Intrinsically disordered features of carbonic anhydrase IX proteoglycan-like domain.

hCA IX is a multi-domain protein belonging to the family of hCAs which are ubiquitous zinc enzymes that catalyze the reversible hydration of CO2 to HCO3- and H+. hCA IX is a tumor-associated enzyme with a limited distribution in normal tissues, but over-expressed in many tumors, and is a promising drug target. Although many studies concerning the CA IX catalytic domain were performed, little is known about the proteoglycan-like (PG-like) domain of hCA IX which has been poorly investigated so far. Here we attempt to fill this gap by providing an overview on the functional, structural and therapeutic studies of the PG-like domain of hCA IX which represents a unique feature within the CA family. The main studies and recent advances concerning PG role in modulating hCA IX catalytic activity as well as in tumor spreading and migration are here reported. Special attention has been paid to the newly discovered disordered features of the PG domain which open new perspectives about its molecular mechanisms of action under physiological and pathological conditions, since disorder is likely involved in mediating interactions with partner proteins. The emerged disordered features of PG domain will be explored for putative diagnostic and therapeutic applications involving CA IX targeting in tumors.

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.

Targeted somatic mutagenesis through CRISPR/Cas9 ribonucleoprotein complexes in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae (Diptera: Tephritidae), is the most destructive insect pest of olive cultivation, causing significant economic and production losses. Here, we present the establishment of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 methodology for gene disruption in this species. We performed targeted mutagenesis of the autosomal gene white (Bo-we), by injecting into early embryos in vitro preassembled and solubilized Cas9 ribonucleoprotein complexes loaded with two gene-specific single-guide RNAs. Gene disruption of Bo-we led to somatic mosaicism of the adult eye color. Large eye patches or even an entire eye lost the iridescent reddish color, indicating the successful biallelic mutagenesis in somatic cells. Cas9 induced either indels in each of the two simultaneously targeted Bo-we sites or a large deletion of the intervening region. This study demonstrates the first efficient implementation of the CRISPR/Cas9 technology in the olive fly, providing new opportunities towards the development of novel genetic tools for its control.

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.

Biochemical, biophysical and molecular dynamics studies on the proteoglycan-like domain of carbonic anhydrase IX.

Human carbonic anhydrase IX (hCA IX) is a tumour-associated enzyme present in a limited number of normal tissues, but overexpressed in several malignant human tumours. It is a transmembrane protein, where the extracellular region consists of a greatly investigated catalytic CA domain and a much less investigated proteoglycan-like (PG) domain. Considering its important role in tumour biology, here, we report for the first time the full characterization of the PG domain, providing insights into its structural and functional features. In particular, this domain has been produced at high yields in bacterial cells and characterized by means of biochemical, biophysical and molecular dynamics studies. Results show that it belongs to the family of intrinsically disordered proteins, being globally unfolded with only some local residual polyproline II secondary structure. The observed conformational flexibility may have several important roles in tumour progression, facilitating interactions of hCA IX with partner proteins assisting tumour spreading and progression.

The zinc - but not cadmium - containing ζ-carbonic from the diatom Thalassiosira weissflogii is potently activated by amines and amino acids.

The activation of the ζ-class carbonic anhydrase (CAs, EC 4.2.1.1) from the diatom Thalassiosira weissflogii (TweCAζ) incorporating both Zn(II) and Cd(II) at the active site, was investigated for the first time, using a panel of natural and non-natural amino acids and amines. CdTweCAζ was completely insensitive to activation, whereas all these compounds were effective activators of the zinc-containing enzyme ZnTweCAζ, with activation constants ranging between 92 nM and 37.9 µM. The most effective ZnTweCAζ activators were l-adrenaline, 1-(2-aminoethyl)-piperazine and 4-(2-aminoethyl)-morpholine, with KAs in the range of 92-150 nM. l-His, l- and d-Tyr and some pyridyl-alkylamines, had KAs in the range of 0.62-0.98 µM, whereas l-/d-DOPA, d-Trp, histamine, serotonin and l-Asn were the next most efficient activators, with KAs in the range of 1.27-3.19 µM. The least effective activators were l-Phe (KA of 15.4 µM) and l-Asp (KA of 37.9 µM). This in vitro study may be useful for a more complete understanding of the activation processes of various CA enzyme families, of which the ζ-class was scarcely investigated.

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.

Protein conformational perturbations in hereditary amyloidosis: Differential impact of single point mutations in ApoAI amyloidogenic variants.

