Post-translational modifications in tumor-associated carbonic anhydrases.

Human carbonic anhydrases IX (hCA IX) and XII (hCA XII) are two proteins associated with tumor formation and development. These enzymes have been largely investigated both from a biochemical and a functional point of view. However, limited data are currently available on the characterization of their post-translational modifications (PTMs) and the functional implication of these structural changes in the tumor environment. In this review, we summarize existing literature data on PTMs of hCA IX and hCA XII, such as disulphide bond formation, phosphorylation, O-/N-linked glycosylation, acetylation and ubiquitination, highlighting, when possible, their specific role in cancer pathological processes.

Biochemical and Structural Insights into the Winged Helix Domain of P150, the Largest Subunit of the Chromatin Assembly Factor 1.

The Chromatin Assembly Factor 1 is a heterotrimeric complex responsible for the nucleosome assembly during DNA replication and DNA repair. In humans, the largest subunit P150 is the major actor of this process. It has been recently considered as a tumor-associated protein due to its overexpression in many malignancies. Structural and functional studies targeting P150 are still limited and only scarce information about this subunit is currently available. Literature data and bioinformatics analysis assisted the identification of a stable DNA binding domain, encompassing residues from 721 to 860 of P150 within the full-length protein. This domain was recombinantly produced and in vitro investigated. An acidic region modulating its DNA binding ability was also identified and characterized. Results showed similarities and differences between the P150 and its yeast homologue, namely Cac-1, suggesting that, although sharing a common biological function, the two proteins may also possess different features.

α-CAs from Photosynthetic Organisms.

Carbonic anhydrases (CAs) are ubiquitous enzymes that catalyze the reversible carbon dioxide hydration reaction. Among the eight different CA classes existing in nature, the α-class is the largest one being present in animals, bacteria, protozoa, fungi, and photosynthetic organisms. Although many studies have been reported on these enzymes, few functional, biochemical, and structural data are currently available on α-CAs isolated from photosynthetic organisms. Here, we give an overview of the most recent literature on the topic. In higher plants, these enzymes are engaged in both supplying CO2 at the Rubisco and determining proton concentration in PSII membranes, while in algae and cyanobacteria they are involved in carbon-concentrating mechanism (CCM), photosynthetic reactions and in detecting or signaling changes in the CO2 level in the environment. Crystal structures are only available for three algal α-CAs, thus not allowing to associate specific structural features to cellular localizations or physiological roles. Therefore, further studies on α-CAs from photosynthetic organisms are strongly needed to provide insights into their structure-function relationship.

Colorectal cancer incidence and mortality after negative fecal immunochemical tests by age 70: A prospective observational study.

Limited endoscopy capacity usually represents the main barrier to the extension of screening to subjects older than 70, given the high positivity rate in this age group. We assessed CRC incidence and mortality by number of previous negative fecal immunochemical tests (FIT) among subjects turning 70. We selected persons aged 70 years who had received their last screening invitation when they were 68 or 69 years old within the population-based screening program in the Veneto region of Italy. Subjects with a positive FIT were excluded. We calculated 10-year cumulative CRC incidence and mortality in cohorts of subjects having performed zero, one, two or three negative FITs over the last three screening rounds before turning 70. Out of 117 858 subjects included in the study (46.4% men), 33.7% had never participated in screening (zero negative FITs), 23.3% had had one-negative FIT, 20.1% two-negative FITs and 22.9% three negative FITs. The 10-year cumulative CRC incidence was 29.7 per 1000 subjects with zero FITs, and respectively, 14.5, 11.7 and 9.6 per 1000 subjects with one, two and three negative FITs. The corresponding figures for 10-year cumulative mortality were 9.3, 3.5, 2.2 and 2.1 per 1000 in the four study cohorts. Figures were roughly double for men than for women for all the study cohorts. In order to use more efficiently limited endoscopy resources, and to minimize the potential harms related to false positive results in the elderly, screening among people aged 70 to 74 might be restricted to those with zero previous negative FITs.

