Plausible role of INPP4A dysregulation in idiopathic pulmonary fibrosis.

INPP4A has been shown to be involved in the regulation of cell proliferation and apoptosis of multiple cell types including fibroblasts. Previous reports from our group have demonstrated the role of inositol polyphosphate 4-phosphatase Type I A (INPP4A) in these functions. Though existing evidences suggest a critical role for INPP4A in the maintenance of lung homeostasis, its role in chronic lung diseases is relatively under explored. In the current study, we made an attempt to understand the regulation of INPP4A in idiopathic pulmonary fibrosis (IPF). Through integration of relevant INPP4A gene expression data from public repositories with our results from in vitro experiments and mouse models, we show that INPP4A is altered in IPF. Interestingly, the direction of the change is dependent both on the disease stage and the region of the lung used. INPP4A was found to be upregulated when analyzed in lung sample representative of the whole lung, but was downregulated in the fibrotic regions of the lung. Similarly, INPP4A was found to be high, compared to controls, only in the early stage of the disease. Though the observed increase in INPP4A was found to be negatively correlated to physiological indices, FVC, and DLCO, of lung function, treatment with anti-INPP4A antibody worsened the condition in bleomycin treated mice. These contrasting results taken together are suggestive of a nuanced regulation of INPP4A in IPF which is dependent on the disease stage, cellular state and extent of fibrosis in the lung region being analyzed.

A salt bridge of the C-terminal carboxyl group regulates PHPT1 substrate affinity and catalytic activity.

PHPT1 is a histidine phosphatase that modulates signaling in eukaryotes through its catalytic activity. Here, we present an analysis of the structure and dynamics of PHPT1 through a combination of solution NMR, molecular dynamics, and biochemical experiments. We identify a salt bridge formed between the R78 guanidinium moiety and the C-terminal carboxyl group on Y125 that is critical for ligand binding. Disruption of the salt bridge by appending a glycine residue at the C-terminus (G126) leads to a decrease in catalytic activity and binding affinity for the pseudo substrate, para-nitrophenylphosphate (pNPP), as well as the active site inhibitor, phenylphosphonic acid (PPA). We show through NMR chemical shift, 15N relaxation measurements, and analysis of molecular dynamics trajectories, that removal of this salt bridge results in an active site that is altered both structurally and dynamically thereby significantly impacting enzymatic function and confirming the importance of this electrostatic interaction.

Clinical features and genetic analysis of 15 Chinese children with dent disease.

OBJECTIVE:  The clinical characteristics, genetic mutation spectrum, treatment strategies and prognoses of 15 children with Dent disease were retrospectively analyzed to improve pediatricians' awareness of and attention to this disease. METHODS:  We analyzed the clinical and laboratory data of 15 Chinese children with Dent disease who were diagnosed and treated at our hospital between January 2017 and May 2023 and evaluated the expression of the CLCN5 and OCRL1 genes. RESULTS:  All 15 patients were male and complained of proteinuria, and the incidence of low-molecular-weight proteinuria (LMWP) was 100.0% in both Dent disease 1 (DD1) and Dent disease 2 (DD2) patients. The incidence of hypercalciuria was 58.3% (7/12) and 66.7% (2/3) in DD1 and DD2 patients, respectively. Nephrocalcinosis and nephrolithiasis were found in 16.7% (2/12) and 8.3% (1/12) of DD1 patients, respectively. Renal biopsy revealed focal segmental glomerulosclerosis (FSGS) in 1 patient, minimal change lesion in 5 patients, and small focal acute tubular injury in 1 patient. A total of 11 mutations in the CLCN5 gene were detected, including 3 missense mutations (25.0%, c.1756C > T, c.1166T > G, and c.1618G > A), 5 frameshift mutations (41.7%, c.407delT, c.1702_c.1703insC, c.137delC, c.665_666delGGinsC, and c.2200delG), and 3 nonsense mutations (25.0%, c.776G > A, c.1609C > T, and c.1152G > A). There was no significant difference in age or clinical phenotype among patients with different mutation types (p > 0.05). All three mutations in the OCRL1 gene were missense mutations (c.1477C > T, c.952C > T, and c.198A > G). CONCLUSION:  Pediatric Dent disease is often misdiagnosed. Protein electrophoresis and genetic testing can help to provide an early and correct diagnosis.

