[Research progress on regulatory mechanism of renal copper homeostasis].

Copper is a vital trace metal element necessary for the functioning of living organisms. It serves as a co-factor or structural component in numerous enzymes, participating in crucial biological metabolic processes. Disruptions in copper homeostasis, whether inherited or acquired, such as copper overload, deficiency, or uneven distribution, can contribute to or exacerbate various diseases, including Menkes disease, Wilson's disease, neurodegenerative disorders, anemia, cardiovascular diseases, kidney diseases and cancer. Recent research has highlighted the close correlation between chronic kidney disease and intracellular copper overload. Therefore, renal cells must establish a well-organized and efficient copper regulation network to maintain intracellular copper homeostasis. This review summarizes the processes of copper uptake, intracellular trafficking, storage, and excretion in renal cells, and elucidates the underlying mechanisms involved, aiming to provide a theoretical foundation and potential therapeutic targets for the fundamental investigation and clinical management of kidney-related diseases.

Physiological Role of ATPase for GABAA Receptor Resensitization.

γ-Aminobutyric acid type A receptors (GABAARs) mediate primarily inhibitory synaptic transmission in the central nervous system. Following fast-paced activation, which provides the selective flow of mainly chloride (Cl-) and less bicarbonate (HCO3-) ions via the pore, these receptors undergo desensitization that is paradoxically prevented by the process of their recovery, referred to as resensitization. To clarify the mechanism of resensitization, we used the cortical synaptoneurosomes from the rat brain and HEK 293FT cells. Here, we describe the effect of γ-phosphate analogues (γPAs) that mimic various states of ATP hydrolysis on GABAAR-mediated Cl- and HCO3- fluxes in response to the first and repeated application of the agonist. We found that depending on the presence of bicarbonate, opened and desensitized states of the wild or chimeric GABAARs had different sensitivities to γPAs. This study presents the evidence that recovery of neuronal Cl- and HCO3- concentrations after desensitization is accompanied by a change in the intracellular ATP concentration via ATPase performance. The transition between the desensitization and resensitization states was linked to changes in both conformation and phosphorylation. In addition, the chimeric ß3 isoform did not exhibit the desensitization of the GABAAR-mediated Cl- influx but only the resensitization. These observations lend a new physiological significance to the ß3 subunit in the manifestation of GABAAR resensitization.

Ycs4 Subunit of Saccharomyces cerevisiae Condensin Binds DNA and Modulates the Enzyme Turnover.

Condensins play a key role in higher order chromosome organization. In budding yeast Saccharomyces cerevisiae, a condensin complex consists of five subunits: two conserved structural maintenance of chromosome subunits, Smc2 and Smc4, a kleisin Brn1, and two HEAT repeat subunits, Ycg1, which possesses a DNA binding activity, and Ycs4, which can transiently associate with Smc4 and thereby disrupt its association with the Smc2 head. We characterized here DNA binding activity of the non-SMC subunits using an agnostic, model-independent approach. To this end, we mapped the DNA interface of the complex using sulfo-NHS biotin labeling. Besides the known site on Ycg1, we found a patch of lysines at the C-terminal domain of Ycs4 that were protected from biotinylation in the presence of DNA. Point mutations at the predicted protein-DNA interface reduced both Ycs4 binding to DNA and the DNA stimulated ATPase activity of the reconstituted condensin, whereas overproduction of the mutant Ycs4 was detrimental for yeast viability. Notably, the DNA binding site on Ycs4 partially overlapped with its interface with SMC4, revealing an intricate interplay between DNA binding, engagement of the Smc2-Smc4 heads, and ATP hydrolysis and suggesting a mechanism for ATP-modulated loading and translocation of condensins on DNA.

The Role of the Rad55-Rad57 Complex in DNA Repair.

