhibitor, with a potency comparable to ovothiol. We also found that both 5-thiohistidine derivatives act as reversible GGT inhibitors compared to the irreversible DON. Finally, we probed the interactions of 5-thiohistidines with GGT by docking analysis and compared them with the 2-thiohistidine ergothioneine, the physiological substrate glutathione, and the DON inhibitor. Overall, our results provide new insight for further development of 5-thiohistidine derivatives as therapeutics for GGT-positive tumors.

Pseudoalteromonas haloplanktis, a psychrotrophilic marine bacterium of biotechnological interest, shows anti-biofilm properties and is particularly relevant for cold storage of vacuum packed seafood. We focused our interest on the activation of cholesterol metabolism in this bacterium as the presence in its genome of a putative 3-ketosteroid-Δ(1) -dehydrogenase. This study reports GC-MS and LC-MS/MS profiles of sterols/steroids and their derivatives found in cell extracts of P. haloplanktis grown in a medium with a low content of cholesterol. Here, for the first time, we suggest that P. haloplanktis produces some intermediates of cholesterol catabolism, putatively identified as 24-hydroxycholest-1,4-dien-3-one-26-oic acid, chol-1,4-dien-3-one-24-oic acid, 26-hydroxycholest-4-en-3-one, and pregn-4-en-3-one-20-carboxylic acid, a finding already reported in other microorganisms. The presence of these compounds, also considered steroid precursors, produced by P. haloplanktis in vacuum packed seafood could be of interest for healthy of consumers, as well as, for biotechnological applications in pharmaceutical industry.

An ecofriendly synthetic pathway for the synthesis of donepezil precursors is described. Alternative energy sources were used for the total synthesis in order to improve yields, regioselectively, and rate of each synthetic step and to reduce the coproduction of waste at the same time. For all products, characterized by an improved structural rigidity respect to donepezil, the inhibitor activity on AChE, the selectivity vs BuChE, the side-activity on BACE-1, and the effect on SHSY-5Y neuroblastoma cells viability were tested. Two potential new lead compounds for a dual therapeutic strategy against Alzheimer’s disease were envisaged.

ObjectiveThe hormone adiponectin exerts beneficial pleiotropic effects on biological and metabolic processes. Although a well-recognized insulin sensitizer, its characteristic has yet to be clearly defined. Myotonic dystrophy type 1 (DM1) is a rare genetic disorder that features muscle wasting and metabolic comorbidity, and patients have an increased risk of developing type 2 diabetes. We analyzed circulating levels of adiponectin and its oligomers to determine whether their expression correlates with metabolic alterations in DM1 patients.Design and methodsWe measured the anthropometric and biochemical features and three insulin resistance (IR) indices (homeostasis model assessment, quantitative insulin sensitivity check index, and McAuley) of 21 DM1 patients and of 82 age-, sex-, and weight-matched controls. In the blood samples of patients and controls, adiponectin levels were measured by ELISA, and its oligomers were characterized by using western blotting and gel filtration. The adiponectin gene was molecularly analyzed in patients.ResultsDM1 patients had significantly higher body mass index, waist circumference, triglycerides (TGs), glucose, tumor necrosis factor α, and IR; conversely, they had significantly lower concentrations of total serum adiponectin with a selective, pronounced decrease of its high molecular weight (HMW) oligomers. There was a strong negative correlation between adiponectin and TGs in DM1 patients.ConclusionsOur results endorse the hypothesis that decreased expression of adiponectin together with a selective reduction of its HMW oligomers contributes to the worsening of IR and its metabolic complications in DM1 patients. These findings suggest that adiponectin and HMW oligomers may serve as biomarkers and are promising therapeutic agents for IR and its consequences in DM1.

The interaction between tetracycline and the archaeal elongation factor 1α from Sulfolobus solfataricus was investigated. The effects produced by this eubacterial antibiotic indicated that this interaction involved the G-domain of the elongation factor 1α from S. solfataricus, although also the M-domain was required. In fact, in the presence of the antibiotic, an increase in the fluorescence quantum yield of the aromatic region was observed for elongation factor 1α from S. solfataricus and its truncated form lacking the C-terminal domain, but not for that lacking also the M-domain. The increase in quantum yield was restored when the G-domain of elongation factor 1α from S. solfataricus was fused to the M and the C-domains of the eubacterial analogue elongation factor Tu. Tetracycline inhibits protein synthesis catalysed by elongation factor 1α from S. solfataricus; this is accompanied by an increase in the GDP/GTP exchange rate and a slight inhibition of the intrinsic GTPase, suggesting that a main effect of the antibiotic was exerted on the GTP-bound form of the enzyme. Furthermore, the mixed inhibition observed for GTPase confirmed that the interaction, besides the G-domain, involved also other region(s) of elongation factor 1α from S. solfataricus. These results can be useful for studying potential side effects arising from the interaction between tetracycline and eukaryotic elongation factors.

