Unlocking Precision Docking for Metalloproteins.

Metalloproteins play a fundamental role in molecular biology, contributing to various biological processes. However, the discovery of high-affinity ligands targeting metalloproteins has been delayed due, in part, to a lack of suitable tools and data. Molecular docking, a widely used technique for virtual screening of small-molecule ligand interactions with proteins, often faces challenges when applied to metalloproteins due to the particular nature of the ligand metal bond. To address these limitations associated with docking metalloproteins, we introduce a knowledge-driven docking approach known as "metalloprotein bias docking" (MBD), which extends the AutoDock Bias technique. We assembled a comprehensive data set of metalloprotein-ligand complexes from 15 different metalloprotein families, encompassing Ca, Co, Fe, Mg, Mn, and Zn metal ions. Subsequently, we conducted a performance analysis of our MBD method and compared it to the conventional docking (CD) program AutoDock4, applied to various metalloprotein targets within our data set. Our results demonstrate that MBD outperforms CD, significantly enhancing accuracy, selectivity, and precision in ligand pose prediction. Additionally, we observed a positive correlation between our predicted ligand free energies and the corresponding experimental values. These findings underscore the potential of MBD as a valuable tool for the effective exploration of metalloprotein-ligand interactions.

Exploring human porphobilinogen synthase metalloprotein by quantum biochemistry and evolutionary methods.

Previous studies have shown the porphobilinogen synthase (PBGS) zinc-binding mechanism and its conservation among the living cells. However, the precise molecular interaction of zinc with the active center of the enzyme is unknown. In particular, quantum chemistry techniques within the density functional theory (DFT) framework have been the key methodology to describe metalloproteins, when one is looking for a compromise between accuracy and computational feasibility. Considering this, we used DFT-based models within the molecular fractionation with conjugate caps scheme to evaluate the binding energy features of zinc interacting with the human PBGS. Besides, phylogenetic and clustering analyses were successfully employed in extracting useful information from protein sequences to identify groups of conserved residues that build the ions-binding site. Our results also report a conservative assessment of the relevant amino acids, as well as the benchmark analysis of the calculation models used. The most relevant intermolecular interactions in Zn2+-PBGS are due to the amino acids CYS0122, CYS0124, CYS0132, ASP0169, SER0168, ARG0221, HIS0131, ASP0120, GLY0133, VAL0121, ARG0209, and ARG0174. Among these residues, we highlighted ASP0120, GLY0133, HIS0131, SER0168, and ARG0209 by co-occurring in all clusters generated by unsupervised clustering analysis. On the other hand, the triple cysteines at 2.5 Å from zinc (CYS0122, CYS0124, and CYS0132) have the highest energy attraction and are absent in the taxa Viridiplantae, Sar, Rhodophyta, and some Bacteria. Additionally, the performance of the DFT-based models shows that the processing time-dependence is more associated with the choice of the basis set than the exchange-correlation functional.

Biochemical metal accumulation effects and metalloprotein metal detoxification in environmentally exposed tropical Perna perna mussels.

Marine bivalves have been widely applied as environmental contamination bioindicators, although studies concerning tropical species are less available compared to temperate climate species. Assessments regarding Perna perna mytilid mussels, in particular, are scarce, even though this is an extremely important species in economic terms in tropical countries, such as Brazil. To this end, Perna perna mytilids were sampled from two tropical bays in Southeastern Brazil, one anthropogenically impacted and one previously considered a reference site for metal contamination. Gill metallothionein (MT), reduced glutathione (GSH), carboxylesterase (CarbE) and lipid peroxidation (LPO) were determined by UV-vis spectrophotometry, and metal and metalloid contents were determined by inductively coupled plasma mass spectrometry (ICP-MS). Metalloprotein metal detoxification routes in heat-stable cellular gill fractions were assessed by size exclusion high performance chromatography (SEC-HPLC) coupled to an ICP-MS. Several associations between metals and oxidative stress endpoints were observed at all four sampling sites through a Principal Component Analysis. As, Cd, Ni and Se contents, in particular, seem to directly affect CarbE activity. MT is implicated in playing a dual role in both metal detoxification and radical oxygen species scavenging. Differential SEC-HPLC-ICP-MS metal-binding profiles, and, thus, detoxification mechanisms, were observed, with probable As-, Cu- and Ni-GSH complexation and binding to low molecular weight proteins. Perna perna mussels were proven adequate tropical bioindicators, and further monitoring efforts are recommended, due to lack of data regarding biochemical metal effects in tropical species. Integrated assessments, as performed herein demonstrate, are invaluable in evaluating contaminated aquatic environments, resulting in more accurate ecological risk assessments.

