Functional characterization of Fur from the strict anaerobe Clostridioides difficile provides insight into its redox-driven regulatory capacity.

Clostridioides (formerly Clostridium) difficile is a leading cause of infectious diarrhea associated with antibiotic therapy. The ability of this anaerobic pathogen to acquire enough iron to proliferate under iron limitation conditions imposed by the host largely determines its pathogenicity. However, since high intracellular iron catalyzes formation of deleterious reactive hydroxyl radicals, iron uptake is tightly regulated at the transcriptional level by the ferric uptake regulator Fur. Several studies relate lacking a functional fur gene in C. difficile cells to higher oxidative stress sensitivity, colonization defect and less toxigenicity, although Fur does not appear to directly regulate either oxidative stress response genes or pathogenesis genes. In this work, we report the functional characterization of C. difficile Fur and describe an additional oxidation sensing Fur-mediated mechanism independent of iron, which affects Fur DNA-binding. Using electrophoretic mobility shift assays, we show that Fur binding to the promoters of fur, feoA and fldX genes, identified as iron and Fur-regulated genes in vivo, is specific and does not require co-regulator metal under reducing conditions. Fur treatment with H2O2 produces dose-dependent soluble high molecular weight species unable to bind to target promoters. Moreover, Fur oligomers are dithiotreitol sensitive, highlighting the importance of some interchain disulfide bond(s) for Fur oligomerization, and hence for activity. Additionally, the physiological electron transport chain NADPH-thioredoxin reductase/thioredoxin from Escherichia coli reduces inactive oligomerized C. difficile Fur that recovers activity. In conjunction with available transcriptomic data, these results suggest a previously underappreciated complexity in the control of some members of the Fur regulon that is based on Fur redox properties and might be fundamental for the adaptive response of C. difficile during infection.

Global Lrp regulator protein from Haloferax mediterranei: Transcriptional analysis and structural characterization.

Haloferax mediterranei, an extreme halophilic archaeon thriving in hypersaline environments, has acquired significant attention in biotechnological and biochemical research due to its remarkable ability to flourish in extreme salinity conditions. Transcription factors, essential in regulating diverse cellular processes, have become focal points in understanding its adaptability. This study delves into the role of the Lrp transcription factor, exploring its modulation of glnA, nasABC, and lrp gene promoters in vivo through ß-galactosidase assays. Remarkably, our findings propose Lrp as the pioneering transcriptional regulator of nitrogen metabolism identified in a haloarchaeon. This study suggests its potential role in activating or repressing assimilatory pathway enzymes (GlnA and NasA). The interaction between Lrp and these promoters is analyzed using Electrophoretic Mobility Shift Assay and Differential Scanning Fluorimetry, highlighting l-glutamine's indispensable role in stabilizing the Lrp-DNA complex. Our research uncovers that halophilic Lrp forms octameric structures in the presence of l-glutamine. The study reveals the three-dimensional structure of the Lrp as a homodimer using X-ray crystallography, confirming this state in solution by Small-Angle X-ray Scattering. These findings illuminate the complex molecular mechanisms driving Hfx. mediterranei's nitrogen metabolism, offering valuable insights about its gene expression regulation and enriching our comprehension of extremophile biology.

Expanding the FurC (PerR) regulon in Anabaena (Nostoc) sp. PCC 7120: Genome-wide identification of novel direct targets uncovers FurC participation in central carbon metabolism regulation.

FurC (PerR, Peroxide Response Regulator) from Anabaena sp. PCC 7120 (also known as Nostoc sp. PCC 7120) is a master regulator engaged in the modulation of relevant processes including the response to oxidative stress, photosynthesis and nitrogen fixation. Previous differential gene expression analysis of a furC-overexpressing strain (EB2770FurC) allowed the inference of a putative FurC DNA-binding consensus sequence. In the present work, more data concerning the regulon of the FurC protein were obtained through the searching of the putative FurC-box in the whole Anabaena sp. PCC 7120 genome. The total amount of novel FurC-DNA binding sites found in the promoter regions of genes with known function was validated by electrophoretic mobility shift assays (EMSA) identifying 22 new FurC targets. Some of these identified targets display relevant roles in nitrogen fixation (hetR and hgdC) and carbon assimilation processes (cmpR, glgP1 and opcA), suggesting that FurC could be an additional player for the harmonization of carbon and nitrogen metabolisms. Moreover, differential gene expression of a selection of newly identified FurC targets was measured by Real Time RT-PCR in the furC-overexpressing strain (EB2770FurC) comparing to Anabaena sp. PCC 7120 revealing that in most of these cases FurC could act as a transcriptional activator.

Responses of Anabaena sp. PCC7120 to lindane: Physiological effects and differential expression of potential lin genes.