Homologous recombination (HR) is a mechanism conserved from bacteria to humans essential for the accurate repair of DNA double-stranded breaks, and maintenance of genome integrity. In eukaryotes, the key DNA transactions in HR are catalyzed by the Rad51 recombinase, assisted by a host of regulatory factors including mediators such as Rad52 and Rad51 paralogs. Rad51 paralogs play a crucial role in regulating proper levels of HR, and mutations in the human counterparts have been associated with diseases such as cancer and Fanconi Anemia. In this review, we focus on the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57, which has served as a model for understanding the conserved role of Rad51 paralogs in higher eukaryotes. Here, we discuss the results from early genetic studies, biochemical assays, and new single-molecule observations that have together contributed to our current understanding of the molecular role of Rad55-Rad57 in HR.

The ATP-hydrolyzing ectoenzyme E-NTPD8 attenuates colitis through modulation of P2X4 receptor-dependent metabolism in myeloid cells.

Extracellular adenosine triphosphate (ATP) released by mucosal immune cells and by microbiota in the intestinal lumen elicits diverse immune responses that mediate the intestinal homeostasis via P2 purinergic receptors, while overactivation of ATP signaling leads to mucosal immune system disruption, which leads to pathogenesis of intestinal inflammation. In the small intestine, hydrolysis of luminal ATP by ectonucleoside triphosphate diphosphohydrolase (E-NTPD)7 in epithelial cells is essential for control of the number of T helper 17 (Th17) cells. However, the molecular mechanism by which microbiota-derived ATP in the colon is regulated remains poorly understood. Here, we show that E-NTPD8 is highly expressed in large-intestinal epithelial cells and hydrolyzes microbiota-derived luminal ATP. Compared with wild-type mice, Entpd8-/- mice develop more severe dextran sodium sulfate-induced colitis, which can be ameliorated by either the depletion of neutrophils and monocytes by injecting with anti-Gr-1 antibody or the introduction of P2rx4 deficiency into hematopoietic cells. An increased level of luminal ATP in the colon of Entpd8-/- mice promotes glycolysis in neutrophils through P2x4 receptor-dependent Ca2+ influx, which is linked to prolonged survival and elevated reactive oxygen species production in these cells. Thus, E-NTPD8 limits intestinal inflammation by controlling metabolic alteration toward glycolysis via the P2X4 receptor in myeloid cells.

A role for condensin in mediating transcriptional adaptation to environmental stimuli.

Nuclear organisation shapes gene regulation; however, the principles by which three-dimensional genome architecture influences gene transcription are incompletely understood. Condensin is a key architectural chromatin constituent, best known for its role in mitotic chromosome condensation. Yet at least a subset of condensin is bound to DNA throughout the cell cycle. Studies in various organisms have reported roles for condensin in transcriptional regulation, but no unifying mechanism has emerged. Here, we use rapid conditional condensin depletion in the budding yeast Saccharomyces cerevisiae to study its role in transcriptional regulation. We observe a large number of small gene expression changes, enriched at genes located close to condensin-binding sites, consistent with a possible local effect of condensin on gene expression. Furthermore, nascent RNA sequencing reveals that transcriptional down-regulation in response to environmental stimuli, in particular to heat shock, is subdued without condensin. Our results underscore the multitude by which an architectural chromosome constituent can affect gene regulation and suggest that condensin facilitates transcriptional reprogramming as part of adaptation to environmental changes.

3D genomics across the tree of life reveals condensin II as a determinant of architecture type.

We investigated genome folding across the eukaryotic tree of life. We find two types of three-dimensional (3D) genome architectures at the chromosome scale. Each type appears and disappears repeatedly during eukaryotic evolution. The type of genome architecture that an organism exhibits correlates with the absence of condensin II subunits. Moreover, condensin II depletion converts the architecture of the human genome to a state resembling that seen in organisms such as fungi or mosquitoes. In this state, centromeres cluster together at nucleoli, and heterochromatin domains merge. We propose a physical model in which lengthwise compaction of chromosomes by condensin II during mitosis determines chromosome-scale genome architecture, with effects that are retained during the subsequent interphase. This mechanism likely has been conserved since the last common ancestor of all eukaryotes.

Fresh Extension of Vibrio cholerae Competence Type IV Pili Predisposes Them for Motor-Independent Retraction.