Polyribonucleotide phosphorilase from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis (PhPNPase) has been purified. This enzyme catalyzes both the RNA polymerisation and degradation reaction, showing the highest activity at temperatures below 40 degrees C. PhPNPase is quite sensitive to heat treatment and it is endowed with remarkable halotolerance.

The dimeric structure of Sulfolobus solfataricus thioredoxin A2 and the basis of its thermostability Alessia Ruggiero, Mariorosario Masullo, Daniela Marasco, Maria Rosaria Ruocco, Pasquale Grimaldi, Paolo Arcari, Adriana Zagari, and Luigi Vitagliano* 1 Istituto di Biostrutture e Bioimmagini, CNR, Via Mezzocannone 16, I-80134 Napoli, Italy 2Dipartimento di Scienze Farmacobiologiche, Universita degli Studi ‘‘Magna Graecia’’ di Catanzaro, Roccelletta di Borgia, I-88021 Catanzaro, Italy 3Dipartimento di Biochimica e Biotecnologie Mediche, Universita degli Studi di Napoli Federico II, Via S. Pansini 5, I-80131 Napoli, Italy 4Dipartimento delle Scienze Biologiche, Sezione di Biostrutture, Universita degli Studi di Napoli Federico II, Via Mezzocannone 16, I-80134 Napoli, Italy 5 CEINGE Biotecnologie Avanzate s.c.a r.l.-Via Comunale Margherita 482, I-80145 Napoli, Italy 6 CNISM Universita degli Studi di Napoli Federico II, Italy

The growth temperature adaptation of six model proteins has been studied in 42 microorganisms belonging to eubacterial and archaeal kingdoms, covering optimum growth temperatures from 7 to 103 degrees C. The selected proteins include three elongation factors involved in translation, the enzymes glyceraldehyde-3-phosphate dehydrogenase and superoxide dismutase, the cell division protein FtsZ. The common strategy of protein adaptation from cold to hot environments implies the occurrence of small changes in the amino acid composition, without altering the overall structure of the macromolecule. These continuous adjustments were investigated through parameters related to the amino acid composition of each protein. The average value per residue of mass, volume and accessible surface area allowed an evaluation of the usage of bulky residues, whereas the average hydrophobicity reflected that of hydrophobic residues. The specific proportion of bulky and hydrophobic residues in each protein almost linearly increased with the temperature of the host microorganism. This finding agrees with the structural and functional properties exhibited by proteins in differently adapted sources, thus explaining the great compactness or the high flexibility exhibited by (hyper)thermophilic or psychrophilic proteins, respectively. Indeed, heat-adapted proteins incline toward the usage of heavier-size and more hydrophobic residues with respect to mesophiles, whereas the cold-adapted macromolecules show the opposite behavior with a certain preference for smaller-size and less hydrophobic residues. An investigation on the different increase of bulky residues along with the growth temperature observed in the six model proteins suggests the relevance of the possible different role and/or structure organization played by protein domains. The significance of the linear correlations between growth temperature and parameters related to the amino acid composition improved when the analysis was collectively carried out on all model proteins.

The interaction between 2',3'-cyclic nucleotide 3'-phosphodiesterase and guanine/adenine nucleotides was investigated. The binding of purine nucleotides to 2',3'-cyclic nucleotide 3'-phosphodiesterase was revealed by both direct and indirect methods. In fact, surface plasmon resonance experiments, triphosphatase activity measurements, and fluorescence experiments revealed that 2',3'-cyclic nucleotide 3'-phosphodiesterase binds purine nucleotide triphosphates with an affinity higher than that displayed for diphosphates; on the contrary, the affinity for both purine monophosphates and pyrimidine nucleotides was negligible. An interpretation of biological experimental data was achieved by a building of 2',3'-cyclic nucleotide 3'-phosphodiesterase N-terminal molecular model. The structural elements responsible for nucleotide binding were identified and potential complexes between the N-terminal domain of CNP-ase and nucleotide were analyzed by docking simulations. Therefore, our findings suggest new functional and structural property of the N-terminal domain of CNPase.

