Golgi maturation-dependent glycoenzyme recycling controls glycosphingolipid biosynthesis and cell growth via GOLPH3.

Glycosphingolipids are important components of the plasma membrane where they modulate the activities of membrane proteins including signalling receptors. Glycosphingolipid synthesis relies on competing reactions catalysed by Golgi-resident enzymes during the passage of substrates through the Golgi cisternae. The glycosphingolipid metabolic output is determined by the position and levels of the enzymes within the Golgi stack, but the mechanisms that coordinate the intra-Golgi localisation of the enzymes are poorly understood. Here, we show that a group of sequentially-acting enzymes operating at the branchpoint among glycosphingolipid synthetic pathways binds the Golgi-localised oncoprotein GOLPH3. GOLPH3 sorts these enzymes into vesicles for intra-Golgi retro-transport, acting as a component of the cisternal maturation mechanism. Through these effects, GOLPH3 controls the sub-Golgi localisation and the lysosomal degradation rate of specific enzymes. Increased GOLPH3 levels, as those observed in tumours, alter glycosphingolipid synthesis and plasma membrane composition thereby promoting mitogenic signalling and cell proliferation. These data have medical implications as they outline a novel oncogenic mechanism of action for GOLPH3 based on glycosphingolipid metabolism.

Chemical Composition, Functional and Anticancer Properties of Carrot.

Plants are a valuable source of drugs for cancer treatment. Daucus carota has been investigated for its health properties. In particular, Daucus carota L. subsp. Sativus, the common edible carrot root, has been found to be rich in bioactive compounds such as carotenoids and dietary fiber and contains many other functional components with significant health-promoting features, while Daucus carota L. subsp. Carrot (Apiacae), also known as wild carrot, has been usually used for gastric ulcer therapy, diabetes, and muscle pain in Lebanon. Here, we review the chemical composition of Daucus carota L. and the functional properties of both edible and wild carrot subspecies. Then, we focus on compounds with anticancer characteristics identified in both Daucus carota subspecies, and we discuss their potential use in the development of novel anticancer therapeutic strategies.

An efficient thermostable organophosphate hydrolase and its application in pesticide decontamination.

In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 10(5) M(-1) s(-1). The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces.

Enlarging the substrate portfolio of the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius.

The enzymatic regioselective hydrolysis of (a) acetylated mono- to tetrasaccharides of different nature, (b) of acetylated aryl glycosides and (c) of different acetylated nucleosides was studied enlarging the portfolio of substrates that can be employed by the thermophilic esterase EST2 from Alicyclobacillus acidocaldarius. The reactions were optimised to the extent that the amount of enzyme needed was lowered of two orders of magnitude with respect to the previously reported reactions, namely from 4000 to 40 U of enzyme per reaction. New additional solvents were screened and dramatic changes in regioselectivity were observed depending on the amount and type of solvent used. For example, in the presence of 10 % DMF, only two α-D-glucose products 6-OH and 4,6-OH (in a 76:24 ratio) were detected, whereas with 25 % DMF, at least four products of similar amount were observed. This versatility adds specific value to the biocatalyst making possible the design of biocatalytic reactions with different hydrophobic ester substrates. As an additional remarkable example, EST2 catalysed with a good yield and high regioselectivity the hydrolysis of p-nitrophenyl ß-D-xylopyranoside triacetate producing only the monoacetylated derivative with acetyl group in 3-O-position, in 2 min. The results with nucleosides as substrates are particularly interesting. The peracetates of 3',5'-di-O-acetylthymidine are converted almost quantitatively (95 %) to the monoacetylated derivative possessing free secondary OH; this regioselectivity is complementary to hydrolysis/alcoholysis reactions catalysed by CAL-B lipase or to other microbial hydrolytic biocatalysts, generally giving products with free primary OH groups. A docking analysis was undertaken with all analysed substrates suggesting a structural interpretation of the results. In most of cases, the best pose of the selected substrate was in line with the observed regioselectivity.

Fluorescence spectroscopy approaches for the development of a real-time organophosphate detection system using an enzymatic sensor.

