Adenosine Triphosphate Hydrolysis Research Articles

Advances in the study of the role of high-frequency mutant subunits of the SWI/SNF complex in tumors.

SWI/SNF (Switch/Sucrose non-fermentable, switch/sucrose non-fermentable) chromatin remodeling complex is a macromolecular complex composed of multiple subunits. It can use the energy generated by the hydrolysis of ATP (Adenosine triphosphate) to destroy the connection between DNA and histones, achieve the breakdown of nucleosomes, and regulate gene expression. SWI/SNF complex is essential for cell proliferation and differentiation, and the abnormal function of its subunits is closely related to tumorigenesis. Among them, ARID1A, an essential non-catalytic subunit of the SWI/SNF complex, can regulate the targeting of the complex through DNA or protein interactions. Moreover, the abnormal function of ARID1A significantly reduces the targeting of SWI/SNF complex to genes and participates in critical intracellular activities such as gene transcription and DNA synthesis. As a catalytic subunit of the SWI/SNF complex, SMARCA4 has ATPase activity that catalyzes the hydrolysis of ATP to produce energy and power the chromatin remodeling complex, which is critical to the function of the SWI/SNF complex. The study data indicate that approximately 25% of cancers have one or more SWI/SNF subunit genetic abnormalities, and at least nine different SWI/SNF subunits have been identified as having repeated mutations multiple times in various cancers, suggesting that mutations affecting SWI/SNF subunits may introduce vulnerabilities to these cancers. Here, we review the mechanism of action of ARID1A and SMARCA4, the two subunits with the highest mutation frequency in the SWI/SNF complex, and the research progress of their targeted therapy in tumors to provide a new direction for precise targeted therapy of clinical tumors.

Role of ENPP1 in cancer pathogenesis: Mechanisms and clinical implications (Review).

Cancer is a significant societal, public health and economic challenge in the 21st century, and is the primary cause of death from disease globally. Ectonucleotide pyrophosphatase/phosphodiesterase (ENPP) serves a crucial role in several biochemical processes, including adenosine triphosphate hydrolysis, purine metabolism and regulation of signaling pathways. Specifically, ENPP1, a type II transmembrane glycoprotein and key member of the ENPP family, may be upregulated in tumor cells and implicated in the pathogenesis of multiple human cancers. The present review provides an overview of the structural, pathological and physiological roles of ENPP1 and discusses the potential mechanisms of ENPP1 in the development of cancers such as breast, colon, gallbladder, liver and lung cancers, and also summarizes the four major signaling pathways in tumors. Furthermore, the present review demonstrates that ENPP1 serves a crucial role in cell migration, proliferation and invasion, and that corresponding inhibitors have been developed and associated with clinical characterization.

KIF18A inhibition: the next big player in the search for cancer therapeutics.

Kinesin-like protein 18A (KIF18A) is a member of the kinesin family of molecular motor proteins, which utilise energy from the hydrolysis of adenosine triphosphate (ATP) to regulate critical cellular processes such as chromosome movement and microtubule dynamics. KIF18A plays a vital role in controlling microtubule length, which is crucial for maintaining proper cell function and division. Notably, increased expression levels of KIF18A have been observed in various types of cancer, indicating its potential involvement in tumour progression. Although preclinical studies have demonstrated that KIF18A is not essential for normal somatic cell division, it appears to be crucial for the survival and division of cancer cells, particularly those exhibiting chromosomal instability. This dependency makes KIF18A a promising target for developing new therapeutic strategies aimed at treating chromosomally unstable cancers. This review delves into the structural and functional aspects of KIF18A, and its role in cancer development, and evaluates current and emerging approaches to targeting KIF18A with innovative cancer treatments.