Nontraditional Roles of Magnesium Ions in Modulating Sav2152: Insight from a Haloacid Dehalogenase-like Superfamily Phosphatase from Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) infection has rapidly spread through various routes. A genomic analysis of clinical MRSA samples revealed an unknown protein, Sav2152, predicted to be a haloacid dehalogenase (HAD)-like hydrolase, making it a potential candidate for a novel drug target. In this study, we determined the crystal structure of Sav2152, which consists of a C2-type cap domain and a core domain. The core domain contains four motifs involved in phosphatase activity that depend on the presence of Mg2+ ions. Specifically, residues D10, D12, and D233, which closely correspond to key residues in structurally homolog proteins, are responsible for binding to the metal ion and are known to play critical roles in phosphatase activity. Our findings indicate that the Mg2+ ion known to stabilize local regions surrounding it, however, paradoxically, destabilizes the local region. Through mutant screening, we identified D10 and D12 as crucial residues for metal binding and maintaining structural stability via various uncharacterized intra-protein interactions, respectively. Substituting D10 with Ala effectively prevents the interaction with Mg2+ ions. The mutation of D12 disrupts important structural associations mediated by D12, leading to a decrease in the stability of Sav2152 and an enhancement in binding affinity to Mg2+ ions. Additionally, our study revealed that D237 can replace D12 and retain phosphatase activity. In summary, our work uncovers the novel role of metal ions in HAD-like phosphatase activity.

Rhizobial-induced phosphatase GmPP2C61A positively regulates soybean nodulation.

Symbiotic nitrogen fixation (SNF) is crucial for legumes, providing them with the nitrogen necessary for plant growth and development. Nodulation is the first step in the establishment of SNF. However, the determinant genes in soybean nodulation and the understanding of the underlying molecular mechanisms governing nodulation are still limited. Herein, we identified a phosphatase, GmPP2C61A, which was specifically induced by rhizobia inoculation. Using transgenic hairy roots harboring GmPP2C61A::GUS, we showed that GmPP2C61A was mainly induced in epidermal cells following rhizobia inoculation. Functional analysis revealed that knockdown or knock-out of GmPP2C61A significantly reduced the number of nodules, while overexpression of GmPP2C61A promoted nodule formation. Additionally, GmPP2C61A protein was mainly localized in the cytoplasm and exhibited conserved phosphatase activity in vitro. Our findings suggest that phosphatase GmPP2C61A serves as a critical regulator in soybean nodulation, highlighting its potential significance in enhancing symbiotic nitrogen fixation.

Identification of PTPN12 Phosphatase as a Novel Negative Regulator of Hippo Pathway Effectors YAP/TAZ in Breast Cancer.

The Hippo pathway plays crucial roles in governing various biological processes during tumorigenesis and metastasis. Within this pathway, upstream signaling stimuli activate a core kinase cascade, involving MST1/2 and LATS1/2, that subsequently phosphorylates and inhibits the transcriptional co-activators YAP and its paralog TAZ. This inhibition modulates the transcriptional regulation of downstream target genes, impacting cell proliferation, migration, and death. Despite the acknowledged significance of protein kinases in the Hippo pathway, the regulatory influence of protein phosphatases remains largely unexplored. In this study, we conducted the first gain-of-functional screen for protein tyrosine phosphatases (PTPs) regulating the Hippo pathway. Utilizing a LATS kinase biosensor (LATS-BS), a YAP/TAZ activity reporter (STBS-Luc), and a comprehensive PTP library, we identified numerous novel PTPs that play regulatory roles in the Hippo pathway. Subsequent experiments validated PTPN12, a master regulator of oncogenic receptor tyrosine kinases (RTKs), as a previously unrecognized negative regulator of the Hippo pathway effectors, oncogenic YAP/TAZ, influencing breast cancer cell proliferation and migration. In summary, our findings offer valuable insights into the roles of PTPs in the Hippo signaling pathway, significantly contributing to our understanding of breast cancer biology and potential therapeutic strategies.