Organophosphates are organic substances that contain a phosphoryl or a thiophosphoryl bond. They are mainly used around the world as pesticides, but can also be used as chemical warfare agents. Their detection is normally entrusted to techniques like GC- and LC-MS that, although sensitive, do not allow their identification on site and in real time. We have approached their identification by exploiting the high-affinity binding of these compounds with the esterase 2 from Alicyclobacillus acidocaldarius. Using an in silico analysis to evaluate the binding affinities of the enzyme with organophosphate inhibitors, like paraoxon, and other organophosphate compounds, like parathion, chlorpyriphos, and other organophosphate thio-derivatives, we have designed fluorescence spectroscopy experiments to study the quenching of the tryptophan residues after esterase 2 binding with the organophosphate pesticides. The changes in the fluorescence signals permitted an immediate and quantitative identification of these compounds from nano- to picomolar concentrations. A fluorescence based polarity-sensitive probe (ANS) was also employed as a means to understand the extent of the interactions involved, as well as to explore other ways to detect organophosphate pesticides. Finally, we designed a framework for the development of a biosensor that exploits fluorescence technology in combination with a sensitive and very stable bio-receptor.

Development of a Qualitative Test to Detect the Presence of Organophosphate Pesticides on Fruits and Vegetables.

BACKGROUND: In recent decades, the use of pesticides in agriculture has increased at a fast pace, highlighting safety problems for the environment and human health, which in turn has made it necessary to develop new detection and decontamination systems for pesticides. METHODS: A new qualitative test capable of detecting the presence of pesticides on fruits and vegetables by using thermostable enzymes was discovered, and the test was carried out on apples and aubergines. The contaminating pesticides were extracted from fruits with acetonitrile and analyzed with a biosensor system based on the thermostable esterase EST2 immobilized on a nitrocellulose filter. This enzyme is irreversibly inhibited mainly in the presence of organophosphates pesticides. Therefore, by observing esterase activity inhibition, we revealed the presence of residual pesticides on the fruits and vegetables. RESULTS: By analyzing the rate of esterase activity inhibition, we predicted that residual pesticides are present on the surface of the fruits. When we cleaned the fruits by washing them in the presence of the phosphotriesterase SsoPox before the detection of the esterase activity on filters, we observed a full recovery of the activity for apples and 30% for aubergines, indicating that the enzymatic decontamination of organophosphates pesticides took place. CONCLUSIONS: The reported method permitted us to assess the pesticides present on the vegetables and their decontamination.

Different Extraction Procedures Revealed the Anti-Proliferation Activity from Vegetable Semi-Purified Sources on Breast Cancer Cell Lines.

Breast cancer (BC) remains the leading cause of mortality in women, despite significant advancements in diagnosis. Thus, the identification of new compounds for its treatment is critical. Phytochemicals are known to exhibit anti-cancer properties. Here, we investigated the anti-proliferation potential of extracts from carrot, Calendula officinalis flower, and Aloe vera on breast cancer vs. epithelial cell lines. Various extraction methods were used, and the proliferative effect of the resulting extracts was assessed by proliferation assay on breast cancer and epithelial cell lines. Carrot, Aloe leaf, and Calendula flower extracts were extracted by hexane and methanol methods, and their semi-purified extracts were able to specifically inhibit the proliferation of breast cancer cell lines. The extract composition was investigated by colorimetric assays, UHPLC-HRMS, and MS/MS analysis. All the extracts contained monogalactosyl-monoacylglycerol (MGMG), while digalactosyl-monoacylglycerol (DGMG) and aloe-emodin were found in Aloe, and glycerophosphocholine (GPC) derivatives were identified in Calendula, except for the isomer 2 detected in carrot, suggesting that their observed different anti-proliferative properties may be associated with the different lipid compounds. Interestingly, Calendula extract was able to strongly inhibit the triple negative breast cancer MDA-MB-231 cell line proliferation (about 20% cell survival), supporting MGMG and GPC derivatives as potential drugs for this BC subtype treatment.

Structural and Phylogenetic Analysis of SARS-CoV-2 Spike Glycoprotein from the Most Widespread Variants.

The SARS-CoV-2 pandemic, reported for the first time at the end of 2019 in the city of Wuhan (China), has spread worldwide in three years; it lead to the infection of more than 500 million people and about six million dead. SARS-CoV-2 has proved to be very dangerous for human health. Therefore, several efforts have been made in studying this virus. In a short time, about one year, the mechanisms of SARS-CoV-2 infection and duplication and its physiological effect on human have been pointed out. Moreover, different vaccines against it have been developed and commercialized. To date, more than 11 billion doses have been inoculated all over the world. Since the beginning of the pandemic, SARS-CoV-2 has evolved; it has done so by accumulating mutations in the genome, generating new virus versions showing different characteristics, and which have replaced the pre-existing variants. In general, it has been observed that the new variants show an increased infectivity and cause milder symptoms. The latest isolated Omicron variants contain more than 50 mutations in the whole genome and show an infectivity 10-folds higher compared to the wild-type strain. Here, we analyse the SARS-CoV-2 variants from a phylogenetic point of view and hypothesize a future scenario for SARS-CoV-2, by following its evolution to date.