Enhancing Molecular‐Level Biological Monitoring with a Smart Self‐Assembling 19F‐Labeled Probe

AbstractReal‐time monitoring of molecular transformations is crucial for advancements in biotechnology. In this study, we introduce a novel self‐assembling 19F‐labeled nuclear magnetic resonance (NMR) probe that disassembles upon interaction with various nucleotides. This interaction not only activates the 19F signals but also produces distinct signatures for each specific component, thereby enabling precise identification and quantification of molecules in evolving samples. We demonstrate the capability of this probe for real‐time monitoring of adenosine triphosphate (ATP) hydrolysis and screening potential enzyme inhibitors. These applications highlight the probe's significant potential in enzyme analysis, drug development, and disease diagnostics.

Enhancing Molecular-Level Biological Monitoring with a Smart Self-Assembling 19F-Labeled Probe.

Real-time monitoring of molecular transformations is crucial for advancements in biotechnology. In this study, we introduce a novel self-assembling 19F-labeled nuclear magnetic resonance (NMR) probe that disassembles upon interaction with various nucleotides. This interaction not only activates the 19F signals but also produces distinct signatures for each specific component, thereby enabling precise identification and quantification of molecules in evolving samples. We demonstrate the capability of this probe for real-time monitoring of adenosine triphosphate (ATP) hydrolysis and screening potential enzyme inhibitors. These applications highlight the probe's significant potential in enzyme analysis, drug development, and disease diagnostics.

Quantifying the reversibility of ATP hydrolysis in rabbit skeletal myosin subfragment 1 using a luciferase assay

BackgroundIt is known that the adenosine triphosphate (ATP) hydrolysis in a number of ATPases is reversible. Radioactive methods are standard in measuring ATPase reversibility. We used myosin as a well-studied model enzyme to develop a luciferase-based assay to quantify the reversibility of ATP hydrolysis. In myosin, ATP bound to the active site is hydrolyzed into adenosine diphosphate (ADP) and inorganic phosphate (Pi) and recombined back into ATP multiple times before products of hydrolysis are released. The reversibility of ATP hydrolysis by skeletal myosin was previously confirmed using radioactively labeled isotopes. Transient kinetics studies indicated that the ATP hydrolysis step is temperature-sensitive, with a dissociation equilibrium constant of 1.6 at 3 °C. Consequently, the association equilibrium constant at this temperature is 0.63. The goal of our work was two-fold, (a) to develop a luciferase-based assay to measure the equilibrium constant of enzymatic ATP hydrolysis, eliminating the need for radioactively labeled Pi, and (b) refine the value of the association equilibrium constant of the myosin ATPase hydrolysis step.MethodsIn this assay, a reaction mixture containing myosin and saturating levels of ADP and Pi was incubated to reach equilibrium, then the reaction was terminated, and the amount of ATP produced by myosin was quantified using the luciferase assay. The equilibrium constant of ATP hydrolysis was defined as the ratio of ATP to ADP bound to myosin.ResultsWe obtained a value of 0.78 ± 0.14 for the association equilibrium constant of ATP hydrolysis at 0 °C. 50 μM ADP bound to myosin is turned into 21.9 ± 3.0 μM ATP. Our result is in excellent agreement with the literature data, supporting the viability of the new methodology.DiscussionThis methodology allows a more accessible and safe option to measure ATP production and reversibility of ATPase enzymes. Standard laboratory equipment is utilized in the assay, and the number of steps in the developed assay is reduced significantly compared to the radioactive method.

Membrane potential stimulates ADP import and ATP export by the mitochondrial ADP/ATP carrier due to its positively charged binding site.

The mitochondrial adenosine 5'-diphosphate (ADP)/adenosine 5'-triphosphate (ATP) carrier imports ADP into the mitochondrion and exports ATP to the cell. Here, we demonstrate that 3.3 positive charges are translocated with the negatively charged substrate in each transport step. They can be assigned to three positively charged residues of the central substrate-binding site and two asparagine/arginine pairs. In this way, the membrane potential stimulates not only the ATP4- export step, as a net -0.7 charge is transported, but also the ADP3- import step, as a net +0.3 charge is transported with the electric field. These positive charge movements also inhibit the import of ATP and export of ADP in the presence of a membrane potential, allowing these nucleotides to be maintained at high concentrations in the cytosol and mitochondrial matrix to drive the hydrolysis and synthesis of ATP, respectively. Thus, this is the mechanism by which the membrane potential drives adenine nucleotide exchange with high directional fluxes to fuel the cellular processes.