Electronic structure benchmark calculations of CO2 fixing elementary chemical steps in RuBisCO using the projector-based embedding approach.

Ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO) is the main enzyme involved in atmospheric carbon dioxide (CO2 ) fixation in the biosphere. This enzyme catalyzes a set of five chemical steps that take place in the same active-site within magnesium (II) coordination sphere. Here, a set of electronic structure benchmark calculations have been carried out on a reaction path proposed by Gready et al. by means of the projector-based embedding approach. Activation and reaction energies for all main steps catalyzed by RuBisCO have been calculated at the MP2, SCS-MP2, CCSD, and CCSD(T)/aug-cc-pVDZ and cc-pVDZ levels of theory. The treatment of the magnesium cation with post-HF methods is explored to determine the nature of its involvement in the mechanism. With the high-level ab initio values as a reference, we tested the performance of a set of density functional theory (DFT) exchange-correlation (xc) functionals in reproducing the reaction energetics of RuBisCO carboxylase activity on a set of model fragments. Different DFT xc-functionals show large variation in activation and reaction energies. Activation and reaction energies computed at the B3LYP level are close to the reference SCS-MP2 results for carboxylation, hydration and protonation reactions. However, for the carbon-carbon bond dissociation reaction, B3LYP and other functionals give results that differ significantly from the ab initio reference values. The results show the applicability of the projector-based embedding approach to metalloenzymes. This technique removes the uncertainty associated with the selection of different DFT xc-functionals and so can overcome some of inherent limitations of DFT calculations, complementing, and potentially adding to modeling of enzyme reaction mechanisms with DFT methods.

C-H Amination via Nitrene Transfer Catalyzed by Mononuclear Non-Heme Iron-Dependent Enzymes.

Expanding the reaction scope of natural metalloenzymes can provide new opportunities for biocatalysis. Mononuclear non-heme iron-dependent enzymes represent a large class of biological catalysts involved in the biosynthesis of natural products and catabolism of xenobiotics, among other processes. Here, we report that several members of this enzyme family, including Rieske dioxygenases as well as α-ketoglutarate-dependent dioxygenases and halogenases, are able to catalyze the intramolecular C-H amination of a sulfonyl azide substrate, thereby exhibiting a promiscuous nitrene transfer reactivity. One of these enzymes, naphthalene dioxygenase (NDO), was further engineered resulting in several active site variants that function as C-H aminases. Furthermore, this enzyme could be applied to execute this non-native transformation on a gram scale in a bioreactor, thus demonstrating its potential for synthetic applications. These studies highlight the functional versatility of non-heme iron-dependent enzymes and pave the way to their further investigation and development as promising biocatalysts for non-native metal-catalyzed transformations.

Engineered small metal-binding protein tag improves the production of recombinant human growth hormone in the periplasm of Escherichia coli.

Fusion proteins play an important role in the production of recombinant proteins in Escherichia coli. They are mostly used for cytoplasmic expression since they can be designed to increase the solubility of the target protein, which then can be easily purified via affinity chromatography. In contrast, fusion proteins are not usually included in construct designs for periplasmic production. Instead, a signal sequence is inserted for protein transport into the periplasm and a C-terminal his-tag added for subsequent purification. Our research group has proposed the small metal-binding protein (SmbP) isolated from the periplasm of Nitrosomonas europaea as a new fusion protein to express recombinant proteins in the cytoplasm or periplasm of E. coli. SmbP also allows purification via immobilized metal affinity chromatography using Ni(II) ions. Recently, we have optimized the periplasmic production of proteins tagged with SmbP by exchanging its native signal peptide with one taken from pectate lyase B (PelB), substantially increasing the amount of protein produced. In this work, we have expressed and purified soluble bioactive human growth hormone (hGH) tagged with PelB-SmbP and obtained the highest periplasmic production reported for this protein so far. Its activity, tested on Nb2-11 cells, was equivalent to commercial growth hormone at 50 ng·mL-1 . Therefore, we strongly recommend the use of PelB-SmbP as a protein tag for the expression and purification of hGH or other possible target proteins in the periplasm of E. coli.

