| 1. |
- Martínez-Carranza, Markel, et al.
(författare)
-
A ribonucleotide reductase from Clostridium botulinum reveals distinct evolutionary pathways to regulation via the overall activity site
- 2020
-
Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 295:46, s. 15576-15587
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is a central enzyme for the synthesis of DNA building blocks. Most aerobic organisms, including nearly all eukaryotes, have class I RNRs consisting of R1 and R2 subunits. The catalytic R1 subunit contains an overall activity site that can allosterically turn the enzyme on or off by the binding of ATP or dATP, respectively. The mechanism behind the ability to turn the enzyme off via the R1 subunit involves the formation of different types of R1 oligomers in most studied species and R1–R2 octamers in Escherichia coli. To better understand the distribution of different oligomerization mechanisms, we characterized the enzyme from Clostridium botulinum, which belongs to a subclass of class I RNRs not studied before. The recombinantly expressed enzyme was analyzed by size-exclusion chromatography, gas-phase electrophoretic mobility macromolecular analysis, EM, X-ray crystallography, and enzyme assays. Interestingly, it shares the ability of the E. coli RNR to form inhibited R1–R2 octamers in the presence of dATP but, unlike the E. coli enzyme, cannot be turned off by combinations of ATP and dGTP/dTTP. A phylogenetic analysis of class I RNRs suggests that activity regulation is not ancestral but was gained after the first subclasses diverged and that RNR subclasses with inhibition mechanisms involving R1 oligomerization belong to a clade separated from the two subclasses forming R1–R2 octamers. These results give further insight into activity regulation in class I RNRs as an evolutionarily dynamic process.
|
|
| 2. |
- Baldassarri, Cecilia, et al.
(författare)
-
Antitrypanosomal Activity of Anthriscus Nemorosa Essential Oils and Combinations of Their Main Constituents
- 2021
-
Ingår i: Antibiotics. - : MDPI. - 2079-6382. ; 10:11
-
Tidskriftsartikel (refereegranskat)abstract
- This study aimed to investigate the susceptibility of Trypanosoma brucei to the Anthriscus nemorosa essential oils (EOs), isolated compounds from these oils, and artificial mixtures of the isolated compounds in their conventional and nanoencapsulated forms. The chemical composition of the essential oils from the aerial parts and roots of Anthriscus nemorosa, obtained from a wild population growing in central Italy, were analyzed by gas chromatography/mass spectrometry (GC/MS). In both cases, the predominant class of compounds was monoterpene hydrocarbons, which were more abundant in the EOs from the roots (81.5%) than the aerial parts (74.0%). The overall results of this work have shed light on the biological properties of A. nemorosa EO from aerial parts (EC50 = 1.17 μg/mL), farnesene (EC50 = 0.84 μg/mL), and artificial mixtures (Mix 3–5, EC50 in the range of 1.27 to 1.58 μg/mL) as relevant sources of antiprotozoal substances. Furthermore, the pool measurements of ADP (adenosine diphosphate) and NTPs (nucleoside triphosphates) in the cultivated bloodstream form of trypanosomes exposed to different concentrations of EOs showed a disturbed energy metabolism, as indicated by increased pools of ADP in comparison to ATP (adenosine triphosphate) and other NTPs. Ultimately, this study highlights the significant efficacy of A. nemorosa EO to develop long-lasting and effective antiprotozoal formulations, including nanoemulsions.
|
|
| 3. |
- Debar, L., et al.
(författare)
-
NUDT6 and NUDT9, two mitochondrial members of the NUDIX family, have distinct hydrolysis activities
- 2023
-
Ingår i: Mitochondrion. - : Elsevier. - 1567-7249 .- 1872-8278. ; 71, s. 93-103
-
Tidskriftsartikel (refereegranskat)abstract
- The 22 members of the NUDIX (NUcleoside DIphosphate linked to another moiety, X) hydrolase superfamily can hydrolyze a variety of phosphorylated molecules including (d)NTPs and their oxidized forms, nucleotide sugars, capped mRNAs and dinucleotide coenzymes such as NADH and FADH. Beside this broad range of enzymatic substrates, the NUDIX proteins can also be found in different cellular compartments, mainly in the nucleus and the cytosol, but also in the peroxisome and in the mitochondria. Here we studied two members of the family, NUDT6 and NUDT9. We showed that NUDT6 is expressed in human cells and localizes exclusively to mito-chondria and we confirmed that NUDT9 has a mitochondrial localization. To elucidate their potential role within this organelle, we investigated the functional consequences at the mitochondrial level of NUDT6-and NUDT9-deficiency and found that the depletion of either of the two proteins results in an increased activity of the res-piratory chain and an alteration of the mitochondrial respiratory chain complexes expression. We demonstrated that NUDT6 and NUDT9 have distinct substrate specificity in vitro, which is dependent on the cofactor used. They can both hydrolyze a large range of low molecular weight compounds such as NAD+(H), FAD and ADPR, but NUDT6 is mainly active towards NADH, while NUDT9 displays a higher activity towards ADPR.
|
|
| 4. |
- Ebenwaldner, Carmen, et al.