Bacteria utilize dynamic appendages, called type IV pili (T4P), to interact with their environment and mediate a wide variety of functions. Pilus extension is mediated by an extension ATPase motor, commonly called PilB, in all T4P. Pilus retraction, however, can occur with the aid of an ATPase motor or in the absence of a retraction motor. While much effort has been devoted to studying motor-dependent retraction, the mechanism and regulation of motor-independent retraction remain poorly characterized. We have previously demonstrated that Vibrio cholerae competence T4P undergo motor-independent retraction in the absence of the dedicated retraction ATPases PilT and PilU. Here, we utilize this model system to characterize the factors that influence motor-independent retraction. We find that freshly extended pili frequently undergo motor-independent retraction, but if these pili fail to retract immediately, they remain statically extended on the cell surface. Importantly, we show that these static pili can still undergo motor-dependent retraction via tightly regulated ectopic expression of PilT, suggesting that these T4P are not broken but simply cannot undergo motor-independent retraction. Through additional genetic and biophysical characterization of pili, we suggest that pilus filaments undergo conformational changes during dynamic extension and retraction. We propose that only some conformations, like those adopted by freshly extended pili, are capable of undergoing motor-independent retraction. Together, these data highlight the versatile mechanisms that regulate T4P dynamic activity and provide additional support for the long-standing hypothesis that motor-independent retraction occurs via spontaneous depolymerization. IMPORTANCE Extracellular pilus fibers are critical to the virulence and persistence of many pathogenic bacteria. A crucial function for most pili is the dynamic ability to extend and retract from the cell surface. Inhibiting this dynamic pilus activity represents an attractive approach for therapeutic interventions; however, a detailed mechanistic understanding of this process is currently lacking. Here, we use the competence pilus of Vibrio cholerae to study how pili retract in the absence of dedicated retraction motors. Our results reveal a novel regulatory mechanism of pilus retraction that is an inherent property of the pilus filament. Thus, understanding the conformational changes that pili adopt under different conditions may be critical for the development of novel therapeutics that aim to target the dynamic activity of these structures.

ATP8a1, an IFT27 binding partner, is dispensable for spermatogenesis and male fertility.

Intraflagellar transport 27 (IFT27) is a key regulator for spermiogenesis and male fertility in mice. ATP8a1, a protein involved in the translocation of phosphatidylserine and phosphatidylethanolamine across lipid bilayers, is the strongest binding partner of IFT27. To investigate the role of ATP8a1 in spermatogenesis and male fertility, the global Atp8a1 knockout mice were analyzed. All mutant mice were fertile, and sperm count and motility were comparable to the control mice. Examination of testis and epididymis by hematoxylin and eosin staining did not reveal major histologic defects. These observations demonstrate that ATP8a1 is not a major spermatogenesis regulator. Given that a tissue-specific paralogue of ATP8a1, ATP8a2, is present, further studies with double-knockout models are warranted to delineate any compensatory functions of the two proteins.

Structural Role of the First Four Transmembrane Helices in ZntA, a P1B-Type ATPase from Escherichia coli.

ZntA from Escherichia coli confers resistance to toxic concentrations of Pb2+, Zn2+, and Cd2+. It is a member of the P1B-ATPase transporter superfamily, which includes the human Cu+-transporting proteins ATP7A and ATP7B. P1B-type ATPases typically have a hydrophilic N-terminal metal-binding domain and eight transmembrane helices. A splice variant of ATP7B was reported, which has 100-fold higher night-specific expression in the pineal gland; it lacks the entire N-terminal domain and the first four transmembrane helices. Here, we report our findings with Δ231-ZntA, a similar truncation we created in ZntA. Δ231-ZntA has no in vivo and greatly reduced in vitro activity. It binds one metal ion per dimer at the transmembrane site, with a 15-19000-fold higher binding affinity, indicating highly significant changes in the dimer structure of Δ231-ZntA relative to that of ZntA. Cd2+ has the highest affinity for Δ231-ZntA, in contrast to ZntA, which has the highest affinity for Pb2+. Site-specific mutagenesis of the metal-binding residues, 392Cys, 394Cys, and 714Asp, showed that there is considerable flexibility at the metal-binding site, with any two of these three residues able to bind Zn2+ and Pb2+ unlike in ZntA. However, Cd2+ binds to only 392Cys and 714Asp, with 394Cys not involved in Cd2+ binding. Three-dimensional homology models show that there is a dramatic difference between the ZntA and Δ231-ZntA dimer structures, which help to explain these observations. Therefore, the first four transmembrane helices in ZntA and P1B-type ATPases play an important role in maintaining the correct dimer structure.