Inhibition of signaling protein ERN1 increases the sensitivity of serine synthesis gene expressions to glucose and glutamine deprivations in U87MG glioblastoma cells.

Objective. Glucose and glutamine supply as well as serine synthesis and endoplasmic reticulum (ER) stress are important factors of glioblastoma growth. Previous studies showed that the knockdown of ERN1 (ER to nucleus signaling 1) suppressed glioblastoma cell proliferation and modified the sensitivity of numerous gene expressions to nutrient deprivations. The present study is aimed to investigate the impact of glucose and glutamine deprivations on the expression of serine synthesis genes in U87MG glioblastoma cells in relation to ERN1 knockdown with the intent to reveal the role of ERN1 signaling pathway on the ER stress-dependent regulation of these gene expressions. Clarification of the regulatory mechanisms of serine synthesis is a great significance for glioblastoma therapy. Methods. The control U87MG glioblastoma cells (transfected by empty vector) and ERN1 knockdown cells (transfected by dominant-negative ERN1) were exposed under glucose and glutamine deprivation conditions for 16 h. RNA was extracted from cells and reverse transcribed. The expression level of PHGDH (phosphoglycerate dehydrogenase), PSAT1 (phosphoserine amino-transferase 1), PSPH (phosphoserine phosphatase), ATF4 (activating transcription factor 4), and SHMT1 (serine hydroxymethyltransferase 1) genes was studied by real-time qPCR and normalized to ACTB. Results. It was found that the expression level of genes responsible for serine synthesis such as PHGDH, PSAT1, PSPH, and transcription factor ATF4 was up-regulated in U87MG glioblastoma cells under glucose and glutamine deprivations. Furthermore, inhibition of ERN1 significantly enhances the impact of glucose and especially glutamine deprivations on these gene expressions. At the same time, the expression of the SHMT1 gene, which is responsible for serine conversion to glycine, was down-regulated in both nutrient deprivation conditions with more significant changes in ERN1 knockdown glioblastoma cells. Conclusion. Taken together, the results of present study indicate that the expression of genes responsible for serine synthesis is sensitive to glucose and glutamine deprivations in gene-specific manner and that suppression of ERN1 signaling significantly modifies the impact of both glucose and glutamine deprivations on PHGDH, PSAT1, PSPH, ATF4, and SHMT1 gene expressions and reflects the ERN1-mediated genome reprograming introduced by nutrient deprivation condition.

Targeting Moonlighting Enzymes in Cancer.

Moonlighting enzymes are multifunctional proteins that perform multiple functions beyond their primary role as catalytic enzymes. Extensive research and clinical practice have demonstrated their pivotal roles in the development and progression of cancer, making them promising targets for drug development. This article delves into multiple notable moonlighting enzymes, including GSK-3, GAPDH, and ENO1, and with a particular emphasis on an enigmatic phosphatase, PTP4A3. We scrutinize their distinct roles in cancer and the mechanisms that dictate their ability to switch roles. Lastly, we discuss the potential of an innovative approach to develop drugs targeting these moonlighting enzymes: target protein degradation. This strategy holds promise for effectively tackling moonlighting enzymes in the context of cancer therapy.

Lysosomal diacylglycerol pyrophosphate phosphatase is not essential in Trypanosoma brucei.

Mg2+-independent phosphatidic acid phosphatase (PAP2), diacylglycerol pyrophosphate phosphatase 1 (Dpp1) is a membrane-associated enzyme in Saccharomyces cerevisiae. The enzyme is responsible for inducing the breakdown of ß-phosphate from diacylglycerol pyrophosphate (DGPP) into phosphatidate (PA) and then removes the phosphate from PA to give diacylglycerol (DAG). In this study through RNAi suppression, we have demonstrated that Trypanosoma brucei diacylglycerol pyrophosphate phosphatase 1 (TbDpp1) procyclic form production is not required for parasite survival in culture. The steady-state levels of triacylglycerol (TAG), the number of lipid droplets, and the PA content are all maintained constant through the inducible down-regulation of TbDpp1. Furthermore, the localization of C-terminally tagged variants of TbDpp1 in the lysosome was demonstrated by immunofluorescence microscopy.