Design, synthesis and biochemical evaluation of novel carbonic anhydrase inhibitors triggered by structural knowledge on hCA VII.

To tackle the challenge of isoform selectivity, we explored the entrance of the cavity for selected druggable human Carbonic Anhydrases (hCAs). Based on X-ray crystallographic studies on the 4-(4-(2-chlorobenzoyl)piperazine-1-carbonyl)benzenesulfonamide in complex with the brain expressed hCA VII (PDB code: 7NC4), a series of 4-(4(hetero)aroylpiperazine-1-carbonyl)benzene-1-sulfonamides has been developed. To evaluate their capability to fit the hCA VII catalytic cavity, the newer benzenesulfonamides were preliminary investigated by means of docking simulations. Then, this series of thirteen benzenesulfonamides was synthesized and tested against selected druggable hCAs. Among them, the 4-(4-(furan-2-carbonyl)piperazine-1-carbonyl)benzenesulfonamide showed remarkable affinity towards hCA VII (Ki: 4.3 nM) and good selectivity over the physiologically widespread hCA I when compared to Topiramate (TPM).

Inhibition of the ß-carbonic anhydrase from the protozoan pathogen Trichomonas vaginalis with sulphonamides.

Sulphonamides and their isosteres are classical inhibitors of the carbonic anhydrase (CAs, EC 4.2.1.1) metalloenzymes. The protozoan pathogen Trichomonas vaginalis encodes two such enzymes belonging to the ß-class, TvaCA1 and TvaCA2. Here we report the first sulphonamide inhibition study of TvaCA1, with a series of simple aromatic/heterocyclic primary sulphonamides as well as with clinically approved/investigational drugs for a range of pathologies (diuretics, antiglaucoma, antiepileptic, antiobesity, and antitumor drugs). TvaCA1 was effectively inhibited by acetazolamide and ethoxzolamide, with KIs of 391 and 283 nM, respectively, whereas many other simple or clinically used sulphonamides were micromolar inhibitors or did not efficiently inhibit the enzyme. Finding more effective TvaCA1 inhibitors may constitute an innovative approach for fighting trichomoniasis, a sexually transmitted infection, caused by T. vaginalis.

Human carbonic anhydrases and post-translational modifications: a hidden world possibly affecting protein properties and functions.

Human carbonic anhydrases (CAs) have become a well-recognized target for the design of inhibitors and activators with biomedical applications. Accordingly, an enormous amount of literature is available on their biochemical, functional and structural aspects. Nevertheless post-translational modifications (PTMs) occurring on these enzymes and their functional implications have been poorly investigated so far. To fill this gap, in this review we have analysed all PTMs occurring on human CAs, as deriving from the search in dedicated databases, showing a widespread occurrence of modification events in this enzyme family. By combining these data with sequence alignments, inspection of 3 D structures and available literature, we have summarised the possible functional implications of these PTMs. Although in some cases a clear correlation between a specific PTM and the CA function has been highlighted, many modification events still deserve further dedicated studies.

Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis.

We report the biochemical and structural characterisation of a beta-carbonic anhydrase (ß-CA) from Trichomonas vaginalis, a unicellular parasite responsible for one of the world's leading sexually transmitted infections, trichomoniasis. CAs are ubiquitous metalloenzymes belonging to eight evolutionarily divergent groups (α, ß, γ, δ, ζ, η, θ, and ι); humans express only α-CAs, whereas many clinically significant pathogens express only ß- and/or γ-CAs. For this reason, the latter two groups of CAs are promising biomedical targets for novel antiinfective agents. The ß-CA from T. vaginalis (TvaCA1) was recombinantly produced and biochemically characterised. The crystal structure was determined, revealing the canonical dimeric fold of ß-CAs and the main features of the enzyme active site. The comparison with the active site of human CA enzymes revealed significant differences that can be exploited for the design of inhibitors selective for the protozoan enzyme with respect to the human ones.