Nucleotide-mediated modulation of chemoselective protein functionalization in a liquid-like condensed phase

Liquid-like protein condensates are ubiquitous in cellular system and are increasingly recognized for their roles in physiological processes. Condensed phase harbors distinctive chemical microenvironment, markedly different than dilute aqueous phase. Herein, we demonstrate chemoselective modification pattern of nucleophilic canonical amino acid sidechains (namely – cysteine, tyrosine and lysine) of the protein towards 4-chloro-7-nitrobenzofurazan in the dilute and condensed phase. We also delineate how the effect of nucleotides and their in situ enzymatic dissociation temporally modulate the protein condensate’s pH and the protein’s corresponding chemoselective modification. We have shown that the pH of the condensate decreases in the presence of nucleoside triphosphate, whereas it increases in the presence of nucleoside monophosphates or phosphate ion. For instance, we find lysine-specific modification gets inhibited in the presence of adenosine triphosphate (ATP), but significantly enhanced in the presence of monophosphates. This feature enables us to gain temporal control over dynamic change in protein functionalization via enzymatic ATP hydrolysis. Overall, this work substantiates the alteration in pH-responsiveness of Brønsted basicity of a protein’s ε-amine in the condensed phase. Furthermore, this environment sensitivity in chemoselective protein functionalization in condensed phase will be important in adaptable protein engineering to the chemical biology of protein phase separation.

Multichannel sensor array based on carbon dots and metal ions system for physiological phosphates sensing

The detection of physiological phosphates is crucial for elucidating metabolic processes and related diagnoses of diseases. Herein, a carbon dots (CDs) modulated multichannel fluorescent sensor array was proposed for detecting and distinguishing physiological phosphates. In this sensor, three metal ions (Nd3+, Ag+, and Fe3+) with significantly different binding forces with phosphate ions were selected to trigger fluorescence quenching of three CDs (BCDs, GCDs, and RCDs). Upon adding physiological phosphates, the fluorescence intensity of CDs changed. Depending on the discrepancy in the content of phosphate units in different physiological phosphates, the sensor array realized the detection and differentiation of five physiological phosphates related to adenosine triphosphate (ATP) hydrolysis, and has good anti-interference ability, which can realize the differentiation of physiological phosphates in complex serum samples.

Salivary Protein Sfapyrase of Spodoptera frugiperda Stimulates Plant Defence Response.

Plants have developed various resistance mechanisms against herbivorous insects through prolonged coevolution. Plant defence responses can be triggered by specific compounds present in insect saliva. Apyrase, a known enzyme that catalyzes the hydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) into adenosine monophosphate (AMP) and inorganic phosphorus, has recently been identified in some herbivorous insects. However, whether insect salivary apyrase induces or inhibits plant responses remains poorly understood. In this study, we identified an apyrase-like protein in the salivary proteome of the fall armyworm, Spodoptera frugiperda, named Sfapyrase. Sfapyrase was primarily expressed in the salivary gland and secreted into plants during insect feeding. Transient expression of Sfapyrase in tobacco and maize enhanced plant resistance and resulted in decreased insect feeding. Knockdown of Sfapyrase through RNA interference led to increased growth and feeding of S. frugiperda. Furthermore, we showed that Sfapyrase activates the jasmonic acid signalling pathway and promotes the synthesis of secondary metabolites, especially benzoxazinoids, thereby enhancing resistance to S. frugiperda. In summary, our findings demonstrated that Sfapyrase acts as a salivary elicitor, inducing maize jasmonic acid defence responses and the production of insect-resistant benzoxazinoids. This study provides valuable insights into plant-insect interactions and offers potential targets for developing innovative insect pest management strategies.