(författare)
-
14-3-3 activated bacterial exotoxins AexT and ExoT share actin and the SH2 domains of CRK proteins as targets for ADP-ribosylation
- 2022
-
Ingår i: Pathogens. - : MDPI. - 2076-0817. ; 11:12
-
Tidskriftsartikel (refereegranskat)abstract
- Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only Aeromonas salmonicida exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins. Structural modeling of AexT indicated a target binding site that shared homology with Pseudomonas aeruginosa Exoenzyme T (ExoT) but not with Exoenzyme S (ExoS). Biochemical analyses confirmed that the catalytic activities of both exotoxins were stimulated by agmatine, indicating that they ADP-ribosylate arginine residues in their targets. Side-by-side comparison of target protein modification showed that AexT had activity toward the SH2 domain of the Crk-like protein (CRKL), a known target for ExoT. We found that both AexT and ExoT ADP-ribosylated actin and in both cases, the modification compromised actin polymerization. Our results indicate that AexT and ExoT are functional homologs that affect cytoskeletal integrity via actin and signaling pathways to the cytoskeleton.
|
|
| 5. |
- Fietze, Tobias, et al.
(författare)
-
HUG Domain Is Responsible for Active Dimer Stabilization in an NrdJd Ribonucleotide Reductase
- 2022
-
Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 61:15, s. 1633-1641
-
Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to the corresponding deoxyribonucleotides. The catalytic activity of most RNRs depends on the formation of a dimer of the catalytic subunits. The active site is located at the interface, and part of the substrate binding site and regulatory mechanisms work across the subunit in the dimer. In this study, we describe and characterize a novel domain responsible for forming the catalytic dimer in several class II RNRs. The 3D structure of the class II RNR from Rhodobacter sphaeroides reveals a so far undescribed α-helical domain in the dimer interface, which is embracing the other subunit. Genetic removal of this HUG domain leads to a severe reduction of activity paired with reduced dimerization capability. In comparison with other described RNRs, the enzyme with this domain is less dependent on the presence of nucleotides to act as allosteric effectors in the formation of dimers. The HUG domain appears to serve as an interlock to keep the dimer intact and functional even at low enzyme and/or effector concentrations.
|
|
| 6. |
- Frezza, Claudio, et al.
(författare)
-
Phytochemical Analysis and Trypanocidal Activity of Marrubium incanum Desr.
- 2020
-
Ingår i: Molecules. - : MDPI. - 1431-5157 .- 1420-3049. ; 25:14
-
Tidskriftsartikel (refereegranskat)abstract
- The rationale inspiring the discovery of lead compounds for the treatment of human parasitic protozoan diseases from natural sources is the well-established use of medicinal plants in various systems of traditional medicine. On this basis, we decided to select an overlooked medicinal plant growing in central Italy, Marrubium incanum Desr. (Lamiaceae), which has been used as a traditional remedy against protozoan diseases, and to investigate its potential against Human African trypanosomiasis (HAT). For this purpose, we assayed three extracts of different polarities obtained from the aerial parts of M. incanum—namely, water (MarrInc-H2O), ethanol (MarrInc-EtOH) and dichloromethane (MarrInc-CH2Cl2)—against Trypanosoma brucei (TC221), with the aim to discover lead compounds for the development of antitrypanosomal drugs. Their selectivity index (SI) was determined on mammalian cells (BALB/3T3 mouse fibroblasts) as a counter-screen for toxicity. The preliminary screening selected the MarrInc-CH2Cl2 extract as the most promising candidate against HAT, showing an IC50 value of 28 μg/mL. On this basis, column chromatography coupled with the NMR spectroscopy of a MarrInc-CH2Cl2 extract led to the isolation and identification of five compounds i.e. 1-α-linolenoyl-2-palmitoyl-3-stearoyl-sn- glycerol (1), 1-linoleoyl-2-palmitoyl-3-stearoyl-sn-glycerol (2), stigmasterol (3), palmitic acid (4), and salvigenin (5). Notably, compounds 3 and 5 were tested on T. brucei, with the latter being five-fold more active than the MarrInc-CH2Cl2 extract (IC50 = 5.41 ± 0.85 and 28 ± 1.4 μg/mL, respectively). Furthermore, the SI for salvigenin was >18.5, showing a preferential effect on target cells compared with the dichloromethane extract (>3.6). Conversely, stigmasterol was found to be inactive. To complete the work, also the more polar MarrInc-EtOH extract was analyzed, giving evidence for the presence of 2″-O-allopyranosyl-cosmosiin (6), verbascoside (7), and samioside (8). Our findings shed light on the phytochemistry of this overlooked species and its antiprotozoal potential, providing evidence for the promising role of flavonoids such as salvigenin for the treatment of protozoal diseases.