Exploration of the residues modulating the catalytic features of human carbonic anhydrase XIII by a site-specific mutagenesis approach.

Carbonic anhydrases (CAs) are ubiquitous metallo-enzymes that catalyse the reversible hydration of carbon dioxide to bicarbonate and proton. In humans there are 15 isoforms among which only 12 are catalytically active. Since active human (h) CAs show different efficiency, the understanding of the molecular determinants affecting it is a matter of debate. Here we investigated, by a site-specific mutagenesis approach, residues modulating the catalytic features of one of the least investigated cytosolic isoform, i.e. hCA XIII. Results showed that residues assisting the formation of an ordered solvent network within the catalytic site as well as those forming a histidine cluster on the protein surface are important to guarantee an efficient proton transfer.

Exploring benzoxaborole derivatives as carbonic anhydrase inhibitors: a structural and computational analysis reveals their conformational variability as a tool to increase enzyme selectivity.

Recent studies identified the benzoxaborole moiety as a new zinc-binding group able to interact with carbonic anhydrase (CA) active site. Here, we report a structural analysis of benzoxaboroles containing urea/thiourea groups, showing that these molecules are very versatile since they can bind the enzyme assuming different binding conformations and coordination geometries of the catalytic zinc ion. In addition, theoretical calculations of binding free energy were performed highlighting the key role of specific residues for protein-inhibitor recognition. Overall, these data are very useful for the development of new inhibitors with higher selectivity and efficacy for various CAs.

Inhibition of carbonic anhydrase IX targets primary tumors, metastases, and cancer stem cells: Three for the price of one.

Human carbonic anhydrase (CA) IX is a tumor-associated protein, since it is scarcely present in normal tissues, but highly overexpressed in a large number of solid tumors, where it actively contributes to survival and metastatic spread of tumor cells. Due to these features, the characterization of its biochemical, structural, and functional features for drug design purposes has been extensively carried out, with consequent development of several highly selective small molecule inhibitors and monoclonal antibodies to be used for different purposes. Aim of this review is to provide a comprehensive state-of-the-art of studies performed on this enzyme, regarding structural, functional, and biomedical aspects, as well as the development of molecules with diagnostic and therapeutic applications for cancer treatment. A brief description of additional pharmacologic applications for CA IX inhibition in other diseases, such as arthritis and ischemia, is also provided.

The Crystal Structure of a hCA VII Variant Provides Insights into the Molecular Determinants Responsible for Its Catalytic Behavior.

Although important progress has been achieved in understanding the catalytic mechanism of Carbonic Anhydrases, a detailed picture of all factors influencing the catalytic efficiency of the various human isoforms is still missing. In this paper we report a detailed structural study and theoretical pKa calculations on a hCA VII variant. The obtained data were compared with those already known for another thoroughly investigated cytosolic isoform, hCA II. Our structural studies show that in hCA VII the network of ordered water molecules, which connects the zinc bound solvent molecule to the proton shuttle His64, is altered compared to hCA II, causing a reduction of the catalytic efficiency. Theoretical calculations suggest that changes in solvent network are related to the difference in pKa of the proton shuttle in the two enzymes. The residue that plays a major role in determining the diverse pKa values of the proton shuttle is the one in position four, namely His for hCA II and Gly for hCA VII. This residue is located on the protein surface, outside of the active site cavity. These findings are in agreement with our previous studies that highlighted the importance of histidines on the protein surface of hCA II (among which His4) as crucial residues for the high catalytic efficiency of this isoform.

How grinding level and brewing method (Espresso, American, Turkish) could affect the antioxidant activity and bioactive compounds in a coffee cup.