Alternating binding and p97-mediated dissociation of SDS22 and I3 recycles active PP1 between holophosphatases

Protein phosphatase-1 catalytic subunit (PP1) joins diverse targeting subunits to form holophosphatases that regulate many cellular processes. Newly synthesized PP1 is known to be transiently sequestered in an inhibitory complex with Suppressor-of-Dis2-number-2 (SDS22) and Inhibitor-3 (I3), which is disassembled by the ATPases Associated with diverse cellular Activities plus (AAA+) protein p97. Here, we show that the SDS22-PP1-I3 complex also acts as a thermodynamic sink for mature PP1 and that cycles of SDS22-PP1-I3 formation and p97-driven disassembly regulate PP1 function and subunit exchange beyond PP1 biogenesis. Förster Resonance energy transfer (FRET) analysis of labeled proteins in vitro revealed that in the p97-mediated disassembly step, both SDS22 and I3 dissociate concomitantly, releasing PP1. In presence of a targeting subunit, for instance Growth Arrest and DNA Damage-inducible protein 34 (GADD34), liberated PP1 formed an active holophosphatase that dephosphorylated its substrate, eukaryotic translation initiation factor 2 alpha (eIF2α). Inhibition of p97 results in displacement of the GADD34 targeting subunit by rebinding of PP1 to SDS22 and I3 indicating that the SDS22-PP1-I3 complex is thermodynamically favored. Likewise, p97 inhibition in cells causes rapid sequestration of PP1 by free SDS22 and I3 at the expense of other subunits. This suggests that PP1 exists in a steady state maintained by spontaneous SDS22-PP1-I3 formation and adenosine triphosphate (ATP) hydrolysis, p97-driven disassembly that recycles active PP1 between different holophosphatase complexes to warrant a dynamic holophosphatase landscape.

Statistical mechanics of active vesicles and the size distribution paradox

Vesicles are the primary modes of communication and transport in cell biology. Conventional wisdom based on thermodynamic equilibrium says that vesicles should have a certain minimum size and size distribution dictated by their thermal fluctuations. However, there is compelling experimental evidence that vesicles exhibit a vast variety of size distributions depending on their formation process and function which cannot be explained by equilibrium statistical mechanics alone. We investigate a non-equilibrium statistical mechanics-based model to understand the role of active membranes on the size distribution of vesicles. Active membranes contain proteins that use external energy sources, such as adenosine triphosphate hydrolysis, and are known to exert forces on the membrane during their activity to carry out different biological functions. The central idea behind our model is that activity, attributed to different sources, impacts vesicle fluctuations in two opposing ways — by active noise which enhances fluctuations, and membrane tension which decreases fluctuations. The interplay of active fluctuations and active tension endows the vesicles with the ability to achieve size distributions that are deemed improbable by equilibrium statistical mechanics. We show that our model for active vesicles, based on linearized curvature elasticity, can reproduce different experimental data for vesicle size distributions available in the literature by varying the activity. Elucidating how these vesicles achieve such diverse size distributions can open avenues for a deeper understanding of physiological and pathological processes and help design vesicles for diagnostics and drug delivery applications.

Prophylactic nicotinamide mononucleotide (NMN) mitigates CSDS-induced depressive-like behaviors in mice via preserving of ATP level in the mPFC

Depression is a prevalent psychiatric disorder with accumulating evidence implicating dysregulation of extracellular adenosine triphosphate (ATP) levels in the medial prefrontal cortex (mPFC). It remains unclear whether facilitating endogenous ATP production and subsequently increasing extracellular ATP level in the mPFC can exert a prophylactic effect against chronic social defeat stress (CSDS)-induced depressive-like behaviors and enhance stress resilience. Here, we found that nicotinamide mononucleotide (NMN) treatment effectively elevated nicotinamide adenine dinucleotide (NAD+) biosynthesis and extracellular ATP levels in the mPFC. Moreover, both the 2-week intraperitoneal (i.p.) injection and 3-week oral gavage of NMN prior to exposure to CSDS effectively prevented the development of depressive-like behavior in mice. These protective effects were accompanied with the preservation of both NAD+ biosynthesis and extracellular ATP level in the mPFC. Furthermore, catalyzing ATP hydrolysis by mPFC injection of the ATPase apyrase negated the prophylactic effects of NMN on CSDS-induced depressive-like behaviors. Prophylactic NMN treatment also prevented the reduction in GABAergic inhibition and the increase in excitability in mPFC neurons projecting to the lateral habenula (LHb). Collectively, these findings demonstrate that the prophylactic effects of NMN on depressive-like behaviors are mediated by preventing extracellular ATP loss in the mPFC, which highlights the potential of NMN supplementation as a novel approach for protecting and preventing stress-induced depression in susceptible individuals.

