Proximity Ligation Assay for the Analysis of Iron-Mediated Protein-Protein Interactions in the Nucleus.

Iron forms essential cofactors used by many nuclear enzymes involved in genome maintenance. However, unchaperoned nuclear iron may represent a threat to the surrounding genetic material as it promotes redox toxicity that may affect DNA integrity. Safely handling intracellular iron implies metal transfer and cofactor assembly processes based on protein-protein interactions. Identifying those interactions commonly occurs via high-throughput approaches using affinity purification or proximity labeling coupled with mass spectrometry analysis. However, these methods do not identify the subcellular location of the interactions. The one-on-one confirmation of proposed nuclear interactions is also challenging. Many approaches used to look at protein interactions are not tailored for looking at the nucleus because the methods used to solubilize nuclear content are harsh enough to disrupt those transient interactions. Here, we describe step-by-step the use of Proximity Ligation Assay (PLA) to analyze iron-mediated protein-protein interactions in the nucleus of cultured human cells. PLA allows the subcellular visualization of the interactions via the in situ detection of the two interacting proteins using fluorescence confocal microscopy. Briefly, cells are fixed, blocked, permeabilized, and incubated with primary antibodies directed to target proteins. Primary antibodies are recognized using PLA probes consisting of one PLUS and one MINUS oligonucleotide-labeled secondary antibody. If the two proteins are close enough (<40 nm), the PLA probes are ligated and used as the template for rolling circle amplification (RCA) with fluorescently labeled oligonucleotides that yield a signal detectable using fluorescence confocal microscopy. A fluorescently labeled membrane-specific stain (WGA) and the DNA-specific probe DAPI are used to identify cellular and nuclear boundaries, respectively. Confocal images are then analyzed using the CellProfiler software to confirm the abundance and localization of the studied protein-protein interactions.

Cysteine Rich Intestinal Protein 2 is a copper-responsive regulator of skeletal muscle differentiation.

Copper (Cu) is an essential trace element required for respiration, neurotransmitter synthesis, oxidative stress response, and transcriptional regulation. Imbalance in Cu homeostasis can lead to several pathological conditions, affecting neuronal, cognitive, and muscular development. Mechanistically, Cu and Cu-binding proteins (Cu-BPs) have an important but underappreciated role in transcription regulation in mammalian cells. In this context, our lab investigates the contributions of novel Cu-BPs in skeletal muscle differentiation using murine primary myoblasts. Through an unbiased synchrotron X-ray fluorescence-mass spectrometry (XRF/MS) metalloproteomic approach, we identified the murine cysteine rich intestinal protein 2 (mCrip2) in a sample that showed enriched Cu signal, which was isolated from differentiating primary myoblasts derived from mouse satellite cells. Immunolocalization analyses showed that mCrip2 is abundant in both nuclear and cytosolic fractions. Thus, we hypothesized that mCrip2 might have differential roles depending on its cellular localization in the skeletal muscle lineage. mCrip2 is a LIM-family protein with 4 conserved Zn2+-binding sites. Homology and phylogenetic analyses showed that mammalian Crip2 possesses histidine residues near two of the Zn2+-binding sites (CX2C-HX2C) which are potentially implicated in Cu+-binding and competition with Zn2+. Biochemical characterization of recombinant human hsCRIP2 revealed a high Cu+-binding affinity for two and four Cu+ ions and limited redox potential. Functional characterization using CRISPR/Cas9-mediated deletion of mCrip2 in primary myoblasts did not impact proliferation, but impaired myogenesis by decreasing the expression of differentiation markers, possibly attributed to Cu accumulation. Transcriptome analyses of proliferating and differentiating mCrip2 KO myoblasts showed alterations in mRNA processing, protein translation, ribosome synthesis, and chromatin organization. CUT&RUN analyses showed that mCrip2 associates with a select set of gene promoters, including MyoD1 and metallothioneins, acting as a novel Cu-responsive or Cu-regulating protein. Our work demonstrates novel regulatory functions of mCrip2 that mediate skeletal muscle differentiation, presenting new features of the Cu-network in myoblasts.

Iron-tracking strategies: Chaperones capture iron in the cytosolic labile iron pool.