SARS-CoV-2: Searching for the Missing Variants.

Structural and phylogenetic analysis of the spike glycoprotein highlighted that the last variants, annotated as omicron, have about 30 mutations compared to the initial version reported in China, while the delta variant, supposed to be the omicron ancestor, shows only 7 mutations. Moreover, the five omicron variants were isolated between November 2021 and January 2022, and the last variant BA.2.75, unofficially named centaurus, was isolated in May 2022. It appears that, since the isolation of the delta variant (October 2020) to the omicron BA.1 (November 2021), there was an interval of one year, whereas the five omicron variants were isolated in three months, and after a successive four months period, the BA.2.75 variant was isolated. So, what is the temporal and phylogenetic correlation among all these variants? The analysis of common mutations among delta and the omicron variants revealed: (i) a phylogenetic correlation among these variants; (ii) the existence of BA.1 and BA.2 omicron variants a few months before being isolated; (iii) at least three possible intermediate variants during the evolution of omicron; (iv) the evolution of the BA.2.12.1, BA.4 and BA.5 variants from omicron BA.2; (v) the centaurus variant evolution from omicron BA.2.12.1.

Five-substrate cocktail as a sensor array for measuring enzyme activity fingerprints of lipases and esterases.

Substrate arrays for measuring enzyme activity fingerprints can be conveniently formulated as cocktails designed such that the reaction products can be separated and quantified by analytical high-performance liquid chromatography (HPLC). Fingerprinting of lipases and esterases, an important class of microbial enzymes, is reported with a cocktail of only five substrates as a practical fingerprinting reagent. An unusually strong C4-esterase activity was thus revealed in a recently discovered microbial esterase.

Melanoma Immunotherapy and Precision Medicine in the Era of Tumor Micro-Tissue Engineering: Where Are We Now and Where Are We Going?

Malignant melanoma still remains a cancer with very poor survival rates, although it is at the forefront of personalized medicine. Most patients show partial responses and disease progressed due to adaptative resistance mechanisms, preventing long-lasting clinical benefits to the current treatments. The response to therapies can be shaped by not only taking into account cancer cell heterogeneity and plasticity, but also by its structural context as well as the cellular component of the tumor microenvironment (TME). Here, we review the recent development in the field of immunotherapy and target-based therapy and how, in the era of tumor micro-tissue engineering, ex-vivo assays could help to enhance our melanoma biology knowledge in its complexity, translating it in the development of successful therapeutic strategies, as well as in the prediction of therapeutic benefits.

Hyperthermophilic phosphotriesterases/lactonases for the environment and human health.

In the last decades the idea to use enzymes for environmental bioremediation has been more and more proposed and, in the light of this, new solutions have been suggested and detailed studies on some classes of enzymes have been performed. In particular, our attention in the last few years has been focused on the enzymes belonging to the amidohydrolase superfamily. Several members of this superfamily are endowed with promiscuous activities. The term 'catalytic promiscuity' describes the capability of an enzyme to catalyse different chemical reactions, called secondary activities, at the active site responsible for the main activity. Recently, a new family of microbial lactonases with promiscuous phosphotriesterase activity, dubbed PTE-Like Lactonase (PLL), has been ascribed to the amidohydrolase superfamily. Among members of this family are enzymes found in the archaea Sulfolobus solfataricus and Sulfolobus acidocaldarius, which show high thermophilicity and thermal resistance. Enzymes showing phosphotriesterase activity are attractive from a biotechnological point of view because they are capable of hydrolysing the organophosphate phosphotriesters (OPs), a class of synthetic compounds employed worldwide both as insecticides and chemical warfare agents. Furthermore, from a basic point of view, studies of catalytic promiscuity offer clues to understand natural evolution of enzymes and to translate this into in vitro adaptation of enzymes to specific human needs. Thermostable enzymes able to hydrolyse OPs are considered good candidates for the set-up of efficient detoxification tools.

Brassicaceae-Derived Anticancer Agents: Towards a Green Approach to Beat Cancer.

Cancer is the main cause of mortality and morbidity worldwide. Although a large variety of therapeutic approaches have been developed and translated into clinical protocols, the toxic side effects of cancer treatments negatively impact patients, allowing cancer to grow. Brassica metabolites are emerging as new weapons for anti-cancer therapeutics. The beneficial role of the consumption of brassica vegetables, the most-used vegetables in the Mediterranean diet, particularly broccoli, in the prevention of chronic diseases, including cardiovascular diseases, diabetes, and obesity, has been well-documented. In this review, we discuss the anti-tumor effects of the bioactive compounds from Brassica vegetables with regard to the compounds and types of cancer against which they show activity, providing current knowledge on the anti-cancer effects of Brassica metabolites against major types of tumors. In addition, we discuss the impacts of industrial and domestic processing on the compounds' functional properties before their consumption as well as the main strategies used to increase the content of health-promoting metabolites in Brassica plants through biofortification. Finally, the impacts of microbiota on the compounds' bioactivity are considered. This information will be helpful for the further development of efficacious anti-cancer drugs.