Hydrolysis rate constants of ATP up to 120 °C and 1.6 GPa: Implications for life at extreme conditions

Extreme environments are habitats for a diverse array of microorganisms, namely extremophiles, that have evolved unique biochemical adaptations to their geological setting. Some examples of extremophiles can be found in the subseafloor or near hydrothermal vents on the ocean floor. Cultivated strains of extremophiles have demonstrated the ability to tolerate temperatures (T) up to 122 °C and pressures (P) up to 125 MPa. Organisms depend on the stability of key metabolites, such as adenosine triphosphate (ATP), to survive and reproduce under these conditions. In order to maintain their intracellular ATP levels, living cells must compensate for the abiotic hydrolysis of ATP, which occurs at a particularly rapid rate at high temperatures. The role of high pressures and high temperatures in abiotic hydrolysis of ATP has been rarely investigated despite the potential for this phenomenon to contribute to limit the adaptation of microorganisms to simultaneous extreme temperatures and pressures. This study presents new data on the effect of pressure on the abiotic hydrolysis of ATP to adenosine diphosphate (ADP) at elevated temperatures. In situ Raman spectra were measured at high pressure and high temperature of the hydrolysis of aqueous disodium ATP solutions in two experimental systems: a hydrothermal diamond anvil cell (HDAC) and a gas-pressurized autoclave. These two systems permitted the determination of hydrolysis rate constants of ATP into ADP up to 1670 MPa at 80 °C, 100 °C, and 120 °C. The data exhibited Arrhenian behavior with a slight decrease in activation energy from 0.5 MPa to 140 MPa. The effect of pressure on ATP hydrolysis rate constants was found to be vanishingly low in the so far known vital range up to 125 MPa. Abiotic hydrolysis rates of ATP showed a pronounced increase at higher pressures. For example, at 100 °C, a rise in pressure from 365 MPa to approximately 1670 MPa results in a nearly tenfold increase in the ATP hydrolysis rate constant. When compared with the typical ATP turnover times reported in living cells, the abiotic ATP hydrolysis rates determined in this study provide insights into the pressure and temperature conditions that could be consistent with living microorganisms.

Genome-Wide Identification and Analysis of Plasma Membrane H+-ATPases Associated with Waterlogging in Prunus persica (L.) Batsch

Plant plasma membrane H+-ATPase is a transport protein that is generally located on the plasma membrane and generates energy by hydrolyzing adenosine triphosphate (ATP) to pump hydrogen ions (H+) in the cytoplasm out of the cell against a concentration gradient. The plasma membrane H+-ATPases in plants are encoded by a multigene family and potentially play a fundamental role in regulating plant responses to various abiotic stresses, thus contributing to plant adaptation under adverse conditions. To understand the characteristics of the plasma membrane H+-ATPase family in peach (Prunus persica), this study analyzed the plasma membrane H+-ATPase family genes in peach. The results showed that there were 27 members of the plasma membrane H+-ATPase family in peach with amino acid sequences ranging from 943 to 1327. Subcellular localization showed that 23 of the 27 members were located on the cell membrane, and the phylogenetic tree analysis indicated that peach plasma membrane H+-ATPase members were divided into five groups. There were four genes with tandem repeat relationships, and six plasma membrane H+-ATPase genes were differentially expressed after 5 days of flooding and under non-flooding conditions based on the RNA-seq and RT-qPCR analyses. This study also investigated the characteristics and possible functions of the plasma membrane H+-ATPase family members in peach. The results provide theoretical support for further studies on their biological functions in peach.