Amyloidoses are devastating diseases characterized by accumulation of misfolded proteins which aggregate in fibrils. Specific gene mutations in Apolipoprotein A I (ApoAI) are associated with systemic amyloidoses. Little is known on the effect of mutations on ApoAI structure and amyloid properties. Here we performed a physico-chemical characterization of L75P- and L174S-amyloidogenic ApoAI (AApoAI) variants to shed light on the effects of two single point mutations on protein stability, proteolytic susceptibility and aggregation propensity. Both variants are destabilized in their N-terminal region and generate fibrils with different morphological features. L75P-AApoAI is significantly altered in its conformation and compactness, whereas a more flexible and pronounced aggregation-competent state is associated to L174S-AApoAI. These observations point out how single point mutations in ApoAI gene evocate differences in the physico-chemical and conformational behavior of the corresponding protein variants, with the common feature of diverting ApoAI from its natural role towards a pathogenic pathway.

Synthesis of isoxazole-containing sulfonamides with potent carbonic anhydrase II and VII inhibitory properties.

Two series of benzenesulfonamide containing isoxazole compounds were prepared by using conventional and microwave (MW) methods. 5-Amino-3-aryl-N-(4-sulfamoylphenyl)isoxazole-4-carboxamide derivatives were synthesized by the reaction of hydroxymoyl chlorides with 2-cyano-N-(4-sulfamoylphenyl)acetamide in the presence of triethylamine. The synthesized 5-amino isoxazoles were reacted with various benzoyl chlorides in order to obtain 5-amidoisoxazoles. The novel compounds were screened in vitro as inhibitors of four human (h) isoforms of the metalloenzyme carbonic anhydrase (CA, EC 4.2.1.1): hCA I, hCA II, hCA IV and hCA VII. The derivatives of the first series were shown to possess excellent inhibitory activity against the cytosolic isoform hCA II, an antiglaucoma drug target, with KIs in the range of 0.5-49.3nM and hCA VII, a recently validated anti-neuropathic pain target with KIs in the range of 4.3-51.9nM.

Discovery of 4-sulfamoyl-phenyl-ß-lactams as a new class of potent carbonic anhydrase isoforms I, II, IV and VII inhibitors: The first example of subnanomolar CA IV inhibitors.

A series of benzenesulfonamides incorporating 1,3,4-trisubstituted-ß-lactam moieties was prepared from sulfanilamide Schiff bases and in situ obtained ketenes, by using the Staudinger cycloaddition reaction. The new compounds were assayed as inhibitors of four human isoforms of the metalloenzyme carbonic anhydrase (hCA, EC 4.2.1.1) involved in various physiological/pathological conditions, hCA I, II, IV and VII. Excellent inhibitory activity was observed against all these isoforms, as follows: hCA I, involved in some eye diseases was inhibited with KIs in the range of 7.3-917nM; hCA II, an antiglaucoma drug target, with KIs in the range of 0.76-163nM. hCA IV, an isoform involved in several pathological conditions such as glaucoma, retinitis pigmentosa and edema was potently inhibited by the lactam-sulfonamides, with KIs in the range of 0.53-51.0nM, whereas hCA VII, a recently validated anti-neuropathic pain target was the most inhibited isoform by these derivatives, with KIs in the range of 0.68-9.1nM. The structure-activity relationship for inhibiting these CAs with the new lactam-sulfonamides is discussed in detail.

Design, synthesis and biological evaluation of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamides as human carbonic anhydrase isoenzymes I, II, VII and XII inhibitors.

A series of N-(5-methyl-isoxazol-3-yl/1,3,4-thiadiazol-2-yl)-4-(3-substitutedphenylureido) benzenesulfonamide derivatives has been designed, synthesized and screened for their in vitro human carbonic anhydrase (hCA; EC 4.2.1.1) inhibition potential. These newly synthesized sulfonamide compounds were assessed against isoforms hCA I, II, VII and XII, with acetazolamide (AAZ) as a reference compound. The majority of these compounds were found quite weak inhibitor against all tested isoforms. Compound 15 showed a modest inhibition potency against hCA I (Ki = 73.7 µM) and hCA VII (Ki = 85.8 µM). Compounds 19 and 25 exhibited hCA II inhibition with Ki values of 96.0 µM and 87.8 µM, respectively. The results of the present study suggest that, although the synthesized derivatives have weak inhibitory potential towards all investigated isoforms, some of them may serve as lead molecules for the further development of selective inhibitors incorporating secondary sulfonamide functionalities, a class of inhibitors for which the inhibition mechanism is poorly understood.