Alfalfa xeno-miR159a regulates bovine mammary epithelial cell proliferation and milk protein synthesis by targeting PTPRF.

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased ß-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis.

Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Context-dependent modification of PFKFB3 in hematopoietic stem cells promotes anaerobic glycolysis and ensures stress hematopoiesis.

Metabolic pathways are plastic and rapidly change in response to stress or perturbation. Current metabolic profiling techniques require lysis of many cells, complicating the tracking of metabolic changes over time after stress in rare cells such as hematopoietic stem cells (HSCs). Here, we aimed to identify the key metabolic enzymes that define differences in glycolytic metabolism between steady-state and stress conditions in murine HSCs and elucidate their regulatory mechanisms. Through quantitative 13C metabolic flux analysis of glucose metabolism using high-sensitivity glucose tracing and mathematical modeling, we found that HSCs activate the glycolytic rate-limiting enzyme phosphofructokinase (PFK) during proliferation and oxidative phosphorylation (OXPHOS) inhibition. Real-time measurement of ATP levels in single HSCs demonstrated that proliferative stress or OXPHOS inhibition led to accelerated glycolysis via increased activity of PFKFB3, the enzyme regulating an allosteric PFK activator, within seconds to meet ATP requirements. Furthermore, varying stresses differentially activated PFKFB3 via PRMT1-dependent methylation during proliferative stress and via AMPK-dependent phosphorylation during OXPHOS inhibition. Overexpression of Pfkfb3 induced HSC proliferation and promoted differentiated cell production, whereas inhibition or loss of Pfkfb3 suppressed them. This study reveals the flexible and multilayered regulation of HSC glycolytic metabolism to sustain hematopoiesis under stress and provides techniques to better understand the physiological metabolism of rare hematopoietic cells.

Significance of hepatitis B virus capsid dephosphorylation via polymerase.

BACKGROUND: It is generally believed that hepatitis B virus (HBV) core protein (HBc) dephosphorylation (de-P) is important for viral DNA synthesis and virion secretion. HBV polymerase contains four domains for terminal protein, spacer, reverse transcriptase, and RNase H activities. METHODS: HBV Polymerase mutants were transfected into HuH-7 cells and assayed for replication and HBc de-P by the Phos-tag gel analysis. Infection assay was performed by using a HepG2-NTCP-AS2 cell line. RESULTS: Here, we show that a novel phosphatase activity responsible for HBc de-P can be mapped to the C-terminal domain of the polymerase overlapping with the RNase H domain. Surprisingly, while HBc de-P is crucial for viral infectivity, it is essential for neither viral DNA synthesis nor virion secretion. The potential origin, significance, and mechanism of this polymerase-associated phosphatase activity are discussed in the context of an electrostatic homeostasis model. The Phos-tag gel analysis revealed an intriguing pattern of "bipolar distribution" of phosphorylated HBc and a de-P HBc doublet. CONCLUSIONS: It remains unknown if such a polymerase-associated phosphatase activity can be found in other related biosystems. This polymerase-associated phosphatase activity could be a druggable target in clinical therapy for hepatitis B.

Quantitative Real-Time Analysis of Living Materials by Stimulated Raman Scattering Microscopy.

Composite materials built in part from living organisms have the potential to exhibit useful autonomous, adaptive, and self-healing behavior. The physicochemical, biological, and mechanical properties of such materials can be engineered through the genetic manipulation of their living components. Successful development of living materials will require not only new methods for design and preparation but also new analytical tools that are capable of real-time noninvasive mapping of chemical compositions. Here, we establish a strategy based on stimulated Raman scattering microscopy to monitor phosphatase-catalyzed mineralization of engineered bacterial films in situ. Real-time label-free imaging elucidates the mineralization process, quantifies both the organic and inorganic components of the material as functions of time, and reveals spatial heterogeneity at multiple scales. In addition, we correlate the mechanical performance of films with the extent of mineralization. This work introduces a promising strategy for quantitatively analyzing living materials, which should contribute to the accelerated development of such materials in the future.