F-actin architecture determines the conversion of chemical energy into mechanical work

Mechanical work serves as the foundation for dynamic cellular processes, ranging from cell division to migration. A fundamental driver of cellular mechanical work is the actin cytoskeleton, composed of filamentous actin (F-actin) and myosin motors, where force generation relies on adenosine triphosphate (ATP) hydrolysis. F-actin architectures, whether bundled by crosslinkers or branched via nucleators, have emerged as pivotal regulators of myosin II force generation. However, it remains unclear how distinct F-actin architectures impact the conversion of chemical energy to mechanical work. Here, we employ in vitro reconstitution of distinct F-actin architectures with purified components to investigate their influence on myosin ATP hydrolysis (consumption). We find that F-actin bundles composed of mixed polarity F-actin hinder network contraction compared to non-crosslinked network and dramatically decelerate ATP consumption rates. Conversely, linear-nucleated networks allow network contraction despite reducing ATP consumption rates. Surprisingly, branched-nucleated networks facilitate high ATP consumption without significant network contraction, suggesting that the branched network dissipates energy without performing work. This study establishes a link between F-actin architecture and myosin energy consumption, elucidating the energetic principles underlying F-actin structure formation and the performance of mechanical work.

Generation and characterization of antagonistic anti-human CD39 nanobodies.

CD39 is the major enzyme controlling the levels of extracellular adenosine triphosphate (ATP) via the stepwise hydrolysis of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP). As extracellular ATP is a strong promoter of inflammation, monoclonal antibodies (mAbs) blocking CD39 are utilized therapeutically in the field of immune-oncology. Though anti-CD39 mAbs are highly specific for their target, they lack deep penetration into the dense tissue of solid tumors, due to their large size. To overcome this limitation, we generated and characterized nanobodies that targeted and blocked human CD39. From cDNA-immunized alpacas we selected 16 clones from seven nanobody families that bind to two distinct epitopes of human CD39. Among these, clone SB24 inhibited the enzymatic activity of CD39. Of note, SB24 blocked ATP degradation by both soluble and cell surface CD39 as a 15kD monomeric nanobody. Dimerization via fusion to an immunoglobulin Fc portion further increased the blocking potency of SB24 on CD39-transfected HEK cells. Finally, we confirmed the CD39 blocking properties of SB24 on human PBMCs. In summary, SB24 provides a new small biological antagonist of human CD39 with potential application in cancer therapy.

Co-delivery of Paclitaxel/Atovaquone/Quercetin to regulate energy metabolism to reverse multidrug resistance in ovarian cancer by PLGA-PEG nanoparticles

Ovarian cancer is a malignant tumor that seriously endangers the lives of women, with chemotherapy being the primary clinical treatment. However, chemotherapy encounters the problem of generating multidrug resistance (MDR), mainly due to drug efflux induced by P-glycoprotein (P-gp), which decreases intracellular accumulation of chemotherapeutic drugs. The drugs efflux mediated by P-gp requires adenosine triphosphate (ATP) hydrolysis to provide energy. Therefore, modulating energy metabolism pathways and inhibiting ATP production may be a potential strategy to reverse MDR. Herein, we developed a PTX-ATO-QUE nanoparticle (PAQNPs) based on a PLGA-PEG nanoplatform capable of loading the mitochondrial oxidative phosphorylation (OXPHOS) inhibitor atovaquone (ATO), the glycolysis inhibitor quercetin (QUE), and the chemotherapeutic drug paclitaxel (PTX) to reverse MDR by inhibiting energy metabolism through multiple pathways. Mechanistically, PAQNPs could effectively inhibit the OXPHOS and glycolytic pathways of A2780/Taxol cells by suppressing the activities of mitochondrial complex III and hexokinase II (HK II), respectively, ultimately decreasing intracellular ATP levels in tumor cells. Energy depletion can effectively inhibit cell proliferation and reduce P-gp activity, increasing the chemotherapeutic drug PTX accumulation in the cells. Moreover, intracellular reactive oxygen species (ROS) is increased with PTX accumulation and leads to chemotherapy-resistant cell apoptosis. Furthermore, PAQNPs significantly inhibited tumor growth in the A2780/Taxol tumor-bearing NCG mice model. Immunohistochemical (IHC) analysis of tumor tissues revealed that P-gp expression was suppressed, demonstrating that PAQNPs are effective in reversing MDR in tumors by inducing energy depletion. In addition, the safety study results, including blood biochemical indices, major organ weights, and H&E staining images, showed that PAQNPs have a favorable in vivo safety profile. In summary, the results suggest that the combined inhibition of the two energy pathways, OXPHOS and glycolysis, can enhance chemotherapy efficacy and reverse MDR in ovarian cancer.

