Β1 Subunit Research Articles

Application of Funnel Metadynamics to the Platelet Integrin αIIbβ3 in Complex with an RGD Peptide.

Integrin αIIbβ3 mediates platelet aggregation by binding the Arginyl-Glycyl-Aspartic acid (RGD) sequence of fibrinogen. RGD binding occurs at a site topographically proximal to the αIIb and β3 subunits, promoting the conformational activation of the receptor from bent to extended states. While several experimental approaches have characterized RGD binding to αIIbβ3 integrin, applying computational methods has been significantly more challenging due to limited sampling and the need for a priori information regarding the interactions between the RGD peptide and integrin. In this study, we employed all-atom simulations using funnel metadynamics (FM) to evaluate the interactions of an RGD peptide with the αIIb and β3 subunits of integrin. FM incorporates an external history-dependent potential on selected degrees of freedom while applying a funnel-shaped restraint potential to limit RGD exploration of the unbound state. Furthermore, it does not require a priori information about the interactions, enhancing the sampling at a low computational cost. Our FM simulations reveal significant molecular changes in the β3 subunit of integrin upon RGD binding and provide a free-energy landscape with a low-energy binding mode surrounded by higher-energy prebinding states. The strong agreement between previous experimental and computational data and our results highlights the reliability of FM as a method for studying dynamic interactions of complex systems such as integrin.

The PDE4 inhibitor apremilast modulates ethanol responses in Gabrb1-S409A knock-in mice via PKA-dependent and independent mechanisms

We previously showed that the PDE4 inhibitor apremilast reduces ethanol consumption in mice by protein kinase A (PKA) and GABAergic mechanisms. Preventing PKA phosphorylation of GABAA β3 subunits partially blocked apremilast-mediated decreases in drinking. Here, we produced Gabrb1-S409A mice to render GABAA β1 subunits resistant to PKA-mediated phosphorylation. Mass spectrometry confirmed the presence of the S409A mutation and lack of changes in β1 subunit expression or phosphorylation at other residues. β1-S409A male and female mice did not differ from wild-type C57BL/6J mice in expression of Gabrb1, Gabrb2, or Gabrb3 subunits or in behavioral characteristics. Apremilast prolonged recovery from ethanol ataxia to a greater extent in Gabrb1-S409A mice but prolonged recovery from zolpidem and propofol to a similar extent in both genotypes. Apremilast shortened recovery from diazepam ataxia in wild-type but prolonged recovery in Gabrb1-S409A mice. In wild-type mice, the PKA inhibitor H89 prevented apremilast modulation of ataxia by ethanol and diazepam, but not by zolpidem. In Gabrb1-S409A mice, inhibiting PKA or EPAC2 (exchange protein directly activated by cAMP) partially reversed apremilast potentiation of ethanol, diazepam, and zolpidem ataxia. Apremilast prevented acute tolerance to ethanol ataxia in both genotypes, but there were no genotype differences in ethanol consumption before or after apremilast. In contrast to results in Gabrb3-S408A/S409A mice, PKA phosphorylation of β1-containing GABAA receptors is not required for apremilast's effects on acute tolerance or on ethanol consumption but is required for its ability to decrease diazepam intoxication. Besides PKA we identified EPAC2 as an additional cAMP-dependent mechanism by which apremilast regulates responses to GABAergic drugs.

MicroRNA-6954-3p Downregulation Contributes to Orofacial Neuropathic Pain in Mice Via Targeting Voltage-Gated Sodium Channel β2 Subunit Protein