Melanoma is the most aggressive skin cancer; its prognosis, particularly in advanced stages, is disappointing largely due to the resistance to conventional anticancer treatments and high metastatic potential. NF-kappa B constitutive activation is a major factor for the apoptosis resistance of melanoma. Several studies suggest a role for the immunophilin FKBP51 in NF-kappa B activation, but the underlying mechanism is still unknown. In the present study, we demonstrate that FKBP51 physically interacts with IKK subunits, and facilitates IKK complex assembly. FKBP51-knockdown inhibits the binding of IKK gamma to the IKK catalytic subunits, IKK-alpha and -beta, and attenuates the IKK catalytic activity. Using FK506, an inhibitor of the FKBP51 isomerase activity, we found that the IKK-regulatory role of FKBP51 involves both its scaffold function and its isomerase activity. Moreover, FKBP51 also interacts with TRAF2, an upstream mediator of IKK activation. Interestingly, both FKBP51 TPR and PPIase domains are required for its interaction with TRAF2 and IKK gamma, whereas only the TPR domain is involved in interactions with IKK alpha and beta. Collectively, these results suggest that FKBP51 promotes NF-kappa B activation by serving as an IKK scaffold as well as an isomerase. Our findings have profound implications for designing novel melanoma therapies based on modulation of FKBP51.

In the present study, we investigated a new approach for studying the interaction between p53 and MDM2/X (where MDM is murine double minute protein). The method is based on the different mobility between the interacting domains of the oncosuppressor p53 and its protein ligands MDM2/X on polyacrylamide gels under native conditions. While the two proteins MDM2/X alone were able to enter the gel, the formation of a binary complex between p53 and MDM2/X prevented the gel entry. The novel technique is reliable for determining the different affinity elicited by MDM2 or MDMX toward p53, and can be useful for analyzing the dissociation power exerted by other molecules on the p53-MDM2/X complex.

The stability against chemical denaturants of the elongation factor EF-1alpha (SsEF-1alpha), a protein isolated from the hyperthermophilic archaeon Sulfolobus solfataricus has been characterized in detail. Indeed, the atypical shape of the protein structure and the unusual living conditions of the host organism prompted us to analyze the effect of urea and guanidine hydrochloride (GuHCl) on the GDP complex of the enzyme (SsEF-1alpha x GDP) by fluorescence and circular dichroism. These studies were also extended to the nucleotide-free form of the protein (nfSsEF-1alpha). Interestingly, the experiments show that the denaturation curves of both SsEF-1alpha forms present a single inflection point, which is indicative of a cooperative unfolding process with no intermediate species. Moreover, the chemically induced unfolding process of both SsEF-1alpha x GDP and nfSsEF-1alpha is fully reversible. Both SsEF-1alpha forms exhibit remarkable stability against urea, but they do not display a strong resistance to the denaturing action of GuHCl. These findings suggest that electrostatic interactions significantly contribute to SsEF-1alpha stability.

Recent studies have shown that elongation factors extracted from archaea/eukarya and from eubacteria exhibit different structural and functional properties. Along this line, it has been demonstrated that, in contrast to EF-Tu, Sulfolobus solfataricus EF-1alpha in complex with GDP (SsEF-1alpha.GDP) does not bind Mg(2+), when the ion is present in the crystallization medium at moderate concentration (5 mM). To further investigate the role that magnesium plays in the exchange process of EF-1alpha and to check the ability of SsEF-1alpha.GDP to bind the ion, we have determined the crystal structure of SsEF-1alpha.GDP in the presence of a nonphysiological concentration (100 mM) of Mg(2+). The analysis of the coordination of Mg(2+) unveils the structural bases for the marginal role played by the ion in the nucleotide exchange process. Furthermore, nucleotide exchange experiments carried out on a truncated form of SsEF-1alpha, consisting only of the nucleotide binding domain, demonstrate that the low affinity of SsEF-1alpha.GDP for Mg(2+) is due to the local architecture of the active site and does not depend on the presence of the other two domains. Finally, considering the available structures of EF-1alpha, a detailed mechanism for the nucleotide exchange process has been traced. Notably, this mechanism involves residues such as His14, Arg95, Gln131, and Glu134, which are strictly conserved in all archaea and eukarya EF-1alpha sequences hitherto reported.