Assessing electron transfer reactions and catalysis in multicopper oxidases with operando X-ray absorption spectroscopy.

Here we propose an experimental setup based on operando X-ray absorption spectroscopy (XAS) to understand why copper-containing oxidoreductase enzymes show exceptional performance as catalysts for the oxygen reduction reaction (ORR). An electrode based on carbon nanoparticles organized in mesoporous structures with bilirubin oxidase (BOD) was developed to be used in a home-made operando XAS electrochemical cell, and we probed the electron transfer under ORR regime. In the presence of molecular oxygen, the BOD cofactor containing 4 copper ions require an overpotential about 150 mV to be reduced as compared to that in the absence of oxygen. A second electron transfer step, which occurs faster than the cofactor reduction, suggests that the cooper ions act as a tridimensional redox active electronic bridges for the electron transfer reaction.

Protein determinants of dissemination and host specificity of metallo-ß-lactamases.

The worldwide dissemination of metallo-ß-lactamases (MBLs), mediating resistance to carbapenem antibiotics, is a major public health problem. The extent of dissemination of MBLs such as VIM-2, SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here, we report that MBL host range is determined by the impact of MBL expression on bacterial fitness. The signal peptide sequence of MBLs dictates their adaptability to each host. In uncommon hosts, inefficient processing of MBLs leads to accumulation of toxic intermediates that compromises bacterial growth. This fitness cost explains the exclusion of VIM-2 and SPM-1 from Escherichia coli and Acinetobacter baumannii, and their confinement to Pseudomonas aeruginosa. By contrast, NDMs are expressed without any apparent fitness cost in different bacteria, and are secreted into outer membrane vesicles. We propose that the successful dissemination and adaptation of MBLs to different bacterial hosts depend on protein determinants that enable host adaptability and carbapenem resistance.

Identification of Biomarkers of Mercury Contamination in Brachyplatystoma filamentosum of the Madeira River, Brazil, Using Metalloproteomic Strategies.

Predator fish can accumulate high levels of mercury, which qualifies them as potential indicators of this toxic metal. The predatory species Brachyplatystoma filamentosum, popularly known as filhote, is among the most consumed species in the Brazilian Amazon. Continuing the metalloproteomic studies of mercury in Amazonian fishes that have been developed in the last 5 years, the present paper provides the data of protein characterization associated with mercury in muscle and liver samples of filhote (Brachyplatystoma filamentosum) collected in the Madeira River, Brazilian Amazon. The mercury concentration in the muscle and liver samples was determined by graphite furnace atomic absorption spectrometry (GFAAS). The protein fraction was extracted in an aqueous medium, and later, a fractional precipitation procedure was performed to obtain the protein pellets. Then, the proteome of the tissue samples of this fish species was separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and a mercury mapping of the protein spots was carried out by GFAAS after acid digestion. Protein spots that had mercury were characterized by mass spectrometry with electrospray ionization in sequence (ESI-MS/MS) after tryptic digestion. It was possible to characterize 11 mercury-associated protein spots that presented biomarker characteristics and could be used to monitor mercury in fish species of the Amazon region. Thus, the metalloproteomic strategies used in the present study allowed us to characterize 11 mercury-associated protein spots. It should be noted that the protein spots identified as GFRP, TMEM186, TMEM57B, and BHMT, which have coordination sites for elements with characteristics of soft acids, such as mercury, can be used as biomarkers of mercury contamination in monitoring studies of this toxic metal in fish species from the Amazon region.

Metallomics Applied to the Study of Neurodegenerative and Mental Diseases.