Cancer-associated point mutations within the extracellular domain of PTPRD affect protein stability and HSPG interaction.

PTPRD, a well-established tumor suppressor gene, encodes the protein tyrosine phosphatase-type D. This protein consists of three immunoglobulin-like (Ig) domains, four to eight fibronectin type 3 (FN) domains, a single transmembrane segment, and two cytoplasmic tandem tyrosine phosphatase domains. PTPRD is known to harbor various cancer-associated point mutations. While it is assumed that PTPRD regulates cellular functions as a tumor suppressor through the tyrosine phosphatase activity in the intracellular region, the function of its extracellular domain (ECD) in cancer is not well understood. In this study, we systematically examined the impact of 92 cancer-associated point mutations within the ECD. We found that 69.6% (64 out of 92) of these mutations suppressed total protein expression and/or plasma membrane localization. Notably, almost all mutations (20 out of 21) within the region between the last FN domain and transmembrane segment affected protein expression and/or localization, highlighting the importance of this region for protein stability. We further found that some mutations within the Ig domains adjacent to the glycosaminoglycan-binding pocket enhanced PTPRD's binding ability to heparan sulfate proteoglycans (HSPGs). This interaction is proposed to suppress phosphatase activity. Our findings therefore suggest that HSPG-mediated attenuation of phosphatase activity may be involved in tumorigenic processes through PTPRD dysregulation.

Parkinsonism Sac domain mutation in Synaptojanin-1 affects ciliary properties in iPSC-derived dopaminergic neurons.

Synaptojanin-1 (SJ1) is a major neuronal-enriched PI(4, 5)P2 4- and 5-phosphatase implicated in the shedding of endocytic factors during endocytosis. A mutation (R258Q) that impairs selectively its 4-phosphatase activity causes Parkinsonism in humans and neurological defects in mice (SJ1RQKI mice). Studies of these mice showed, besides an abnormal assembly state of endocytic factors at synapses, the presence of dystrophic nerve terminals selectively in a subset of nigro-striatal dopamine (DA)-ergic axons, suggesting a special lability of DA neurons to the impairment of SJ1 function. Here we have further investigated the impact of SJ1 on DA neurons using iPSC-derived SJ1 KO and SJ1RQKI DA neurons and their isogenic controls. In addition to the expected enhanced clustering of endocytic factors in nerve terminals, we observed in both SJ1 mutant neuronal lines increased cilia length. Further analysis of cilia of SJ1RQDA neurons revealed abnormal accumulation of the Ca2+ channel Cav1.3 and of ubiquitin chains, suggesting a defect in the clearing of ubiquitinated proteins at the ciliary base, where a focal concentration of SJ1 was observed. We suggest that SJ1 may contribute to the control of ciliary protein dynamics in DA neurons, with implications on cilia-mediated signaling.

Insight into the function of voltage-sensing phosphatase in hindgut-derived pseudoplacenta of a viviparous teleost Xenotoca eiseni.