The voltage-gated sodium channel β2 subunit protein (SCN2B) plays a crucial role in neuropathic pain. However, the role and mechanisms of SCN2B in orofacial neuropathic pain are still unclear. This study aimed to investigate the upstream regulatory mechanisms of SCN2B in the trigeminal ganglion (TG) underlying orofacial neuropathic pain. Chronic constriction injury of the infraorbital nerve (CCI-ION) of mice was performed to establish the model of orofacial neuropathic pain. Von Frey filament test was performed to detect the head withdrawal threshold (HWT) of mice. Quantitative reverse transcription-polymerase chain, western blotting (WB), fluorescence in situ hybridization, and immunofluorescence (IF) staining were used to detect the expression and distribution of SCN2B and miR-6954-3p in the TG of mice. A luciferase activity assay was carried out to prove the binding between SCN2B messenger ribonucleic acid (mRNA) and miR-6954-3p. After the CCI-ION surgery, the levels of Scn2b mRNA and protein significantly increased and miR-6954-3p decreased in the TG of mice with decreasing HWT. IF staining revealed that SCN2B was expressed specifically in the TG neurons. Silencing SCN2B in the TG of CCI-ION mice significantly increased the HWT. Importantly, the 3′-untranslated region of Scn2b mRNA was proved to bind with miR-6954-3p. Fluorescence in situ hybridization and IF staining demonstrated that miR-6954-3p was expressed in TG neurons and co-expressed with SCN2B. Furthermore, intraganglionic injection of miR-6954-3p agomir into the TG of CCI-ION mice resulted in the downregulation of SCN2B and increased the HWT. These findings suggest that the downregulation of miR-6954-3p in the TG promotes orofacial neuropathic pain by promoting SCN2B expression following trigeminal nerve injury. PerspectiveThis study points to the important role of SCN2B in orofacial neuropathic pain. Furthermore, miR-6954-3p is proven to regulate the expression of SCN2B by binding to the 3′-untranslated region of Scn2b mRNA. These findings indicate that SCN2B and miR-6954-3p are potential therapeutic targets for the treatment of orofacial neuropathic pain.

CDK7/CDK9 mediates transcriptional activation to prime paraptosis in cancer cells

BackgroundParaptosis is a programmed cell death characterized by cytoplasmic vacuolation, which has been explored as an alternative method for cancer treatment and is associated with cancer resistance. However, the mechanisms underlying the progression of paraptosis in cancer cells remain largely unknown.MethodsParaptosis-inducing agents, CPYPP, cyclosporin A, and curcumin, were utilized to investigate the underlying mechanism of paraptosis. Next-generation sequencing and liquid chromatography-mass spectrometry analysis revealed significant changes in gene and protein expressions. Pharmacological and genetic approaches were employed to elucidate the transcriptional events related to paraptosis. Xenograft mouse models were employed to evaluate the potential of paraptosis as an anti-cancer strategy.ResultsCPYPP, cyclosporin A, and curcumin induced cytoplasmic vacuolization and triggered paraptosis in cancer cells. The paraptotic program involved reactive oxygen species (ROS) provocation and the activation of proteostatic dynamics, leading to transcriptional activation associated with redox homeostasis and proteostasis. Both pharmacological and genetic approaches suggested that cyclin-dependent kinase (CDK) 7/9 drive paraptotic progression in a mutually-dependent manner with heat shock proteins (HSPs). Proteostatic stress, such as accumulated cysteine-thiols, HSPs, ubiquitin-proteasome system, endoplasmic reticulum stress, and unfolded protein response, as well as ROS provocation primarily within the nucleus, enforced CDK7/CDK9–Rpb1 (RNAPII subunit B1) activation by potentiating its interaction with HSPs and protein kinase R in a forward loop, amplifying transcriptional regulation and thereby exacerbating proteotoxicity leading to initiate paraptosis. The xenograft mouse models of MDA-MB-231 breast cancer and docetaxel-resistant OECM-1 head and neck cancer cells further confirmed the induction of paraptosis against tumor growth.ConclusionsWe propose a novel regulatory paradigm in which the activation of CDK7/CDK9–Rpb1 by nuclear proteostatic stress mediates transcriptional regulation to prime cancer cell paraptosis.Graphical

Protein interactome analysis of ATP1B1 in alveolar epithelial cells using Co-Immunoprecipitation mass spectrometry and parallel reaction monitoring assay