Biochemical imbalances, provoked by aging or a secondary illness, might directly affect the brain, causing severe problems, such as loss of memory or alteration of behavior patterns. Brain disorders are usually classified as injuries (such as stroke, hematomas, and concussions), tumors, and neurodegenerative (such as Parkinson's and Alzheimer's diseases) and mental (such as depression, bipolar disorder, schizophrenia) diseases. As the pathophysiology of these illnesses is not completely established and multiple factors are involved, metallomics, a bioanalytical strategy that allows the detection of metal ions and metalloproteins in diverse biological matrices, is of extreme relevance in identifying which elements are affected by a disease and/or treatment. Thus, determining which element ions suffer disturbances in their homeostasis during the disease progress is relevant to understand the biochemical changes and propose new drug targets. In addition, it is well known that oxidative stress plays an important role in the development of pathological neurodegenerative and mental diseases, which may be caused by metal ion dyshomeostasis, so it is also important to understand endogenous antioxidant metalloprotein and metalloenzyme mechanisms in this regard. In this context, recent applications of metallomics in the study of neurodegenerative and mental disorders are discussed in this chapter, as well as future trends in this research area.

Novel method for metalloproteins determination in human breast milk by size exclusion chromatography coupled to inductively coupled plasma mass spectrometry.

Levels of essential metals in human breast milk (HBM) have been determined by different analytical techniques, but there is few woks about human whey milk fractions. However, the current trend lies in metalloproteomic and identification of different metalloproteins. In this sense, native separative techniques (N-PAGE and SEC) coupled to ICP-MS provide us with valuable information. Besides it is necessary the development of new methodologies in order to determine with accuracy and precision the profile of such metals and metalloproteins in the different whey protein fractions of HBM. Thus, the aim of this work was to develop a new method for metals and metalloproteins determination by SEC-ICP-MS in whey protein fractions of HBM. Human whey fractions were obtained of HBM samples by ultracentrifugation. Then, protein fractions of whey milk were separated by SEC coupled to ICP-MS for metalloproteins and Mn, Co, Cu and Se quantification. Besides, protein profile of whey milk was determined by N-PAGE and computer assisted image analysis. SEC-ICP-MS results indicated that first and second protein fractions showed detectable levels of the Mn, Co, Cu, and Se. Protein profile determined by N-PAGE and image analysis showed that molecular weight of protein fractions ranged between 68,878-1,228.277 Da. In this work, metalloproteins were analyzed by SEC coupled to ICP-MS, with adequate sensitivity and accuracy. Our study has shown the presence of Mn, Co, Cu and Se bound to two protein fractions in whey milk of HBM. Metals levels analyzed were within the ranges reported in the literature.

Artificial Metalloproteins Containing Co4O4 Cubane Active Sites.

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII-OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e-/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII-OH2 groups. With this variant, multi-e-/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to thin film water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e-/multi-H+ reactivity.

Haloferax volcanii Proteome Response to Deletion of a Rhomboid Protease Gene.

Rhomboids are conserved intramembrane serine proteases involved in cell signaling processes. Their role in prokaryotes is scarcely known and remains to be investigated in Archaea. We previously constructed a rhomboid homologue deletion mutant (ΔrhoII) in Haloferax volcanii, which showed reduced motility, increased novobiocin sensitivity, and an N- glycosylation defect. To address the impact of rhoII deletion on H. volcanii physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. A total of 1847 proteins were identified (45.8% of H. volcanii predicted proteome), from which 103 differed in amount. Additionally, the mutant strain evidenced 99 proteins with altered electrophoretic migration, which suggested differential post-translational processing/modification. Integral membrane proteins that evidenced variations in concentration, electrophoretic migration, or semitryptic cleavage in the mutant were considered as potential RhoII targets. These included a PrsW protease homologue (which was less stable in the mutant strain), a predicted halocyanin, and six integral membrane proteins potentially related to the mutant glycosylation (S-layer glycoprotein, Agl15) and cell adhesion/motility (flagellin1, HVO_1153, PilA1, and PibD) defects. This study investigated for the first time the impact of a rhomboid protease on the whole proteome of an organism.