Microwave-assisted extraction, HPLC analysis, and inhibitory effects on carbonic anhydrase I, II, VA, and VII isoforms of 14 blueberry Italian cultivars.

The multi-component fingerprint and the biological evaluation of plant-derived material are indispensable for the pharmaceutical field, in food quality control procedures, and in all plant-based products. We investigated the quantitative content of biologically active compounds (anthocyanins and chlorogenic acid) of microwave-assisted blueberry extracts from 14 different Italian cultivars, using validated high-performance liquid chromatography-photodiode array detector (HPLC-PDA) method and routinely instrument configuration. The carbonic anhydrase (CA, EC 4.2.1.1) inhibition profiles against several pharmacologically relevant CA isoforms of blueberry extracts and some bioactive compounds were also investigated. The various cultivars showed a highly variable content in anthocyanins and chlorogenic acid, and their CA inhibitory effects were also highly variable. Overall these data prove that antioxidant natural products found in blueberries may be useful for designing pharmacological agents in which various CAs are involved, e.g., antiobesity, antitumor, or anticonvulsants agents.

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.

The structural comparison between membrane-associated human carbonic anhydrases provides insights into drug design of selective inhibitors.

Carbonic anhydrase isoform XIV (CA XIV) is the last member of the human (h) CA family discovered so far, being localized in brain, kidneys, colon, small intestine, urinary bladder, liver, and spinal cord. It has recently been described as a possible drug target for treatment of epilepsy, some retinopathies as well as some skin tumors. Human carbonic anhydrase (hCA) XIV is a membrane-associated protein consisting of an N-terminal extracellular domain, a putative transmembrane region, and a small cytoplasmic tail. In this article, we report the expression, purification, and the crystallographic structure of the entire extracellular domain of this enzyme. The analysis of the structure revealed the typical α-CA fold, in which a 10-stranded ß-sheet forms the core of the molecule, while the comparison with all the other membrane associated isoforms (hCAs IV, IX, and XII) allowed to identify the diverse oligomeric arrangement and the sequence and structural differences observed in the region 127-136 as the main factors to consider in the design of selective inhibitors for each one of the membrane associated α-CAs.

Intrinsically disordered Prosystemin discloses biologically active repeat motifs.

The in-depth studies over the years on the defence barriers by tomato plants have shown that the Systemin peptide controls the response to a wealth of environmental stress agents. This multifaceted stress reaction seems to be related to the intrinsic disorder of its precursor protein, Prosystemin (ProSys). Since latest findings show that ProSys has biological functions besides Systemin sequence, here we wanted to assess if this precursor includes peptide motifs able to trigger stress-related pathways. Candidate peptides were identified in silico and synthesized to test their capacity to trigger defence responses in tomato plants against different biotic stressors. Our results demonstrated that ProSys harbours several repeat motifs which triggered plant immune reactions against pathogens and pest insects. Three of these peptides were detected by mass spectrometry in plants expressing ProSys, demonstrating their effective presence in vivo. These experimental data shed light on unrecognized functions of ProSys, mediated by multiple biologically active sequences which may partly account for the capacity of ProSys to induce defense responses to different stress agents.

Biochemical, structural, and computational studies of a γ-carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei.

Melioidosis is a severe disease caused by the highly pathogenic gram-negative bacterium Burkholderia pseudomallei. Several studies have highlighted the broad resistance of this pathogen to many antibiotics and pointed out the pivotal importance of improving the pharmacological arsenal against it. Since γ-carbonic anhydrases (γ-CAs) have been recently introduced as potential and novel antibacterial drug targets, in this paper, we report a detailed characterization of BpsγCA, a γ-CA from B. pseudomallei by a multidisciplinary approach. In particular, the enzyme was recombinantly produced and biochemically characterized. Its catalytic activity at different pH values was measured, the crystal structure was determined and theoretical pKa calculations were carried out. Results provided a snapshot of the enzyme active site and dissected the role of residues involved in the catalytic mechanism and ligand recognition. These findings are an important starting point for developing new anti-melioidosis drugs targeting BpsγCA.

Tomato Prosystemin Is Much More than a Simple Systemin Precursor.