AimsAlveolar epithelial barrier integrity is essential for lung homeostasis. Na, K-ATPase β1 subunit (ATP1B1) involves alveolar edema fluid clearance and alveolar epithelial barrier stability. However, the underlying molecular mechanism of ATP1B1 in alveolar epithelial cells still needs to be understood. Main methodsWe utilized Co-Immunoprecipitation mass spectrometry proteomic analysis, protein-protein interaction (PPI) analysis, enrichment analysis, and parallel reaction monitoring (PRM) analysis to investigate proteins interacting with ATP1B1 in A549 cells. Key findingsA total of 159 proteins were identified as significant proteins interacting with ATP1B1 in A549 cells. Ribosomal and heat shock proteins were major constituents of the two main functional modules based on the PPI network. Enrichment analysis showed that significant proteins were involved in protein translation, posttranslational processing, and function regulation. Moreover, 10 proteins of interest were verified by PRM, and fold changes in 6 proteins were consistent with proteomics results. Finally, HSP90AB1, EIF4A1, TUBB4B, HSPA8, STAT1, and PLEC were considered candidates for binding to ATP1B1 to function in alveolar epithelial cells. SignificanceOur study provides new insights into the role of ATP1B1 in alveolar epithelial cells and indicates that six proteins, in particular HSP90AB1, may be key proteins interacting with and regulating ATP1B1, which might be potential targets for the treatment of acute respiratory distress syndrome.

Systematic proteomics analysis revealed different expression of laminin interaction proteins in breast cancer: lower in luminal subtype and higher in claudin-low subtype.

Breast cancer is a major public health concern. Proteomics enables identification of proteins with aberrant properties. Here, we identified proteins with abnormal expression levels in breast cancer tissues and systematically analyzed and validated the data to locate potential diagnostic and therapeutic targets. Protein expression level in breast cancer tissues and para-carcinoma tissues were detected by Isobaric Tags for Relative and Absolute Quantification (iTRAQ) technology and further screened through Gene Expression Profiling Interactive Analysis (GEPIA) database. Cellular components, protein domain and Reactome pathway analysis were performed to screen functional targets. Abnormal expression levels of functional targets were validated by Oncomine database, quantitative real time polymerase chain reaction (qRT-PCR) and proteomics detection. Protein correlation analysis was performed to explain the abnormal expression levels of potential targets in breast cancer. Overall, 207 and 207 proteins were up- and down-regulated, respectively, in breast cancer tissues, and approximately 50% were also detected in the GEPIA database. The overlapping proteins were mainly extracellular proteins containing epidermal growth factor-like domain in leukocyte adhesion molecule (EGF-Lam) domain and enriched in laminin interaction pathway. Moreover, the downregulated laminin interaction proteins could be functional targets, which were also validated through Oncomine-Richardson and Oncomine-Curtis database. However, the lower expression level of laminin interaction proteins only fit for luminal breast cancer cells with no or low metastasis ability because the proteins achieved higher expression level in more invasive claudin-low breast cancer cells. In addition, when compared with corresponding in situ carcinoma tissues, above-mentioned proteins also showed higher expression levels in invasive carcinoma tissues. Finally, we have revealed the negative correlation between the laminin interaction proteins and the claudins. The laminin interaction protein, especially for laminins with β1 and γ1 subunits and their integrin receptors with α1 and α6 subunits, showed lower expression levels in luminal breast cancer with no or lower metastatic ability, but showed higher expression levels in claudin-low breast cancer with higher metastatic ability; and their higher expression could be related to the low claudin expression.

β1 integrins regulate cellular behaviour and cardiomyocyte organization during ventricular wall formation.

The mechanisms regulating the cellular behaviour and cardiomyocyte organization during ventricular wall morphogenesis are poorly understood. Cardiomyocytes are surrounded by extracellular matrix (ECM) and interact with ECM via integrins. This study aims to determine whether and how β1 integrins regulate cardiomyocyte behaviour and organization during ventricular wall morphogenesis in the mouse. We applied mRNA deep sequencing and immunostaining to determine the expression repertoires of α/β integrins and their ligands in the embryonic heart. Integrin β1 subunit (β1) and some of its ECM ligands are asymmetrically distributed and enriched in the luminal side of cardiomyocytes, and fibronectin surrounds cardiomyocytes, creating a network for them. Itgb1, which encodes the β1, was deleted via Nkx2.5Cre/+ to generate myocardial-specific Itgb1 knockout (B1KO) mice. B1KO hearts display an absence of a trabecular zone but a thicker compact zone. The levels of hyaluronic acid and versican, essential for trabecular initiation, were not significantly different between control and B1KO. Instead, fibronectin, a ligand of β1, was absent in the myocardium of B1KO hearts. Furthermore, B1KO cardiomyocytes display a random cellular orientation and fail to undergo perpendicular cell division, be organized properly, and establish the proper tissue architecture to form trabeculae. Mosaic clonal lineage tracing showed that Itgb1 regulates cardiomyocyte transmural migration and proliferation autonomously. β1 is asymmetrically localized in the cardiomyocytes, and some of its ECM ligands are enriched along the luminal side of the myocardium, and fibronectin surrounds cardiomyocytes. β1 integrins are required for cardiomyocytes to attach to the ECM network. This engagement provides structural support for cardiomyocytes to maintain shape, undergo perpendicular division, and establish cellular organization. Deletion of Itgb1 leads to loss of β1 and fibronectin and prevents cardiomyocytes from engaging the ECM network, resulting in failure to establish tissue architecture to form trabeculae.