Cells express hundreds of iron-dependent enzymes that rely on the iron cofactors heme, iron-sulfur clusters, and mono-or di-nuclear iron centers for activity. Cells require systems for both the assembly and the distribution of iron cofactors to their cognate enzymes. Proteins involved in the binding and trafficking of iron ions in the cytosol, called cytosolic iron chaperones, have been identified and characterized in mammalian cells. The first identified iron chaperone, poly C-binding protein 1 (PCBP1), has also been studied in mice using genetic models of conditional deletion in tissues specialized for iron handling. Studies of iron trafficking in mouse tissues have necessitated the development of new approaches, which have revealed new roles for PCBP1 in the management of cytosolic iron. These approaches can be applied to investigate use of other nutrient metals in mammals.

Unique underlying principles shaping copper homeostasis networks.

Copper is essential in cells as a cofactor for key redox enzymes. Bacteria have acquired molecular components that sense, uptake, distribute, and expel copper ensuring that cuproenzymes are metallated and steady-state metal levels are maintained. Toward preventing deleterious reactions, proteins bind copper ions with high affinities and transfer the metal via ligand exchange, warranting that copper ions are always complexed. Consequently, the directional copper distribution within cell compartments and across cell membranes requires specific dynamic interactions and metal exchange between cognate holo-apo protein partners. These metal exchange reactions are determined by thermodynamic and kinetics parameters and influenced by mass action. Then, copper distribution can be conceptualized as a molecular system of singular interacting elements that maintain a physiological copper homeostasis. This review focuses on the impact of copper high-affinity binding and exchange reactions on the homeostatic mechanisms, the conceptual models to describe the cell as a homeostatic system, the various molecule functions that contribute to copper homeostasis, and the alternative system architectures responsible for copper homeostasis in model bacteria.

The Two-Component System CopRS Maintains Subfemtomolar Levels of Free Copper in the Periplasm of Pseudomonas aeruginosa Using a Phosphatase-Based Mechanism.

Two-component systems control periplasmic Cu+ homeostasis in Gram-negative bacteria. In characterized systems such as Escherichia coli CusRS, upon Cu+ binding to the periplasmic sensing region of CusS, a cytoplasmic phosphotransfer domain of the sensor phosphorylates the response regulator CusR. This drives the expression of efflux transporters, chaperones, and redox enzymes to ameliorate metal toxic effects. Here, we show that the Pseudomonas aeruginosa two-component sensor histidine kinase CopS exhibits a Cu-dependent phosphatase activity that maintains CopR in a nonphosphorylated state when the periplasmic Cu levels are below the activation threshold of CopS. Upon Cu+ binding to the sensor, the phosphatase activity is blocked and the phosphorylated CopR activates transcription of the CopRS regulon. Supporting the model, mutagenesis experiments revealed that the ΔcopS strain exhibits maximal expression of the CopRS regulon, lower intracellular Cu+ levels, and increased Cu tolerance compared to wild-type cells. The invariant phosphoacceptor residue His235 of CopS was not required for the phosphatase activity itself but was necessary for its Cu dependency. To sense the metal, the periplasmic domain of CopS binds two Cu+ ions at its dimeric interface. Homology modeling of CopS based on CusS structure (four Ag+ binding sites) clearly supports the different binding stoichiometries in the two systems. Interestingly, CopS binds Cu+/2+ with 3 × 10-14 M affinity, pointing to the absence of free (hydrated) Cu+/2+ in the periplasm.IMPORTANCE Copper is a micronutrient required as cofactor in redox enzymes. When free, copper is toxic, mismetallating proteins and generating damaging free radicals. Consequently, copper overload is a strategy that eukaryotic cells use to combat pathogens. Bacteria have developed copper-sensing transcription factors to control copper homeostasis. The cell envelope is the first compartment that has to cope with copper stress. Dedicated two-component systems control the periplasmic response to metal overload. This paper shows that the sensor kinase of the copper-sensing two-component system present in Pseudomonadales exhibits a signal-dependent phosphatase activity controlling the activation of its cognate response regulator, distinct from previously described periplasmic Cu sensors. Importantly, the data show that the system is activated by copper levels compatible with the absence of free copper in the cell periplasm. These observations emphasize the diversity of molecular mechanisms that have evolved in bacteria to manage the copper cellular distribution.

The Medicago truncatula Yellow Stripe1-Like3 gene is involved in vascular delivery of transition metals to root nodules.