ATP synthase: Evolution, energetics, and membrane interactions.

The synthesis of ATP, life's "universal energy currency," is the most prevalent chemical reaction in biological systems and is responsible for fueling nearly all cellular processes, from nerve impulse propagation to DNA synthesis. ATP synthases, the family of enzymes that carry out this endless task, are nearly as ubiquitous as the energy-laden molecule they are responsible for making. The F-type ATP synthase (F-ATPase) is found in every domain of life and has facilitated the survival of organisms in a wide range of habitats, ranging from the deep-sea thermal vents to the human intestine. Accordingly, there has been a large amount of work dedicated toward understanding the structural and functional details of ATP synthases in a wide range of species. Less attention, however, has been paid toward integrating these advances in ATP synthase molecular biology within the context of its evolutionary history. In this review, we present an overview of several structural and functional features of the F-type ATPases that vary across taxa and are purported to be adaptive or otherwise evolutionarily significant: ion channel selectivity, rotor ring size and stoichiometry, ATPase dimeric structure and localization in the mitochondrial inner membrane, and interactions with membrane lipids. We emphasize the importance of studying these features within the context of the enzyme's particular lipid environment. Just as the interactions between an organism and its physical environment shape its evolutionary trajectory, ATPases are impacted by the membranes within which they reside. We argue that a comprehensive understanding of the structure, function, and evolution of membrane proteins-including ATP synthase-requires such an integrative approach.

Comprehensive classification of ABC ATPases and their functional radiation in nucleoprotein dynamics and biological conflict systems.

ABC ATPases form one of the largest clades of P-loop NTPase fold enzymes that catalyze ATP-hydrolysis and utilize its free energy for a staggering range of functions from transport to nucleoprotein dynamics. Using sensitive sequence and structure analysis with comparative genomics, for the first time we provide a comprehensive classification of the ABC ATPase superfamily. ABC ATPases developed structural hallmarks that unambiguously distinguish them from other P-loop NTPases such as an alternative to arginine-finger-based catalysis. At least five and up to eight distinct clades of ABC ATPases are reconstructed as being present in the last universal common ancestor. They underwent distinct phases of structural innovation with the emergence of inserts constituting conserved binding interfaces for proteins or nucleic acids and the adoption of a unique dimeric toroidal configuration for DNA-threading. Specifically, several clades have also extensively radiated in counter-invader conflict systems where they serve as nodal nucleotide-dependent sensory and energetic components regulating a diversity of effectors (including some previously unrecognized) acting independently or together with restriction-modification systems. We present a unified mechanism for ABC ATPase function across disparate systems like RNA editing, translation, metabolism, DNA repair, and biological conflicts, and some unexpected recruitments, such as MutS ATPases in secondary metabolism.

MksB, an alternate condensin from Mycobacterium smegmatis is involved in DNA binding and condensation.