Nutrient absorption is essential for animal survival and development. Our previous study on zebrafish reported that nutrient absorption in lysosome-rich enterocytes (LREs) is promoted by the voltage-sensing phosphatase (VSP), which regulates phosphoinositide (PIP) homeostasis via electrical signaling in biological membranes. However, it remains unknown whether this VSP function is shared by different absorptive tissues in other species. Here, we focused on the function of VSP in a viviparous teleost Xenotoca eiseni, whose intraovarian embryos absorb nutrients from the maternal ovarian fluid through a specialized hindgut-derived pseudoplacental structure called trophotaenia. Xenotoca eiseni VSP (Xe-VSP) is expressed in trophotaenia epithelium, an absorptive tissue functionally similar to zebrafish LREs. Notably, the apical distribution of Xe-VSP in trophotaenia epithelial cells closely resembles zebrafish VSP (Dr-VSP) distribution in zebrafish LREs, suggesting a shared role for VSP in absorptive tissues between the two species. Electrophysiological analysis using a heterologous expression system revealed that Xe-VSP preserves functional voltage sensors and phosphatase activity with the leftward shifted voltage sensitivity compared with zebrafish VSP (Dr-VSP). We also identified a single amino acid variation in the S4 helix of Xe-VSP as one of the factors contributing to the leftward shifted voltage sensitivity. This study highlights the biological variation and significance of VSP in various animal species, as well as hinting at the potential role of VSP in nutrient absorption in X. eiseni trophotaenia.NEW & NOTEWORTHY We investigate the voltage-sensing phosphatase (VSP) in Xenotoca eiseni, a viviparous fish whose intraovarian embryos utilize trophotaenia for nutrient absorption. Although X. eiseni VSP (Xe-VSP) shares key features with known VSPs, its distinct voltage sensitivity arises from species-specific amino acid variation. Xe-VSP in trophotaenia epithelium suggests its involvement in nutrient absorption, similar to VSP in zebrafish enterocytes and potentially in species with similar absorptive cells. Our findings highlight the potential role of VSP across species.

Structural change of the cytoplasmic N-terminus and S1 segment of voltage-sensing phosphatase reported by Anap.

BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. RESULTS: We detected robust voltage-dependent signals from widely distributed positions including N-terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage-sensor domain in the down-state. We also found that the mechanical coupling between the voltage-sensor and phosphatase domains affects the depolarization-induced optical signals but not the hyperpolarization-induced signals. CONCLUSIONS: These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage-sensor domain transition as well as its coupling with the effector.

Identification of inositol monophosphatase as a broad-spectrum antiviral target of ivermectin.

Ivermectin has broad-spectrum antiviral activities. Despite the failure in clinical application of COVID-19, it can serve as a lead compound for the development of more effective broad-spectrum antivirals, for which a better understanding of its antiviral mechanisms is essential. We thus searched for potential novel targets of ivermectin in host cells by label-free thermal proteomic profiling using Huh-7 cells. Inositol monophosphatase (IMPase) was found among the proteins with shifted thermal stability by ivermectin. Ivermectin could inhibit IMPase activity and reduce cellular myo-inositol and phosphatidylinositol-4-phosphate levels. On the other hand, inositol could impair the antiviral activity of ivermectin and lithium, an IMPase inhibitor with known antiviral activity. As phosphatidylinositol phosphate is crucial for the replication of many RNA viruses, inhibition of cellular myo-inositol biosynthesis may be an important antiviral mechanism of ivermectin. Hence, inhibition of IMPase could serve as a potential target for broad-spectrum antiviral development.

Exploring MTH1 inhibitory potential of Thymoquinone and Baicalin for therapeutic targeting of breast cancer.

Cancers frequently have increased ROS levels due to disrupted redox balance, leading to oxidative DNA and protein damage, mutations, and apoptosis. The MTH1 protein plays a crucial role by sanitizing the oxidized dNTP pools. Hence, cancer cells rely on MTH1 to prevent the integration of oxidized dNTPs into DNA, preventing DNA damage and allowing cancer cell proliferation. We have discovered Thymoquinone (TQ) and Baicalin (BC) as inhibitors of MTH1 using combined docking and MD simulation approaches complemented by experimental validations via assessing binding affinity and enzyme inhibition. Docking and MD simulations studies revealed an efficient binding of TQ and BC to the active site pocket of the MTH1, and the resultant complexes are appreciably stable. Fluorescence measurements estimated a strong binding affinity of TQ and BC with Ka 3.4 ×106 and 1.0 ×105, respectively. Treating breast cancer cells with TQ and BC significantly inhibited the growth and proliferation (IC50 values 28.3 µM and 34.8 µM) and induced apoptosis. TQ and BC increased the ROS production in MCF7 cells, imposing substantial oxidative stress on cancer cells and leading to cell death. Finally, TQ and BC are proven strong MTH1 inhibitors, offering promising prospects for anti-cancer therapy.