Dmxl1 Is an Essential Mammalian Gene that Is Required for V-ATPase Assembly and Function In Vivo.

The proton pumping V-ATPase drives essential biological processes, such as acidification of intracellular organelles. Critically, the V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting kidney intercalated cells (ICs). However, little is known about the molecular regulation of V-ATPase in mammals. We identified a novel interactor of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 (Dmxl1), aka Rabconnectin-3A. The yeast homologue of Dmxl1, Rav1p, is part of a complex that catalyzes the reversible assembly of the domains. We, therefore,hypothesized that Dmxl1 is a mammalian V-ATPase assembly factor. Here, we generated kidney IC-specific Dmxl1 knockout (KO) mice, which had high urine pH, like B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting showed decreased B1 expression and B1 (V1) and a4 (VO) subunits were more intracellular and less colocalized in Dmxl1 KO ICs. In parallel, subcellular fractionation revealed less V1 associated B1 in the membrane fraction of KO cells relative to the cytosol. Furthermore, a proximity ligation assay performed using probes against B1 and a4 V-ATPase subunits also revealed decreased association. We propose that loss of Dmxl1 reduces V-ATPase holoenzyme assembly, thereby inhibiting proton pumping function. Dmxl1 may recruit the V1 domain to the membrane and facilitate assembly with the VO domain and in its absence V1 may be targeted for degradation. We conclude that Dmxl1 is a bona fide mammalian V-ATPase assembly factor.

ATP6V1B1 regulates ovarian cancer progression and cisplatin sensitivity through the mTOR/autophagy pathway.

Early detection and effective chemotherapy for ovarian cancer, a serious gynecological malignancy, require further progress. This study aimed to investigate the molecular mechanism of ATPase H+-Transporting V1 Subunit B1 (ATP6V1B1) in ovarian cancer development and chemoresistance. Our data show that ATP6V1B1 is upregulated in ovarian cancer and correlated with decreased progression-free survival. Gain- and loss-of-function experiments demonstrated that ATP6V1B1 promotes the proliferation, migration, and invasion of ovarian cancer cells in vitro, while ATP6V1B1 knockout inhibits tumor growth in vivo. In addition, knocking down ATP6V1B1 increases the sensitivity of ovarian cancer cells to cisplatin. Mechanistic studies showed that ATP6V1B1 regulates the activation of the mTOR/autophagy pathway. Overall, our study confirmed the oncogenic role of ATP6V1B1 in ovarian cancer and revealed that ATP6V1B1 promotes ovarian cancer progression via the mTOR/autophagy axis.

Deletion of Tmem268 in mice suppresses anti-infectious immune responses by downregulating CD11b signaling

Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit β4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome–lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in β2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268–CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.

Neonicotinoids differentially modulate nicotinic acetylcholine receptors in immature and antral follicles in the mouse ovary†.