Gastrokine 1 (GKN1) is a stomach-specific protein expressed in normal gastric tissue but absent in gastric cancer. GKN1 plays a major role in maintaining gastric mucosa integrity and is characterized by the presence of a BRICHOS domain consisting of about 100 amino acids also found in several unrelated proteins associated with major human diseases like BRI2, related to familial British and Danish dementia and surfactant protein C (SP-C), associated with respiratory distress syndrome. It was reported that recombinant BRICHOS domains from BRI2 and SP-C precursor (proSP-C) prevent fibrils formation of amyloid-beta peptide (A β ), that is the major component of extracellular amyloid deposits in Alzheimer's disease. Here we investigated on the interaction between human recombinant GKN1 (rGKN1) and A β peptide (1–40) that derives from the partial hydrolysis of the amyloid precursor protein (APP). GKN1 prevented amyloid aggregation and fibrils formation by inhibiting A β (1–40) polymerization, as evaluated by SDS-PAGE, thioflavin-T binding assay and gel filtration experiments. Mass spectrometry showed the formation of a prevailing 1:1 complex between GKN1 and A β (1–40). SPR analysis of GKN1/A β interaction led to calculate a dissociation constant ( K D ) of 34 μM. Besides its interaction with A β (1–40), GKN1 showed also to interact with APP as evaluated by confocal microscopy and Ni-NTA pull-down. Data strongly suggest that GKN1 has anti-amyloidogenic properties thus functioning as a chaperone directed against unfolded segments and with the ability to recognize amyloidogenic polypeptides and prevent their aggregation.

A recombinant γ-glutamyl-cysteine ligase from the psychrophile Pseudoalteromonas haloplanktis (rPhGshA II) was produced and characterised. This enzyme catalyses the first step of glutathione biosynthesis by forming γ-glutamyl-cysteine from glutamate and cysteine in an ATP-dependent reaction. The other ATP-dependent enzyme, glutathione synthetase (rPhGshB), involved in the second step of the biosynthesis, was already characterised. rPhGshA II is a monomer of 58 kDa and its activity was characterised through a direct radioisotopic method, measuring the rate of ATP hydrolysis. The enzyme was active even at cold temperatures in a moderately alkaline buffer containing a high concentration of Mg++; 2-aminobutyrate could replace cysteine, although a lower activity was detected. The reaction rate of rPhGshA II at 15 °C was higher than that reported for rPhGshB, thus suggesting that formation of γ-glutamyl-cysteine was not the rate limiting step of glutathione biosynthesis in P. haloplanktis. rPhGshA II had different affinities for its substrates, as evaluated on the basis of the KM values for ATP (0.093 mM), glutamate (2.8 mM) and cysteine (0.050 mM). Reduced glutathione acted as an inhibitor of rPhGshA II, probably through the binding to an enzyme pocket different from the active site. Also the oxidised form of glutathione inhibited the enzyme with a more complex inhibition profile, due to the complete mono-glutathionylation of rPhGshA II on Cys 386, as proved by mass spectrometry data. When compared to rPhGshB, rPhGshA II possessed more typical features of a psychrophilic enzyme, as it was endowed with lower thermodependence and higher heat sensitivity. In conclusion, this work extends the knowledge on glutathione biosynthesis in the first cold-adapted source; however, another possible redundant γ-glutamyl-cysteine ligase (PhGshA I), not yet characterised, could participate in the biosynthesis of this cellular thiol in P. haloplanktis. © 2014 Elsevier Masson SAS. All rights reserved.

The crystal structure of elongation factor 1alpha from the archaeon Sulfolobus solfataricus in complex with GDP (SsEF-1alpha.GDP) at 1.8 A resolution is reported. As already known for the eubacterial elongation factor Tu, the SsEF-1alpha.GDP structure consists of three different structural domains. Surprisingly, the analysis of the GDP-binding site reveals that the nucleotide- protein interactions are not mediated by Mg(2+). Furthermore, the residues that usually co-ordinate Mg(2+) through water molecules in the GTP-binding proteins, though conserved in SsEF-1alpha, are located quite far from the binding site. [(3)H]GDP binding experiments confirm that Mg(2+) has only a marginal effect on the nucleotide exchange reaction of SsEF-1alpha, although essential to GTPase activity elicited by SsEF-1alpha. Finally, structural comparisons of SsEF- 1alpha.GDP with yeast EF-1alpha in complex with the nucleotide exchange factor EF-1beta shows that a dramatic rearrangement of the overall structure of EF-1alpha occurs during the nucleotide exchange.