BACKGROUND: Depending on geographical origin and cultural traditions, different brewing procedures are used all over the world to prepare a cup of coffee. In this work, we explored how three grinding levels of coffee powder and three coffee preparation methods - filtration (American), boiling (Turkish) and extraction under pressure (Espresso) - affect healthy compounds and physicochemical attributes in coffee served to consumers. RESULTS: Grinding level slightly affected the quality of coffee, whereas the preparation method significantly influenced all in-cup attributes. When the content per cup was compared, the American coffee presented higher values of antioxidant activity and total phenol content than espresso and Turkish coffees. Caffeine content was 316, 112 and 64 mg for the American, Turkish and espresso coffee cup, respectively. CONCLUSION: One American, three Turkish and five Espresso coffee cups contain similar amount of caffeine of 316, 336 and 320 mg, respectively which are below the maximum daily consumption (400 mg per day) suggested by the European Food Safety Authority. The extraction method affects the intake of bioactive and antioxidant substances with specific properties. © 2017 Society of Chemical Industry.

Insights into the role of reactive sulfhydryl groups of Carbonic Anhydrase III and VII during oxidative damage.

Carbonic anhydrases (CAs) III and VII are two cytosolic isoforms of the α-CA family which catalyze the physiological reaction of carbon dioxide hydration to bicarbonate and proton. Despite these two enzymes share a 49% sequence identity and present a very similar three-dimensional structure, they show profound differences when comparing the specific activity for CO2 hydration reaction, with CA VII being much more active than CA III. Recently, CA III and CA VII have been proposed to play a new role as scavenger enzymes in cells where oxidative damage occurs. Here, we will examine functional and structural features of these two isoforms giving insights into their newly proposed protective role against oxidative stress.

The anticonvulsant sulfamide JNJ-26990990 and its S,S-dioxide analog strongly inhibit carbonic anhydrases: solution and X-ray crystallographic studies.

JNJ-26990990 ((benzo[b]thien-3-yl)methyl)sulfamide, a sulfamide derivative structurally related to the antiepileptic drug zonisamide, was reported to be devoid of carbonic anhydrase (CA, EC 4.2.1.1) inhibitory properties. Here we report that JNJ-26990990 and its S,S-dioxide analog significantly inhibit six human (h) isoforms, hCA I, II, VII, IX, XII and XIV, involved in crucial physiological processes. Inhibition and X-ray crystallographic data for the binding of the two compounds to these enzymes show significant similarity with the zonisamide inhibitory pattern. These findings prompted us to reconsider the structural/pharmacological requirements for designing effective antiepileptics possessing zinc-binding groups of the sulfamide, sulfamate or sulfonamide type in their molecules.

How the variance of some extraction variables may affect the quality of espresso coffees served in coffee shops.

BACKGROUND: To improve the quality of espresso coffee, the variables under the control of the barista, such as grinding grade, coffee quantity and pressure applied to the coffee cake, as well as their variance, are of great importance. A nonlinear mixed effect modeling was used to obtain information on the changes in chemical attributes of espresso coffee (EC) as a function of the variability of extraction conditions. RESULTS: During extraction, the changes in volume were well described by a logistic model, whereas the chemical attributes were better fit by a first-order kinetic. The major source of information was contained in the grinding grade, which accounted for 87-96% of the variance of the experimental data. The variability of the grinding produced changes in caffeine content in the range of 80.03 mg and 130.36 mg when using a constant grinding grade of 6.5. CONCLUSION: The variability in volume and chemical attributes of EC is large. Grinding had the most important effect as the variability in particle size distribution observed for each grinding level had a profound effect on the quality of EC. Standardization of grinding would be of crucial importance for obtaining all espresso coffees with a high quality. © 2015 Society of Chemical Industry.

The zinc coordination pattern in the η-carbonic anhydrase from Plasmodium falciparum is different from all other carbonic anhydrase genetic families.