Metalloproteomics Approach to Analyze Mercury in Breast Milk and Hair Samples of Lactating Women in Communities of the Amazon Basin, Brazil.

Mercury is a potentially toxic element that is present in the environment of the Brazilian Amazon and is responsible for adverse health effects in humans. This study sought to assess possible protein biomarkers of mercury exposure in breast milk samples from lactating women in the Madeira and Negro Rivers in the Brazilian Amazon. The mercury content of hair samples of lactating women was determined, and the proteome of breast milk samples was obtained using two-dimensional electrophoresis after protein precipitation with acetone. Mercury measurements of protein spots obtained via protein fractionation were performed by graphite furnace atomic absorption spectrometry (GFAAS), and it was observed that mercury is linked to proteins with molecular masses in the range of 14-26 kDa. The total mercury concentration was also determined by GFAAS in unprocessed milk, lyophilized milk, and protein pellets, with the purpose of determining the mercury mass balance in relation to the concentration of this element in milk and pellets. Approximately 85 to 97% of mercury present in the lyophilized milk from samples of lactating women of the Madeira River is bound in the protein fraction. From lactating women of the Negro River, approximately 49% of the total mercury is bound in the protein fraction, and a difference of 51% is bound in the lipid fraction.

CFTR modulates RPS27 gene expression using chloride anion as signaling effector.

In Cystic Fibrosis (CF), the impairment of the CFTR channel activity leads to a variety of alterations, including differential gene expression. However, the CFTR signaling mechanisms remain unclear. Recently, culturing IB3-1 CF cells under different intracellular Cl- concentrations ([Cl-]i), we observed several Cl--dependent genes and further characterized one of them as RPS27. Thus, we hypothesized that Cl- might act as a signaling effector for CFTR signaling. Here, to test this idea, we study RPS27 expression in T84 cells modulating the CFTR activity by using CFTR inhibitors. First, we observed that incubation of T84 cells with increasing concentrations of the CFTR inhibitors CFTR(inh)-172 or GlyH-101 determined a progressive increase in the relative [Cl-]i (using the Cl- fluorescent probe SPQ). The [Cl-]i rise was concomitant with a dose-dependent down-regulation of RPS27. These results imply that CFTR inhibition produce Cl- accumulation and that RPS27 expression can be modulated by CFTR inhibition. Therefore, Cl- behaves as a signaling effector for CFTR in the modulation of RPS27 expression. In addition, the IL-1ß receptor antagonist IL1RN or the JNK inhibitor SP600125, both restored the down-regulation of RPS27 induced by CFTRinh-172, implying a role of autocrine IL-1ß and JNK signaling downstream of Cl- in RPS27 modulation.

Melanotransferrin: New Homolog Genes and Their Differential Expression during Intestinal Regeneration in the Sea Cucumber Holothuria glaberrima.

Melanotransferrin (MTf) is a protein associated with oncogenetic, developmental, and immune processes which function remains unclear. The MTf gene has been reported in numerous vertebrate and invertebrate species, including echinoderms. We now report the finding of four different MTfs in the transcriptome of the sea cucumber Holothuria glaberrima. Sequence studies and phylogenetic analyses were done to ascertain the similarities among the putative proteins and their relationship with other transferrin family members. The genes were shown to be differentially expressed in various holothurian organs and to respond differently when the animals were challenged with the immune system activator lipopolysaccharide (LPS). Moreover, the four genes were found to be highly overexpressed during the early stages of intestinal regeneration. The finding of four different genes in the holothurian is particularly surprising, because only one MTf gene has been reported in all other animal species sequenced to date. This finding, combined with the increase expression during intestinal regeneration, suggests a new possible function of MTf in organ regenerative processes.

Improved QM/MM Linear-Interaction Energy Model for Substrate Recognition in Zinc-Containing Metalloenzymes.