Symbiotic nitrogen fixation carried out in legume root nodules requires transition metals. These nutrients are delivered by the host plant to the endosymbiotic nitrogen-fixing bacteria living within the nodule cells, a process in which vascular transport is essential. As members of the Yellow Stripe-Like (YSL) family of metal transporters are involved in root to shoot transport, they should also be required for root to nodule metal delivery. The genome of the model legume Medicago truncatula encodes eight YSL proteins, four of them with a high degree of similarity to Arabidopsis thaliana YSLs involved in long-distance metal trafficking. Among them, MtYSL3 is a plasma membrane protein expressed by vascular cells in roots and nodules and by cortical nodule cells. Reducing the expression level of this gene had no major effect on plant physiology when assimilable nitrogen was provided in the nutrient solution. However, nodule functioning was severely impaired, with a significant reduction of nitrogen fixation capabilities. Further, iron and zinc accumulation and distribution changed. Iron was retained in the apical region of the nodule, while zinc became strongly accumulated in the nodule veins in the ysl3 mutant. These data suggest a role for MtYSL3 in vascular delivery of iron and zinc to symbiotic nitrogen fixation.

The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells.

Among the 12 P-type ATPases encoded by the genome of Mycobacterium tuberculosis (Mtb), CtpF responds to the greatest number of stress conditions, including oxidative stress, hypoxia, and infection. CtpF is the mycobacterial homolog of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) of higher eukaryotes. Its expression is regulated by the global regulator of latency, DosR. However, the role that CtpF plays in the mycobacterial plasma membrane remains unknown. In this study, different functional analyses showed that CtpF is associated with calcium pumping from mycobacterial cells. Specifically, Mtb CtpF expression in Mycobacterium smegmatis cells prevents Ca2+ accumulation compared with wild type (WT) cells. In addition, plasma membrane vesicles from recombinant membranes, in which the direction of ion transport is inverted, accumulate more Ca2+ compared with vesicles obtained from the WT strain. This findings support the hypothesis that CtpF contributes to calcium efflux from mycobacterial cells. Accordingly, Mtb cells defective in ctpF (MtbΔctpF) accumulate more Ca2+ compared with WT cells, while the Ca2+-dependent ATPase activity is significantly lower in the mutant cells. Interestingly, the deletion of ctpF in Mtb impairs the tolerance of the bacteria to oxidative and nitrosative stress. Overall, our results indicate that CtpF is associated with calcium pumping from mycobacterial cells and the response to oxidative stress.

The interplay of the metallosensor CueR with two distinct CopZ chaperones defines copper homeostasis in Pseudomonas aeruginosa.

Copper homeostasis in pathogenic bacteria is critical for cuproprotein assembly and virulence. However, in vivo biochemical analyses of these processes are challenging, which has prevented defining and quantifying the homeostatic interplay between Cu+-sensing transcriptional regulators, chaperones, and sequestering molecules. The cytoplasm of Pseudomonas aeruginosa contains a Cu+-sensing transcriptional regulator, CueR, and two homologous metal chaperones, CopZ1 and CopZ2, forming a unique system for studying Cu+ homeostasis. We found here that both chaperones exchange Cu+, albeit at a slow rate, reaching equilibrium after 3 h, a time much longer than P. aeruginosa duplication time. Therefore, they appeared as two separate cellular Cu+ pools. Although both chaperones transferred Cu+ to CueR in vitro, experiments in vivo indicated that CopZ1 metallates CueR, eliciting the translation of Cu+ efflux transporters involved in metal tolerance. Although this observation was consistent with the relative Cu+ affinities of the three proteins (CopZ1 < CueR < CopZ2), in vitro and in silico analyses also indicated a stronger interaction between CopZ1 and CueR that was independent of Cu+ In contrast, CopZ2 function was defined by its distinctly high abundance during Cu2+ stress. Under resting conditions, CopZ2 remained largely in its apo form. Metal stress quickly induced CopZ2 expression, and its holo form predominated, reaching levels commensurate with the cytoplasmic Cu+ levels. In summary, these results show that CopZ1 acts as chaperone delivering Cu+ to the CueR sensor, whereas CopZ2 functions as a fast-response Cu+-sequestering storage protein. We propose that equivalent proteins likely play similar roles in most bacterial systems.

Nicotianamine Synthase 2 Is Required for Symbiotic Nitrogen Fixation in Medicago truncatula Nodules.

Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.

Copper homeostasis networks in the bacterium Pseudomonas aeruginosa.