Enzyme Promiscuous Activity: How to Define it and its Evolutionary Aspects.

Enzymes are among the most studied biological molecules because better understanding enzymes structure and activity will shed more light on their biological processes and regulation; from a biotechnological point of view there are many examples of enzymes used with the aim to obtain new products and/or to make industrial processes less invasive towards the environment. Enzymes are known for their high specificity in the recognition of a substrate but considering the particular features of an increasing number of enzymes this is not completely true, in fact, many enzymes are active on different substrates: this ability is called enzyme promiscuity. Usually, promiscuous activities have significantly lower kinetic parameters than to that of primary activity, but they have a crucial role in gene evolution. It is accepted that gene duplication followed by sequence divergence is considered a key evolutionary mechanism to generate new enzyme functions. In this way, promiscuous activities are the starting point to increase a secondary activity in the main activity and then get a new enzyme. The primary activity can be lost or reduced to a promiscuous activity. In this review we describe the differences between substrate and enzyme promiscuity, and its rule in gene evolution. From a practical point of view the knowledge of promiscuity can facilitate the in vitro progress of proteins engineering, both for biomedical and industrial applications. In particular, we report cases regarding esterases, phosphotriesterases and cytochrome P450.

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).

Evolution in the amidohydrolase superfamily: substrate-assisted gain of function in the E183K mutant of a phosphotriesterase-like metal-carboxylesterase.

The recent specialization for utilization of pesticides reported for Pseudomonas diminuta phosphotriesterase (pPTE) strongly suggests that this activity evolved from an enzyme endowed with promiscuous phosphotriesterase activity. Such a putative "generalist" enzyme was recently proposed to be a member of the new phoshotriesterase-like lactonase family (PLL). The promiscuous carboxylesterase and phosphodiesterase activities detected in pPTE and PLLs in turn paved the way for the prediction of the existence in nature of PTE-like enzymes with predominant carboxylesterase or phosphodiesterase activities. An "in silico" analysis of the related Mesorhizobium loti ORF MLL7664 and the biochemical characterization demonstrated its prominent carboxylesterase and low phosphotriesterase specificity. On the basis of sequence similarity with the phosphotriesterase homology protein from Escherichia coli and the carboxylesterase activity, we called it phosphotriesterase-like carboxylesterase (MloPLC). The carboxylesterase activity is strictly dependent on divalent cations, and as such MloPLC is the first phosphotriesterase-like metal-carboxylesterase characterized to date. In related enzymes of the amidohydrolase superfamily either glutamate or carboxylated lysine substitutes for MloPLC glutamate 183 and the residue appear invariantly involved in maintaining the structural integrity of the binuclear metal center. Accordingly, we changed Glu-183 to lysine or glutamine. All the tested activities were completely abolished in the E183Q mutant, while only a residual phosphotriesterase activity could be detected in the E183K mutant. Surprisingly, in the latter mutant a parallel 650-fold specificity increase in bis-p-nitrophenyl-phosphate (BpNP-P) was observed, turning MloPLC from a carboxylesterase into a phosphodiesterase. Chemical, structural, and kinetic data strongly suggested that K183 is not carboxylated and that the gain of the new function is assisted by the substrate.

Structural determinants of the high thermal stability of SsoPox from the hyperthermophilic archaeon Sulfolobus solfataricus.

Organophosphates (OPs) constitute the largest class of insecticides used worldwide and certain of them are potent nerve agents. Consequently, enzymes degrading OPs are of paramount interest, as they could be used as bioscavengers and biodecontaminants. Looking for a stable OPs catalyst, able to support industrial process constraints, a hyperthermophilic phosphotriesterase (PTE) (SsoPox) was isolated from the archaeon Sulfolobus solfataricus and was found to be highly thermostable. The solved 3D structure revealed that SsoPox is a noncovalent dimer, with lactonase activity against "quorum sensing signals", and therefore could represent also a potential weapon against certain pathogens. The structural basis of the high thermostability of SsoPox has been investigated by performing a careful comparison between its structure and that of two mesophilic PTEs from Pseudomonas diminuta and Agrobacterium radiobacter. In addition, the conformational stability of SsoPox against the denaturing action of temperature and GuHCl has been determined by means of circular dichroism and fluorescence measurements. The data suggest that the two fundamental differences between SsoPox and the mesophilic counterparts are: (a) a larger number of surface salt bridges, also involved in complex networks; (b) a tighter quaternary structure due to an optimization of the interactions at the interface between the two monomers.