Systemin (Sys) is an octadecapeptide, which upon wounding, is released from the carboxy terminus of its precursor, Prosystemin (ProSys), to promote plant defenses. Recent findings on the disordered structure of ProSys prompted us to investigate a putative biological role of the whole precursor deprived of the Sys peptide. We produced transgenic tomato plants expressing a truncated ProSys gene in which the exon coding for Sys was removed and compared their defense response with that induced by the exogenous application of the recombinant truncated ProSys (ProSys(1-178), the Prosystemin sequence devoid of Sys region). By combining protein structure analyses, transcriptomic analysis, gene expression profiling and bioassays with different pests, we demonstrate that truncated ProSys promotes defense barriers in tomato plants through a hormone-independent defense pathway, likely associated with the production of oligogalacturonides (OGs). Both transgenic and plants treated with the recombinant protein showed the modulation of the expression of genes linked with defense responses and resulted in protection against the lepidopteran pest Spodoptera littoralis and the fungus Botrytis cinerea. Our results suggest that the overall function of the wild-type ProSys is more complex than previously shown, as it might activate at least two tomato defense pathways: the well-known Sys-dependent pathway connected with the induction of jasmonic acid biosynthesis and the successive activation of a set of defense-related genes, and the ProSys(1-178)-dependent pathway associated with OGs production leading to the OGs mediate plant immunity.

Sultam based Carbonic Anhydrase VII inhibitors for the management of neuropathic pain.

We report a series of compounds 1-17 derived from the antiepileptic drug Sulthiame (SLT) from which both the benzenesulfonamide and the sultam moiety were retained. All compounds were tested in vitro for their inhibition activity against the human (h) Carbonic Anhydrase (CA; EC 4.2.1.1) I, II, VII, IX and XII isoforms. Among the series, derivatives 1 and 11 showed great enhancement of both inhibition potency and selectivity towards the hCA VII isoform, when compared to the reference SLT drug. The binding mode of 11 within the hCA VII active site was deciphered by means of X-ray crystallography and revealed the sultam moiety being exposed to the rim of the active site. In vivo experiments on a model of neuropathic pain induced by oxaliplatin clearly showed 11 being an effective pain relieving agent and therefore worth of further exploitation towards the validation of the hCA VII as new target for the management of neuropathies.

Not Only Systemin: Prosystemin Harbors Other Active Regions Able to Protect Tomato Plants.

Prosystemin is a 200-amino acid precursor expressed in Solanaceae plants which releases at the C-terminal part a peptidic hormone called Systemin in response to wounding and herbivore attack. We recently showed that Prosystemin is not only a mere scaffold of Systemin but, even when deprived of Systemin, is biologically active. These results, combined with recent discoveries that Prosystemin is an intrinsically disordered protein containing disordered regions within its sequence, prompted us to investigate the N-terminal portions of the precursor, which contribute to the greatest disorder within the sequence. To this aim, PS1-70 and PS1-120 were designed, produced, and structurally and functionally characterized. Both the fragments, which maintained their intrinsic disorder, were able to induce defense-related genes and to protect tomato plants against Botrytis cinerea and Spodoptera littoralis larvae. Intriguingly, the biological activity of each of the two N-terminal fragments and of Systemin is similar but not quite the same and does not show any toxicity on experimental non-targets considered. These regions account for different anti-stress activities conferred to tomato plants by their overexpression. The two N-terminal fragments identified in this study may represent new promising tools for sustainable crop protection.

Zeta-carbonic anhydrases show CS2 hydrolase activity: A new metabolic carbon acquisition pathway in diatoms?

CDCA1 is a very peculiar member of the Carbonic Anhydrase (CA) family. It has been the first enzyme to show an efficient utilization of Cd(II) ions in Nature and a unique adaptation capability to live on the surface ocean. Indeed, in this environment, which is extremely depleted in essential metal ions, CDCA1 can utilize Zn(II) or Cd(II) as catalytic metal to support the metabolic needs of fast growing diatoms. In this paper we demonstrate a further catalytic versatility of this enzyme by using a combination of X-ray crystallography, molecular dynamics simulations and enzymatic experiments. First we identified the CO2 binding site and the way in which this substrate travels from the environment to the enzyme active site. Then, starting from the observation of a structural similarity with the substrate entry route of CS2 hydrolase from Acidanius A1-3, we hypothesized and demonstrated that also CS2 is a substrate for CDCA1. This finding is new and unexpected since until now only few CS2 hydrolases have been characterized, and none of them is reported to have any CO2 hydratase action. The physiological implications of this supplementary catalytic activity still remain to be unveiled. We suggest here that it could represent another ability of diatoms expressing CDCA1 to adapt to the external environment. Indeed, the ability of this enzyme to convert CS2 could represent an alternative source of carbon acquisition for diatoms, in addition to CO2.