Bacterial copper (Cu+) homeostasis enables both precise metallation of diverse cuproproteins and control of variable metal levels. To this end, protein networks mobilize Cu+ to cellular targets with remarkable specificity. However, the understanding of these processes is rather fragmented. Here, we use genome-wide transcriptomic analysis by RNA-Seq to characterize the response of Pseudomonas aeruginosa to external 0.5 mm CuSO4, a condition that did not generate pleiotropic effects. Pre-steady-state (5-min) and steady-state (2-h) Cu+ fluxes resulted in distinct transcriptome landscapes. Cells quickly responded to Cu2+ stress by slowing down metabolism. This was restored once steady state was reached. Specific Cu+ homeostasis genes were strongly regulated in both conditions. Our system-wide analysis revealed induction of three Cu+ efflux systems (a P1B-ATPase, a porin, and a resistance-nodulation-division (RND) system) and of a putative Cu+-binding periplasmic chaperone and the unusual presence of two cytoplasmic CopZ proteins. Both CopZ chaperones could bind Cu+ with high affinity. Importantly, novel transmembrane transporters probably mediating Cu+ influx were among those largely repressed upon Cu+ stress. Compartmental Cu+ levels appear independently controlled; the cytoplasmic Cu+ sensor CueR controls cytoplasmic chaperones and plasma membrane transporters, whereas CopR/S responds to periplasmic Cu+ Analysis of ΔcopR and ΔcueR mutant strains revealed a CopR regulon composed of genes involved in periplasmic Cu+ homeostasis and its putative DNA recognition sequence. In conclusion, our study establishes a system-wide model of a network of sensors/regulators, soluble chaperones, and influx/efflux transporters that control the Cu+ levels in P. aeruginosa compartments.

Pma1 is an alkali/alkaline earth metal cation ATPase that preferentially transports Na(+) and K(+) across the Mycobacterium smegmatis plasma membrane.

Mycobacterium smegmatis Pma1 is the orthologue of M. tuberculosis P-type ATPase cation transporter CtpF, which is activated under stress conditions, such as hypoxia, starvation and response to antituberculous and toxic substances. The function of Pma1 in the mycobacterial processes across the plasma membrane has not been characterised. In this work, bioinformatic analyses revealed that Pma1 likely contains potential sites for, Na(+), K(+) and Ca(2+) binding and transport. Accordingly, RT-qPCR experiments showed that M. smegmatis pma1 transcription is stimulated by sub-lethal doses of Na(+), K(+) and Ca(2+); in addition, the ATPase activity of plasma membrane vesicles in recombinant Pma1-expressing M. smegmatis cells is stimulated by treatment with these cations. In contrast, M. smegmatis cells homologously expressing Pma1 displayed tolerance to high doses of Na(+) and K(+) but not to Ca(2+) ions. Consistently, the recombinant protein Km embedded in plasma membrane demonstrated that Ca(2+) has more affinity for Pma1 than Na(+) and K(+) ions; furthermore, the estimation of Vmax/Km suggests that Na(+) and K(+) ions are more efficiently translocated than Ca(2+). Thus, these results strongly suggest that Pma1 is a promiscuous alkali/alkaline earth cation ATPase that preferentially transports Na(+) and/or K(+) across the mycobacterial plasma membrane.

CtpA, a putative Mycobacterium tuberculosis P-type ATPase, is stimulated by copper (I) in the mycobacterial plasma membrane.

The transport of heavy-metal ions across the plasma membrane is essential for mycobacterial intracellular survival; in this context, P-type ATPases are pivotal for maintenance of ionic gradients and the plasma membrane homeostasis of mycobacteria. To date, the copper ion transport that is mediated by P-type ATPases in mycobacteria is poorly understood. In this work, the ion-specific activation of CtpA, a putative plasma membrane Mycobacterium tuberculosis P-type ATPase, with different heavy-metal cations was assessed. Mycobacterium smegmatis mc(2)155 cells heterologously expressing the M. tuberculosis ctpA gene displayed an increased tolerance to toxic levels of the Cu(2+) ion (4 mM) compared to control cells, suggesting that CtpA is possibly involved in the copper detoxification of mycobacterial cells. In contrast, the tolerance of M. smegmatis recombinant cells against other heavy-metal divalent cations, such as Co(2+), Mn(2+), Ni(2+) and Zn(2+), was not detected. In addition, the ATPase activity of plasma membrane vesicles that were obtained from M. smegmatis cells expressing CtpA was stimulated by Cu(+) (4.9 nmol of Pi released/mg of protein.min) but not by Cu(2+) ions; therefore, Cu(2+) reduction to Cu(+) inside mycobacterial cells is suggested. Finally, the plasma membrane vesicles of M. smegmatis that were enriched with CtpA exhibited an optimal activity at 37 °C and pH 7.9; the apparent kinetic parameters of the enzyme were a K(1/2) of 4.68 × 10(-2) µM for Cu(+), a Vmax of 10.3 U/mg of protein, and an h value of 1.91.