One of the essential challenges in the description of receptor-drug interactions in the presence of various polyvalent cations (such as zinc, magnesium, or iron) is the accurate assessment of the electronic effects due to cofactor binding. The effects can range from partial electronic polarization of the proximal atoms in a receptor and bound substrate to long-range effects related to partial charge transfer and electronic delocalization effects between the cofactor and the drug. Here, we examine the role of the explicit account for electronic effects for a panel of small-molecule inhibitors binding to the zinc-aminopeptidase PfA-M1, an essential target for antimalarial drug development. Our study on PfA-M1:inhibitor interactions at the QM level reveals that the partial charge and proton transfer due to bound zinc ion are important mechanisms in the inhibitors' recognition and catalysis. The combination of classical MD simulations with a posteriori QM/MM corrections with novel DFTB parameters for the zinc cation and the linear-interaction energy (LIE) approach offers by far the most accurate estimates for the PfA-M1:inhibitor binding affinities, opening the door for future inhibitor design.

Zinc as Allosteric Ion Channel Modulator: Ionotropic Receptors as Metalloproteins.

Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators.

Investigation of thermostable metalloproteins in Perna perna mussels from differentially contaminated areas in Southeastern Brazil by bioanalytical techniques.

Metallomic studies regarding environmental contamination by metals are of value in elucidating metal uptake, trafficking, accumulation and metabolism in biological systems. Many proven bioindicator species, such as bivalves, have not yet, however, been well-characterized regarding their metalloprotein expression in response to environmental contaminants. In this context, the aim of the present study was to investigate metalloprotein expressions in the thermostable protein fraction of muscle tissue and digestive glands from mussels (Perna perna) from three differentially metal-contaminated sites in Southeastern Brazil in comparison with a reference site. The thermostable protein fractions were analyzed by SDS-PAGE and SEC-HPLC-ICP-MS. Metal content was also determined in both the crude and the purified extracts. Several inter-organ differences were observed, which is to be expected, while inter-site differences regarding thermostable protein content were also verified, indicating accumulation of these elements in muscle tissue and digestive glands and disruption of homeostasis of essential elements, with detoxification attempts by metal-bound proteins, since all metalloproteins present in both matrices eluted bound to at least one non-essential metal. These results are also noteworthy with regard to the adopted reference site, that also seems to be contaminated by toxic metals.

The Chloride Anion Acts as a Second Messenger in Mammalian Cells - Modifying the Expression of Specific Genes.

BACKGROUND/AIMS: Cystic Fibrosis (CF) is caused by mutations in the CFTR gene, encoding a cAMP-activated chloride (Cl-) channel. We have previously demonstrated that the expression of several genes can be modulated by the CFTR activity; among them, SRC, MTND4, CISD1, and IL1B. However, the CFTR signalling mechanism involved in the expression of CFTR-dependent genes is unknown. The aim of this work was to determine if intracellular chloride (Cl-)i might function as a second messenger modulating the expression of specific genes. METHODS: Differential display (DD) was applied to IB3-1 cells (CF cells), cultured under conditions that produce different intracellular Cl- concentrations ([Cl-]i), to analyse their expression profile. RESULTS: Several differentially expressed gene products were observed by using DD, suggesting the presence of chloride-dependent gene expression. Two cDNA fragments, derived from differentially expressed mRNAs and showing opposed response to Cl-' were isolated, cloned, sequenced and its Cl- dependency validated by reverse transcription quantitative-PCR (RT-qPCR). We identified the gene RPS27, which encodes the multifunctional ribosomal protein RPS27, also known as metallopanstimulin-1 (MPS-1), and the gene GLRX5, encoding glutaredoxin-related protein 5, as chloride-dependent genes. RPS27 was negatively regulated with increased [Cl-]i, approximately from 25-75 mM Cl- (EC50 = 46 ± 7 mM), and positively regulated from 75-125 mM Cl- (EC50 = 110 ± 11 mM) (biphasic response). In contrast, GLRX5 was positively modulated by [Cl-]i, showing a typical sigmoidal dose-response curve from 0-50 mM Cl-, reaching a plateau after 50 mM Cl- (EC50 ∼ 34 mM). CONCLUSION: The results suggest the existence of chloride-dependent genes. The Cl- anion, therefore, might act as a second messenger for channels or receptors able to modulate the intracellular Cl- concentration, regulating in turn the expression of specific genes.