Lindane (γ-HCH) is an organochlorine pesticide that causes huge environmental concerns worldwide due to its recalcitrance and toxicity. The use of the cyanobacterium Anabaena sp. PCC 7120 in aquatic lindane bioremediation has been suggested but information relative to this process is scarce. In the present work, data relative to the growth, pigment composition, photosynthetic/respiration rate, and oxidative stress response of Anabaena sp. PCC 7120 in the presence of lindane at its solubility limit in water are shown. In addition, lindane degradation experiments revealed almost a total disappearance of lindane in the supernatants of Anabaena sp. PCC 7120 culture after 6 days of incubation. The diminishing in lindane concentration was in concordance with an increase in the levels of trichlorobenzene inside the cells. Furthermore, to identify potential orthologs of the linA, linB, linC, linD, linE, and linR genes from Sphingomonas paucimobilis B90A in Anabaena sp. PCC 7120, a whole genome screening was performed allowing the identification of five putative lin orthologs (all1353 and all0193 putative orthologs of linB, all3836 putative orthologs of linC, and all0352 and alr0353 putative orthologs of linE and linR, respectively) which could be involved in the lindane degradation pathway. Differential expression analysis of these genes in the presence of lindane revealed strong upregulation of one of the potential lin genes of Anabaena sp. PCC 7120.

Unbalancing Zur (FurB)-mediated homeostasis in Anabaena sp. PCC7120: Consequences on metal trafficking, heterocyst development and biofilm formation.

Zinc is required for the activity of many enzymes and plays an essential role in gene regulation and redox homeostasis. In Anabaena (Nostoc) sp. PCC7120, the genes involved in zinc uptake and transport are controlled by the metalloregulator Zur (FurB). Comparative transcriptomics of a zur mutant (Δzur) with the parent strain unveiled unexpected links between zinc homeostasis and other metabolic pathways. A notable increase in the transcription of numerous desiccation tolerance-related genes, including genes involved in the synthesis of trehalose and the transference of saccharide moieties, among many others, was detected. Biofilm formation analysis under static conditions revealed a reduced capacity of Δzur filaments to form biofilms compared to the parent strain, and such capacity was enhanced when Zur was overexpressed. Furthermore, microscopy analysis revealed that zur expression is required for the correct formation of the envelope polysaccharide layer in the heterocyst, as Δzur cells showed reduced staining with alcian blue compared to Anabaena sp. PCC7120. We suggest that Zur is an important regulator of the enzymes involved in the synthesis and transport of the envelope polysaccharide layer, influencing heterocyst development and biofilm formation, both relevant processes for cell division and interaction with substrates in its ecological niche.

FurC (PerR) from Anabaena sp. PCC7120: a versatile transcriptional regulator engaged in the regulatory network of heterocyst development and nitrogen fixation.

FurC (PerR) from Anabaena sp. PCC7120 was previously described as a key transcriptional regulator involved in setting off the oxidative stress response. In the last years, the cross-talk between oxidative stress, iron homeostasis and nitrogen metabolism is becoming more and more evident. In this work, the transcriptome of a furC-overexpressing strain was compared with that of a wild-type strain under both standard and nitrogen-deficiency conditions. The results showed that the overexpression of furC deregulates genes involved in several categories standing out photosynthesis, iron transport and nitrogen metabolism. The novel FurC-direct targets included some regulatory elements that control heterocyst development (hetZ and asr1734), genes directly involved in the heterocyst envelope formation (devBCA and hepC) and genes which participate in the nitrogen fixation process (nifHDK and nifH2, rbrA rubrerythrin and xisHI excisionase). Likewise, furC overexpression notably impacts the mRNA levels of patA encoding a key protein in the heterocyst pattern formation. The relevance of FurC in these processes is bringing out by the fact that the overexpression of furC impairs heterocyst development and cell growth under nitrogen step-down conditions. In summary, this work reveals a new player in the complex regulatory network of heterocyst formation and nitrogen fixation.

Thioredoxin Dependent Changes in the Redox States of FurA from Anabaena sp. PCC 7120.

FurA is a multifunctional regulator in cyanobacteria that contains five cysteines, four of them arranged into two CXXC motifs. Lack of a structural zinc ion enables FurA to develop disulfide reductase activity. In vivo, FurA displays several redox isoforms, and the oxidation state of its cysteines determines its activity as regulator and its ability to bind different metabolites. Because of the relationship between FurA and the control of genes involved in oxidative stress defense and photosynthetic metabolism, we sought to investigate the role of type m thioredoxin TrxA as a potential redox partner mediating dithiol-disulfide exchange reactions necessary to facilitate the interaction of FurA with its different ligands. Both in vitro cross-linking assays and in vivo two-hybrid studies confirmed the interaction between FurA and TrxA. Light to dark transitions resulted in reversible oxidation of a fraction of the regulator present in Anabaena sp. PCC7120. Reconstitution of an electron transport chain using E. coli NADPH-thioredoxin-reductase followed by alkylation of FurA reduced cysteines evidenced the ability of TrxA to reduce FurA. Furthermore, the use of site-directed mutants allowed us to propose a plausible mechanism for FurA reduction. These results point to TrxA as one of the redox partners that modulates FurA performance.