|
|
| 7. |
- Hofer, Anders
(författare)
-
Targeting the nucleotide metabolism of Trypanosoma brucei and other trypanosomatids
- 2023
-
Ingår i: FEMS Microbiology Reviews. - : Oxford University Press. - 0168-6445 .- 1574-6976. ; 47:3
-
Forskningsöversikt (refereegranskat)abstract
- African sleeping sickness, Chagas disease, and leishmaniasis are life-threatening diseases that together affect millions of people around the world and are caused by different members of the protozoan family Trypanosomatidae. The most studied member of the family is Trypanosoma brucei, which is spread by tsetse flies and causes African sleeping sickness. Nucleotide metabolism in T. brucei and other trypanosomatids is significantly different from that of mammals and was recognized as a target for chemotherapy already in the 1970–1980s. A more thorough investigation of the nucleotide metabolism in recent years has paved the way for identifying nucleoside analogues that can cure T. brucei brain infections in animal models. Specific features of T. brucei nucleotide metabolism include the lack of de novo purine biosynthesis, the presence of very efficient purine transporters, the lack of salvage pathways for CTP synthesis, unique enzyme localizations, and a recently discovered novel pathway for dTTP synthesis. This review describes the nucleotide metabolism of T. brucei, highlights differences and similarities to other trypanosomatids, and discusses how to exploit the parasite-specific features for drug development.
|
|
| 8. |
- Krakovka, Sascha, et al.
(författare)
-
Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery
- 2022
-
Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 298:6
-
Tidskriftsartikel (refereegranskat)abstract
- Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis–infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.
|
|
| 9. |
- Marchetti, Fabio, et al.
(författare)
-
Zinc(II) Complex with Pyrazolone-Based Hydrazones is Strongly Effective against Trypanosoma brucei Which Causes African Sleeping Sickness
- 2022
-
Ingår i: Inorganic Chemistry. - : American Chemical Society (ACS). - 0020-1669 .- 1520-510X. ; 61:34, s. 13561-13575
-
Tidskriftsartikel (refereegranskat)abstract
- Two pyrazolone-based hydrazones H2L′ [in general, H2L′ in detail, H2L1 = 5-methyl-2-phenyl-4-(2-phenyl-1-(2-(4-(trifluoromethyl)phenyl)hydrazineyl)ethyl)-2,4-dihydro-3H-pyrazol-3-one, H2L2 = (Z)-5-methyl-2-phenyl-4-(2-phenyl-1-(2-(pyridin-2-yl)hydrazineyl)ethylidene)-2,4-dihydro-3H-pyrazol-3-one] were reacted with Zn(II) and Cu(II) acceptors affording the complexes [Zn(HL1)2(MeOH)2], [Cu(HL1)2], and [M(HL2)2] (M = Cu or Zn). X-ray and DFT studies showed the free proligands to exist in the N-H,N-H tautomeric form and that in [Zn(HL1)2(MeOH)2], zinc is six-coordinated by the N,O-chelated (HL1) ligand and other two oxygen atoms of coordinated methanol molecules, while [Cu(HL1)2] adopts a square planar geometry with the two (HL1) ligands in anti-conformation. Finally, the [M(HL2)2] complexes are octahedral with the two (HL2) ligands acting as κ-O,N,N-donors in planar conformation. Both the proligands and metal complexes were tested against the parasite Trypanosoma brucei and Balb3T3 cells. The Zn(II) complexes were found to be very powerful, more than the starting proligands, while maintaining a good safety level. In detail, H2L1 and its Zn(II) complex have high selective index (55 and >100, respectively) against T. brucei compared to the mammalian Balb/3T3 reference cells. These results encouraged the researchers to investigate the mechanism of action of these compounds that have no structural relations with the already known drugs used against T. brucei. Interestingly, the analysis of NTP and dNTP pools in T. brucei treated by H2L1 and its Zn(II) complex showed that the drugs had a strong impact on the CTP pools, making it likely that CTP synthetase is the targeted enzyme.
|
|
| 10. |
- Németh, Brigitta, et al.
(författare)
-
The maturase HydF enables [FeFe] hydrogenase assembly via transient, cofactor-dependent interactions
- 2020
-
Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 295:33, s. 11891-11901
-
Tidskriftsartikel (refereegranskat)abstract
- [FeFe] hydrogenases have attracted extensive attention in the field of renewable energy research because of their remarkable efficiency for H(2)gas production. H(2)formation is catalyzed by a biologically unique hexanuclear iron cofactor denoted the H-cluster. The assembly of this cofactor requires a dedicated maturation machinery including HydF, a multidomain [4Fe4S] cluster protein with GTPase activity. HydF is responsible for harboring and delivering a precatalyst to the apo-hydrogenase, but the details of this process are not well understood. Here, we utilize gas-phase electrophoretic macromolecule analysis to show that a HydF dimer forms a transient interaction complex with the hydrogenase and that the formation of this complex depends on the cofactor content on HydF. Moreover, Fourier transform infrared, electron paramagnetic resonance, and UV-visible spectroscopy studies of mutants of HydF show that the isolated iron-sulfur cluster domain retains the capacity for binding the precatalyst in a reversible fashion and is capable of activating apo-hydrogenase inin vitroassays. These results demonstrate the central role of the iron-sulfur cluster domain of HydF in the final stages of H-cluster assembly,i.e.in binding and delivering the precatalyst.
|
|