Mycobacterium tuberculosis P-type ATPases: possible targets for drug or vaccine development.

Tuberculosis (TB) has been the biggest killer in the human history; currently, Mycobacterium tuberculosis (Mtb) kills nearly 2 million people each year worldwide. The high prevalence of TB obligates the identification of new therapeutic targets and the development of anti-TB vaccines that can control multidrug resistance and latent TB infections. Membrane proteins have recently been suggested as key targets for bacterial viability. Current studies have shown that mycobacteria P-type ATPases may play critical roles in ion homeostasis and in the response of mycobacteria to toxic substances in the intraphagosomal environment. In this review, we bring together the genomic, transcriptomic, and structural aspects of the P-type ATPases that are relevant during active and latent Mtb infections, which can be useful in determining the potential of these ATPases as drug targets and in uncovering their possible roles in the development of new anti-TB attenuated vaccines.

The DosR dormancy regulator of Mycobacterium tuberculosis stimulates the Na(+)/K (+) and Ca (2+) ATPase activities in plasma membrane vesicles of mycobacteria.

The latency global regulator DosR regulon of Mycobacterium tuberculosis, which is stimulated by hypoxia, comprises approximately fifty genes including ctpF (Rv1997), which encodes a putative alkali/alkaline earth ion transporter of the plasma membrane. In this work, the influence of hypoxia and M. tuberculosis DosR on the ATPase activity of mycobacterial plasma membrane was assessed. We performed bioinformatic analyses which indicated that the pma1 gene product is the M. smegmatis ortholog of the M. tuberculosis cation transporter CtpF. In addition, a possible Na(+), K(+) and/or Ca(2+) pumping mediated by Pma1 was also predicted. Enzymatic analyses indicated that the basal ATPase activity of plasma membrane vesicles from M. smegmatis cells cultured under hypoxia and over-expressing DosR, decreased 30 and 40 % respectively in comparison to oxygenated cells. In contrast, the specific Na(+)/K(+) and Ca(2+) ATPase activities of the plasma membrane increased 2.8- and 3.5-fold, respectively, under hypoxia, similar to that observed for cells over-expressing the DosR regulator. In agreement, RT-qPCR experiments demonstrated that the transcription level of the pma1 gene increased under hypoxia at levels similar to that of M. smegmatis cells over-expressing the M. tuberculosis DosR regulator. The entire findings suggest that hypoxia stimulates Na(+)/K(+) and Ca(2+) ATPase activities in the mycobacterial plasma membrane, and this is possibly mediated by the dormancy regulator DosR.

In silico identification and characterization of the ion transport specificity for P-type ATPases in the Mycobacterium tuberculosis complex.

BACKGROUND: P-type ATPases hydrolyze ATP and release energy that is used in the transport of ions against electrochemical gradients across plasma membranes, making these proteins essential for cell viability. Currently, the distribution and function of these ion transporters in mycobacteria are poorly understood. RESULTS: In this study, probabilistic profiles were constructed based on hidden Markov models to identify and classify P-type ATPases in the Mycobacterium tuberculosis complex (MTBC) according to the type of ion transported across the plasma membrane. Topology, hydrophobicity profiles and conserved motifs were analyzed to correlate amino acid sequences of P-type ATPases and ion transport specificity. Twelve candidate P-type ATPases annotated in the M. tuberculosis H37Rv proteome were identified in all members of the MTBC, and probabilistic profiles classified them into one of the following three groups: heavy metal cation transporters, alkaline and alkaline earth metal cation transporters, and the beta subunit of a prokaryotic potassium pump. Interestingly, counterparts of the non-catalytic beta subunits of Hydrogen/Potassium and Sodium/Potassium P-type ATPases were not found. CONCLUSIONS: The high content of heavy metal transporters found in the MTBC suggests that they could play an important role in the ability of M. tuberculosis to survive inside macrophages, where tubercle bacilli face high levels of toxic metals. Finally, the results obtained in this work provide a starting point for experimental studies that may elucidate the ion specificity of the MTBC P-type ATPases and their role in mycobacterial infections.