2-oxoglutarate modulates the affinity of FurA for the ntcA promoter in Anabaena sp. PCC 7120.

2-oxoglutarate (2-OG) is a central metabolite that acts as a signaling molecule informing about the status of the carbon/nitrogen balance of the cell. In recent years, some transcriptional regulators and even two-component systems have been described as 2-OG sensors. In the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7120, two master regulators, NtcA and FurA, are deeply involved in the regulation of nitrogen metabolism. Both of them show a complex intertwined regulatory circuit to achieve a suitable regulation of nitrogen fixation. In this work, 2-OG is found to bind FurA, modulating the specific binding of FurA to the ntcA promoter. This study provides evidence of a new additional control point in the complex network controlled by the NtcA and FurA proteins.

Agentes dopaminérgicos centrales análogos N-aralquil metoxilados del 2-aminoindano / Central dopaminergic agents analogues of N-aralkyl methoxylated of 2-aminoindane

RESUMEN La neurotransmisión dopaminérgica interviene en los mecanismos que involucran los procesos motores, cognoscitivos, conductuales y neurocrinos y su mal funcionamiento la involucra en los trastornos neurodegenerativos que afectan al sistema nervioso central (SNC), tales como en la enfermedad de Parkinson y la enfermedad de Huntington, entre otras. Con el propósito de encontrar una solución terapéutica a estas patologías, en publicaciones anteriores hemos reportado la síntesis, la evaluación farmacológica y el estudio teórico computacional de los compuestos análogos mono y dihidroxilados (sobre el anillo indano) del N-aralquil-2-aminoindano 4-8, análogos 4,7-dimetoxi-2-aminoindano-N-aralquil, bajo sus formas metoxiladas sobre el anillo bencénico del fragmento aralquil 9 y el derivado fenólico 10, así como también los análogos diclorados del N-aralquil-2-aminoindano 11 con actividades dopaminérgicas centrales. En el presente trabajo se sintetizaron los clorhidratos del 2-aminoindano- N-[2-(mono o dimetoxi)-fenil)-1-metil-etil] 12-15 y su evaluación farmacológica mostraron respuestas agonísticas como potenciales agentes antihuntington y antipárkinson.

SUMMARY Dopaminergic neurotransmission is implicated in mechanisms that involve motor, cognoscitive, conductual and neurocrine process, and its malfunction involucrates it in neurodegenerative disorders affecting central nervous system (CNS), like Parkinson's disease and Huntington's disease, among others. On the purpose of finding some therapeutic for these pathologies, in previous researches we have reported synthesis, pharmacological evaluation and theoretical computational study of compounds analogues mono or di hydroxilated (on indane ring) of N-aralkyl-2-aminoindane 4-8, analogues 4,7-dimethoxy-2-aminoindane-N-aralkyl, under its methoxylated forms on benzene ring of aralkyl fragment 9 and phenolic derivate 10, also dichlorade analogs of N-aralkyl-2-aminoindane 11 with central dopaminergic activities. In this work were synthesized hydrochlorides of 2-aminoindane-N-[(mono or di methoxy)-phenyl-1-methyl-ethyl] (12-15) and its pharmacologic evaluation showed agonistic responses as potential agents anti Huntington and/or anti Parkinson.

Novedoso profármaco derivado de la lilolidina con actividad dopaminérgica central / Novel prodrug derivative from lilolidine with central dopaminergic activity

La desregulación de la neurotransmisión dopaminérgica central ha sido relacionada con enfermedades neurodegenerativas, tales como la enfermedad de Parkinson y la Corea de Huntington. En las últimas décadas son muchos los compuestos con actividad dopaminérgica central que se han diseñado, sintetizado y evaluado farmacológicamente y, a pesar de estos esfuerzos, no se ha logrado obtener un fármaco efectivo capaz de mejorar o curar estas patologías. Con el fin de contribuir en esta búsqueda primordial, en el presente trabajo se describe el diseño, la síntesis y la evaluación farmacológica del derivado de la lilolidina, clorhidrato de 6-(2-aminoindanil)-N-(2,4,5,6-tetrahidro-1H-pirrolo[3,2,1-ij]quinolina) 4 (MAIL), con el propósito de restablecer la homeostasis de la transmisión dopaminérgica en la enfermedad de Parkinson y/o la Corea de Huntington. Para ello, se utilizaron las diferentes estrategias nigroesclásicas y heterocíclicas enmarcadas en la síntesis orgánica a través de una ruta convergente. Asimismo, se realizó la evaluación farmacológica preliminar, al determinar el comportamiento estereotipado en ratas Sprague-Dawley cuando les fue administrado el derivado de la lilolidina, por las vías ICV (intracerebroventricular) e IE (intraestriatal). Los resultados obtenidos del compuesto 4 mostraron una acción central como agonista, a través de mecanismos dopaminérgicos.