Functional and structural features of the oxyanion hole in a thermophilic esterase from Alicyclobacillus acidocaldarius.

Recent mutagenic and molecular modelling studies suggested a role for glycine 84 in the putative oxyanion loop of the carboxylesterase EST2 from Alicyclobacillus acidocaldarius. A 114 times decrease of the esterase catalytic activity of the G84S mutant was observed, without changes in the thermal stability. The recently solved three-dimensional (3D) structure of EST2 in complex with a HEPES molecule permitted to demonstrate that G84 (together with G83 and A156) is involved in the stabilization of the oxyanion through a hydrogen bond from its main chain NH group. The structural data in this case did not allowed us to rationalize the effect of the mutation, since this hydrogen bond was predicted to be unaltered in the mutant. Since the mutation could shed light on the role of the oxyanion loop in the HSL family, experiments to elucidate at the mechanistic level the reasons of the observed drop in k (cat) were devised. In this work, the kinetic and structural features of the G84S mutant were investigated in more detail. The optimal temperature and pH for the activity of the mutated enzyme were found significantly changed (T = 65 degrees C and pH = 5.75). The catalytic constants K (M) and V(max) were found considerably altered in the mutant, with ninefold increased K (M) and 14-fold decreased V(max), at pH 5.75. At pH 7.1, the decrease in k (cat) was much more dramatic. The measurement of kinetic constants for some steps of the reaction mechanism and the resolution of the mutant 3D structure provided evidences that the observed effects were partly due to the steric hindrance of the S84-OH group towards the ester substrate and partly to its interference with the nucleophilic attack of a water molecule on the second tetrahedral intermediate.

A proteomic approach to study Escherichia coli. Acetyl esterase interactors unveil a sequence motif involved in protein-protein interaction.

E. coli acetyl esterase (Aes) and beta-cysthationase (MalY) interact, probably with the same mechanism, with the N-terminus of transcriptional activator of maltose regulon (MalT). In order to investigate the basic mechanism of this interaction, we used both a proteomic and a bioinformatic approach. Affinity-based mass spectrometry experiments with purified Aes protein as bait allowed to fish twenty-three, apparently specific, interactors from crude extracts of E. coli cells grown in different conditions. The group of interactors appeared quite heterogeneous, comprising Aes itself, some molecular chaperons, metabolic enzymes, and several proteins of unknown function. Among the identified proteins, two are in some way related to the maltose metabolism and two are related to the lipopolysaccharide metabolism. By superposing the structures of the Alicyclobacillus acidocaldarius EST2, an Aes homolog, and MalY, a region of structural similarity was discovered that allowed detecting a short stretch of nine residues with sequence similarity among EST2, AES and MalY. Degenerated sequence consensuses derived from the alignment were used to analyse the E. coli proteome in the Swiss Prot database and permitted to retrieve sequences of Aes interactors already known or detected in this study. Most of these interactors (14 out of 25) contain the expected consensus. A site-directed mutant of Aes R179A made in the consensus sequence resulted in complete loss of interaction. Based on the analysis of the available three-dimensional structures and mutagenic and structural data inferred from literature, we predict a role of this motif in protein-protein interaction.

Innovative Biocatalysts as Tools to Detect and Inactivate Nerve Agents.

Pesticides and warfare nerve agents are frequently organophosphates (OPs) or related compounds. Their acute toxicity highlighted more than ever the need to explore applicable strategies for the sensing, decontamination and/or detoxification of these compounds. Herein, we report the use of two different thermostable enzyme families capable to detect and inactivate OPs. In particular, mutants of carboxylesterase-2 from Alicyclobacillus acidocaldarius and of phosphotriesterase-like lactonases from Sulfolobus solfataricus and Sulfolobus acidocaldarius, have been selected and assembled in an optimized format for the development of an electrochemical biosensor and a decontamination formulation, respectively. The features of the developed tools have been tested in an ad-hoc fabricated chamber, to mimic an alarming situation of exposure to a nerve agent. Choosing ethyl-paraoxon as nerve agent simulant, a limit of detection (LOD) of 0.4 nM, after 5 s of exposure time was obtained. Furthermore, an optimized enzymatic formulation was used for a fast and efficient environmental detoxification (>99%) of the nebulized nerve agent simulants in the air and on surfaces. Crucial, large-scale experiments have been possible thanks to production of grams amounts of pure (>90%) enzymes.