The structural maintenance of chromosomes (SMC) proteins play a vital role in genome stability and chromosome organization in all domains of life. Previous reports show that smc deletion causes decondensation of chromosome and an increased frequency of anucleated cells in bacteria. However, smc deletion in both Mycobacterium smegmatis and Mycobacterium tuberculosis did not affect chromosome condensation or the frequency of anucleated cells. In an attempt to understand this difference in M. smegmatis, we investigated the function of MksB (MsMksB), an alternate SMC-like protein. Like other bacterial SMCs, MsMksB is also an elongated homodimer, in which a central hinge domain connects two globular ATPase head domains via two coiled-coil arms. We show that full-length MsMksB binds to different topological forms of DNA without any preferences. However, the hinge and headless domains prefer binding to single-stranded DNA (ssDNA) and linear double-stranded DNA (dsDNA), respectively. The binding of MsMksB to DNA was independent of ATP as its ATP hydrolysis deficient mutant was also proficient in DNA binding. Further, the cytological profiling studies revealed that only the full-length MsMksB and none of its structural domains could condense the bacterial chromosome. This observation indicates the plausibility of the concerted action of different structural domains of SMC to bind and condense the chromosome. Moreover, MsMksB exhibited DNA stimulated ATPase activity, in addition to its intrinsic ATPase activity. Taken together, we have elucidated the function of an alternate bacterial condensin protein MksB and its structural domains in DNA binding and condensation.

The Bacillus subtilis dCMP deaminase ComEB acts as a dynamic polar localization factor for ComGA within the competence machinery.

Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase ComGA during the state of competence. We show that ComEB plays an important role during competence, possibly because it is necessary for the recruitment of GomGA to the cell pole. ComEB localizes to the cell poles even upon expression during exponential phase, indicating that it can serve as polar marker. ComEB is also a deoxycytidylate monophosphate (dCMP) deaminase, for the function of which a conserved cysteine residue is important. However, cysteine-mutant ComEB is still capable of natural transformation, while a comEB deletion strain is highly impaired in competence, indicating that ComEB confers two independent functions. Single-molecule tracking (SMT) reveals that both proteins exchange at the cell poles between bound and unbound in a time scale of a few milliseconds, but turnover of ComGA increases during DNA uptake, whereas the mobility of ComEB is not affected. Our data reveal a highly dynamic role of ComGA during DNA uptake and an unusual role for ComEB as a mediator of polar localization, localizing by diffusion-capture on an extremely rapid time scale and functioning as a moonlighting enzyme.

The P5-type ATPase Spf1 is required for development and virulence of the rice blast fungus Pyricularia oryzae.

Pyricularia oryzae (synonym Magnaporthe oryzae) is a plant pathogen causing major yield losses in cultivated rice and wheat. The P-type ATPases play important roles in cellular processes of fungi, plants, and animals via transporting specific substrates through ATP hydrolysis. Here, we characterized the roles of a P5-ATPase, Spf1, in the development and virulence of P. oryzae. Deletion of SPF1 led to decreased hyphal growth and conidiation, delayed spore germination and appressorium formation, reduced penetration and invasive hyphal extension, and attenuated virulence. Appressorium turgor, however, was not affected by deletion of SPF1. The co-localization of Spf1-GFP and an endoplasmic reticulum (ER) marker protein, Lhs1-DsRed2, indicated that Spf1 is an ER-localized P5-ATPase. An ER stress factor, 0.5 µg/ml tunicamycin (TUNI), inhibited the growth of ∆spf1, but another ER stress factor, 5 mM dithiothreitol (DTT), promoted the growth of ∆spf1. Treatment with chemicals for oxidative stress (5 mM H2O2 and 0.8 mM paraquat) also promoted the growth of ∆spf1. Gene expression assays showed that unfolded protein response (UPR) components KAR2, OST1, PMT1, ERV29, PDI1, SCJ1, SEC61, a Ca2+ channel-related P-type ATPase gene PMR1, and a calcineurin-dependent transcription factor CRZ1 were significantly up-regulated in ∆spf1, suggesting activation of UPR in the mutant. These lines of experimental evidence indicate that SPF1 is involved in some basal ER mechanisms of P. oryzae including UPR pathway and responses to ER related stresses, therefore, affecting fungal development and virulence. However, the detailed mechanism between Spf1 and virulence still awaits future researches.

GATA Binding Protein 3 Boosts Extracellular ATP Hydrolysis and Inhibits Metastasis of Breast Cancer by Up-regulating Ectonucleoside Triphosphate Diphosphohydrolase 3.