In this Letter we reinvestigate the sequence analysis and report a homology model of the carbonic anhydrase (CA, EC 4.2.1.1) from the protozoan parasite Plasmodium falciparum, recently reported by us to belong to a new genetic family, the η-CA class. Our findings show that the metal ion coordination pattern of this CA is unique among all five other genetic families encoding for such enzymes, comprising two His and one Gln residues, in addition to the water molecule/hydroxide ion acting as nucleophile in the catalytic cycle. Although the η- and α-CAs present the same 3D fold, strongly suggesting the first ones to be evolutionary derived from the last, there are significant differences between the two families to allow optimism for the drug design of selective inhibitors for the parasite over the host enzymes. The preliminary studies reported here are relevant for drug design campaigns of anti-plasmodium CA inhibitors but further work by X-ray crystallography should validate the proposed model.

Crystal structure of the most catalytically effective carbonic anhydrase enzyme known, SazCA from the thermophilic bacterium Sulfurihydrogenibium azorense.

Two thermostable α-carbonic anhydrases (α-CAs) isolated from thermophilic Sulfurihydrogenibium spp., namely SspCA (from S. yellowstonensis) and SazCA (from S. azorense), were shown in a previous work to possess interesting complementary properties. SspCA was shown to have an exceptional thermal stability, whereas SazCA demonstrated to be the most active α-CA known to date for the CO2 hydration reaction. Here we report the crystallographic structure of SazCA and the identification of the structural features responsible for its high catalytic activity, by comparing it with SspCA structure. These data are of relevance for the design of engineered proteins showing higher stability and catalytic activity than other α-CAs known to date.

Thermostable Carbonic Anhydrases in Biotechnological Applications.

Carbonic anhydrases are ubiquitous metallo-enzymes which catalyze the reversible hydration of carbon dioxide in bicarbonate ions and protons. Recent years have seen an increasing interest in the utilization of these enzymes in CO2 capture and storage processes. However, since this use is greatly limited by the harsh conditions required in these processes, the employment of thermostable enzymes, both those isolated by thermophilic organisms and those obtained by protein engineering techniques, represents an interesting possibility. In this review we will provide an extensive description of the thermostable carbonic anhydrases so far reported and the main processes in which these enzymes have found an application.

Thyroid hormone inhibition in L6 myoblasts of IGF-I-mediated glucose uptake and proliferation: new roles for integrin αvß3.

Thyroid hormones L-thyroxine (T4) and 3,3',5-triiodo-L-thyronine (T3) have been shown to initiate short- and long-term effects via a plasma membrane receptor site located on integrin αvß3. Also insulin-like growth factor type I (IGF-I) activity is known to be subject to regulation by this integrin. To investigate the possible cross-talk between T4 and IGF-I in rat L6 myoblasts, we have examined integrin αvß3-mediated modulatory actions of T4 on glucose uptake, measured through carrier-mediated 2-deoxy-[3H]-D-glucose uptake, and on cell proliferation stimulated by IGF-I, assessed by cell counting, [3H]-thymidine incorporation, and fluorescence-activated cell sorting analysis. IGF-I stimulated glucose transport and cell proliferation via the cell surface IGF-I receptor (IGFIR) and, downstream of the receptor, by the phosphatidylinositol 3-kinase signal transduction pathway. Addition of 0.1 nM free T4 caused little or no cell proliferation but prevented both glucose uptake and proliferative actions of IGF-I. These actions of T4 were mediated by an Arg-Gly-Asp (RGD)-sensitive pathway, suggesting the existence of crosstalk between IGFIR and the T4 receptor located near the RGD recognition site on the integrin. An RGD-sequence-containing integrin inhibitor, a monoclonal antibody to αvß3, and the T4 metabolite tetraiodothyroacetic acid all blocked the inhibition by T4 of IGF-I-stimulated glucose uptake and cell proliferation. Western blotting confirmed roles for activated phosphatidylinositol 3-kinase and extracellular regulated kinase 1/2 (ERK1/2) in the effects of IGF-I and also showed a role for ERK1/2 in the actions of T4 that modified the effects of IGF-I. We conclude that thyroid hormone inhibits IGF-I-stimulated glucose uptake and cell proliferation in L6 myoblasts.