Structural basis for CFTR inhibition by CFTRinh-172

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that regulates electrolyte and fluid balance in epithelial tissues. While activation of CFTR is vital to treating cystic fibrosis, selective inhibition of CFTR is a potential therapeutic strategy for secretory diarrhea and autosomal dominant polycystic kidney disease. Although several CFTR inhibitors have been developed by high-throughput screening, their modes of action remain elusive. In this study, we determined the structure of CFTR in complex with the inhibitor CFTRinh-172 to an overall resolution of 2.7 Å by cryogenic electron microscopy. We observe that CFTRinh-172 binds inside the pore near transmembrane helix 8, a critical structural element that links adenosine triphosphate hydrolysis with channel gating. Binding of CFTRinh-172 stabilizes a conformation in which the chloride selectivity filter is collapsed, and the pore is blocked from the extracellular side of the membrane. Single-molecule fluorescence resonance energy transfer experiments indicate that CFTRinh-172 inhibits channel gating without compromising nucleotide-binding domain dimerization. Together, these data reconcile previous biophysical observations and provide a molecular basis for the activity of this widely used CFTR inhibitor.

Unravelling HetC as a peptidase-based ABC exporter driving functional cell differentiation in the cyanobacterium Nostoc PCC 7120.

The export of peptides or proteins is essential for a variety of important functions in bacteria. Among the diverse protein-translocation systems, peptidase-containing ABC transporters (PCAT) are involved in the maturation and export of quorum-sensing or antimicrobial peptides in Gram-positive bacteria and of toxins in Gram-negative organisms. In the multicellular and diazotrophic cyanobacterium Nostoc PCC 7120, the protein HetC is essential for the differentiation of functional heterocysts, which are micro-oxic and non-dividing cells specialized in atmospheric nitrogen fixation. HetC shows similarities to PCAT systems, but whether it actually acts as a peptidase-based exporter remains to be established. In this study, we show that the N-terminal part of HetC, encompassing the peptidase domain, displays a cysteine-type protease activity. The conserved catalytic residues conserved in this family of proteases are essential for the proteolytic activity of HetC and the differentiation of heterocysts. Furthermore, we show that the catalytic residue of the ATPase domain of HetC is also essential for cell differentiation. Interestingly, HetC has a cyclic nucleotide-binding domain at its N-terminus which can bind ppGpp in vitro and which is required for its function in vivo. Our results indicate that HetC is a peculiar PCAT that might be regulated by ppGpp to potentially facilitate the export of a signaling peptide essential for cell differentiation, thereby broadening the scope of PCAT role in Gram-negative bacteria.IMPORTANCEBacteria have a great capacity to adapt to various environmental and physiological conditions; it is widely accepted that their ability to produce extracellular molecules contributes greatly to their fitness. Exported molecules are used for a variety of purposes ranging from communication to adjust cellular physiology, to the production of toxins that bacteria secrete to fight for their ecological niche. They use export machineries for this purpose, the most common of which energize transport by hydrolysis of adenosine triphosphate. Here, we demonstrate that such a mechanism is involved in cell differentiation in the filamentous cyanobacterium Nostoc PCC 7120. The HetC protein belongs to the ATP-binding cassette transporter superfamily and presumably ensures the maturation of a yet unknown substrate during export. These results open interesting perspectives on cellular signaling pathways involving the export of regulatory peptides, which will broaden our knowledge of how these bacteria use two cell types to conciliate photosynthesis and nitrogen fixation.