Despite remarkable advancements in our understanding of breast cancer, it remains the leading cause of cancer deaths in women. Distant recurrence and metastasis is the main reason for death due to breast cancer. It is well recognized that the GATA binding protein 3 (GATA3), a transcription factor, is a tumor suppressor in breast cancer. To date, the mechanistic molecular details of GATA3 remain elusive, because, as a transcription factor, it is not a direct executor in physiological and pathological processes. Here, we demonstrate that GATA3 reduces the ATP level in the breast cancer microenvironment and inhibits breast cancer metastasis by up-regulating ectonucleoside triphosphate diphosphohydrolase 3 (ENTPD3). The extracellular ATP concentration is significantly higher in tumor tissues than in normal tissues and promotes the migration of cancer cells from the primary site. ENTPD3 hydrolyzes ATP in tumor microenvironment and suppresses breast cancer metastasis. Furthermore, ENTPD3 inhibits epithelial-to-mesenchymal transition, a key program responsible for the development of metastatic disease. These findings provide novel insights into the tumor suppressor activity of GATA3.

Sec17 (α-SNAP) and Sec18 (NSF) restrict membrane fusion to R-SNAREs, Q-SNAREs, and SM proteins from identical compartments.

Membrane fusion at each organelle requires conserved proteins: Rab-GTPases, effector tethering complexes, Sec1/Munc18 (SM)-family SNARE chaperones, SNAREs of the R, Qa, Qb, and Qc families, and the Sec17/α-SNAP and ATP-dependent Sec18/NSF SNARE chaperone system. The basis of organelle-specific fusion, which is essential for accurate protein compartmentation, has been elusive. Rab family GTPases, SM proteins, and R- and Q-SNAREs may contribute to this specificity. We now report that the fusion supported by SNAREs alone is both inefficient and promiscuous with respect to organelle identity and to stimulation by SM family proteins or complexes. SNARE-only fusion is abolished by the disassembly chaperones Sec17 and Sec18. Efficient fusion in the presence of Sec17 and Sec18 requires a tripartite match between the organellar identities of the R-SNARE, the Q-SNAREs, and the SM protein or complex. The functions of Sec17 and Sec18 are not simply negative regulation; they stimulate fusion with either vacuolar SNAREs and their SM protein complex HOPS or endoplasmic reticulum/cis-Golgi SNAREs and their SM protein Sly1. The fusion complex of each organelle is assembled from its own functionally matching pieces to engage Sec17/Sec18 for fusion stimulation rather than inhibition.

PilT and PilU are homohexameric ATPases that coordinate to retract type IVa pili.

Bacterial type IV pili are critical for diverse biological processes including horizontal gene transfer, surface sensing, biofilm formation, adherence, motility, and virulence. These dynamic appendages extend and retract from the cell surface. In many type IVa pilus systems, extension occurs through the action of an extension ATPase, often called PilB, while optimal retraction requires the action of a retraction ATPase, PilT. Many type IVa systems also encode a homolog of PilT called PilU. However, the function of this protein has remained unclear because pilU mutants exhibit inconsistent phenotypes among type IV pilus systems and because it is relatively understudied compared to PilT. Here, we study the type IVa competence pilus of Vibrio cholerae as a model system to define the role of PilU. We show that the ATPase activity of PilU is critical for pilus retraction in PilT Walker A and/or Walker B mutants. PilU does not, however, contribute to pilus retraction in ΔpilT strains. Thus, these data suggest that PilU is a bona fide retraction ATPase that supports pilus retraction in a PilT-dependent manner. We also found that a ΔpilU mutant exhibited a reduction in the force of retraction suggesting that PilU is important for generating maximal retraction forces. Additional in vitro and in vivo data show that PilT and PilU act as independent homo-hexamers that may form a complex to facilitate pilus retraction. Finally, we demonstrate that the role of PilU as a PilT-dependent retraction ATPase is conserved in Acinetobacter baylyi, suggesting that the role of PilU described here may be broadly applicable to other type IVa pilus systems.