Central dopaminergic neurotransmission deregulation has been related with neurodegenerative sicknesses, like Parkinson’s disease and Huntington’s Chorea. During the last decades, many compounds with dopaminergic activity have been designed, synthesized and pharmacologically evaluated, but despite all these efforts, an effective drug able to improve or cure these pathologies has not been achieved. With the purpose to contribute in this essential search, this work describes the design, synthesis and pharmacological evaluation of the lilolidine derivative, 6-(2-aminoindanyl)-N-(2,4,5,6-tetrahydro-1H-pyrrolo[3,2,1-ij]quinoline hydrochloride) 4 (MAIL), with the purpose of restoring the homeostasis of dopaminergic transmission in Parkinson’s disease and/or Huntington’s Chorea. To perform that, different organic synthesis classic and heterocyclic strategies were employed through a convergent route. Also, a preliminar pharmacological evaluation was done, where the stereotyped behavior of Sprague-Dawley rats was studied after the ICV (intracerebroventricular) and IE (intrastriatum) administration of the lilodine derivative. The results obtained of compound 4 showed a central dopaminergic agonist activity through dopaminergic mechanisms.

[Novel central dopaminergic agents derived from atypical di-substituted 2-aminoindane-4, 7. Synthesis and central pharmacological profile]. / Novedosos agentes dopaminérgicos centrales derivados del 2-aminoindano-4,7 disustituido atipico. Síntesis y perfil farmacológico central.

In recent decades, many compounds with central dopaminergic activity have been designed, synthesized and evaluated pharmacologically. However, it has not been possible to obtain a drug able to improve or cure diseases involving dopaminergic regulation in the central nervous system, such as Parkinson's disease and schizophrenia, among others. Taking into consideration the term "atypical pharmacophore" and from the compound 5, the aralkyl fragment was incorporated, and the compounds 10, 11, 13a-h and 14a-h were synthesized. Both the compounds 10 and 13a-h under its methoxylated form and the compounds 11 and 14a-h under the phenolic form, were evaluated to determine their pharmacologically agonistic and antagonistic effects on central dopaminergic activity. For this, the effect of intracerebroventricular injection of said compounds on the hydromineral balance and stereotyped behavior in rats, was determined. The results of the preliminary pharmacological evaluation show a centrally acting action through dopamine mechanisms, in which the compounds 10, 11, 13d-h and 14a showed responses as agonists, whereas compounds 14b-h, had responses as antagonists.

Synthesis, dopaminergic profile, and molecular dynamics calculations of N-aralkyl substituted 2-aminoindans.

Brain dopaminergic system has a crucial role in the etiology of several neuropsychiatric disorders, including Parkinson's disease, depression, and schizophrenia. Several dopaminergic drugs are used to treat these pathologies, but many problems are attributed to these therapies. Within this context, the search for new more efficient dopaminergic agents with less adverse effects represents a vast research field. The aim of the present study was to synthesize N-[2-(4,5-dihydroxyphenyl)-methyl-ethyl]-4,5-dihydroxy-2-aminoindan hydrobromide (3), planned to be a dopamine ligand, and to evaluate its dopaminergic action profile. This compound was assayed as a diastereoisomeric mixture in two experimental models: stereotyped behavior (gnaw) and renal urinary response, after central administration. The pharmacological results showed that compound 3 significantly blocked the apomorphine-induced stereotypy and dopamine-induced diuresis and natriuresis in rats. Thus, compound 3 demonstrated an inhibitory effect on dopaminergic-induced behavior and renal action. N-[2-(-Methyl-ethyl)]-4,5-dihydroxy-2-aminoindan hydrobromide (4) was previously reported as an inotropic agent, and in the present work it was also re-evaluated as a diastereoisomeric mixture for its possible central action on the behavior parameters such as stereotypy and dopamine-induced diuresis and natriuresis in rats. Our results indicate that compound 4 produces an agonistic response, possibly through dopaminergic mechanisms. To better understand the experimental results we performed molecular dynamics simulations of two complexes: compound 3/D(2)DAR (dopamine receptor) and compound 4/D(2)DAR. The differential binding mode obtained for these complexes could explain the antagonist and agonist activity obtained for compounds 3 and 4, respectively.