Neonicotinoids are the most widely used insecticides in the world. They are synthetic nicotine derivatives that act as nicotinic acetylcholine receptor agonists. Although parent neonicotinoids have low affinity for the mammalian nicotinic acetylcholine receptor, they can be activated in the environment and the body to positively charged metabolites with high affinity for the mammalian nicotinic acetylcholine receptor. Imidacloprid, the most popular neonicotinoid, and its bioactive metabolite desnitro-imidacloprid differentially interfere with ovarian antral follicle physiology in vitro, but their effects on ovarian nicotinic acetylcholine receptor subunit expression are unknown. Furthermore, ovarian nicotinic acetylcholine receptor subtypes have yet to be characterized in the ovary. Thus, this work tested the hypothesis that ovarian follicles express nicotinic acetylcholine receptors and their expression is differentially modulated by imidacloprid and desnitro-imidacloprid in vitro. We used polymerase chain reaction, RNA in situ hybridization, and immunohistochemistry to identify and localize nicotinic acetylcholine receptor subunits (α2, 4, 5, 6, 7 and β1, 2, 4) expressed in neonatal ovaries (NO) and antral follicles. Chrnb1 was expressed equally in NO and antral follicles. Chrna2 and Chrnb2 expression was higher in antral follicles compared to NO and Chrna4, Chrna5, Chrna6, Chrna7, and Chrnb4 expression was higher in NO compared to antral follicles. The α subunits were detected throughout the ovary, especially in oocytes and granulosa cells. Imidacloprid and desnitro-imidacloprid dysregulated the expression of multiple nicotinic acetylcholine receptor subunits in NO, but only dysregulated one subunit in antral follicles. These data indicate that mammalian ovaries contain nicotinic acetylcholine receptors, and their susceptibility to imidacloprid and desnitro-imidacloprid exposure varies with the stage of follicle maturity.

The assembly factor Dmxl1 is required for H+(V)-ATPase assembly and function in mammals in vivo

The V-ATPase (proton pump) generates electrochemical gradients across cell membranes, driving essential biological processes. Critically, the two V-ATPase domains, V1 and VO, must assemble to produce a functional holoenzyme. V-ATPase dysfunction results in serious pathologies such as cancer, neurodegeneration, and diabetes, as well as systemic acidosis caused by reduced activity of proton-secreting intercalated cells (IC) in the kidney. However, very little is known about the molecular regulation of V-ATPase in mammals. We have identified a novel interactor of the IC-enriched B1 subunit of the mammalian V-ATPase, Drosophila melanogaster X chromosomal gene-like 1 protein (Dmxl1), aka Rabconnectin-3A (Rbcn3A). The yeast homologue of Dmxl1, Rav1p, is part of a protein complex that catalyzes the reversible assembly of the V1 and VO domains, thus regulating proton pumping activity. We, therefore, hypothesized that Dmxl1 is an assembly factor for the V-ATPase in mammalian cells. We generated kidney IC-specific Dmxl1 knockout (KO) mice and they had a high urine pH, similar to B1 V-ATPase KO mice, suggesting impaired V-ATPase function. Western blotting of kidney lysates showed decreased B1 V-ATPase expression, but other V-ATPase subunits were normal. B1 (V1 domain) and a4 (VO domain) subunits were immunolocalized in ICs and apical localization quantified using FIJI. Both B1 and a4 were more intracellular in Dmxl1 KO ICs relative to control mice. A Pearson’s correlation analysis of B1 and a4 staining revealed a significant decrease in colocalization in Dmxl1 KO ICs, suggesting reduced B1/a4 assembly. In parallel, subcellular fractionation experiments revealed less V1 domain-associated B1 subunit in the membrane fraction relative to the cytosol. Furthermore, a Proximity Ligation Assay (PLA) was performed using probes against B1 and a4 to confirm decreased assembly of the two domains in KO ICs. When the two probes are within 40 μM of each other, like when the domains are assembled, this results in a fluorescent PLA punctum. As expected, we observed a significant decrease in the area of PLA puncta per cell in KO animals relative to control animals. These data demonstrate that the V-ATPase is less assembled in Dmxl1 KO ICs, relative to controls. We propose that loss of Dmxl1 reduces assembly of the V-ATPase holoenzyme, thereby inhibiting its proton pumping function. Dmxl1 may mediate recruitment of the V1 domain to the membrane and facilitate its assembly with the transmembrane VO domain. In the absence of Dmxl1, V1 is instead targeted for degradation, explaining the inability of these mice to properly acidify their urine. We conclude that Dmxl1 is a bona-fide mammalian V-ATPase assembly factor that functions similarly to its yeast homolog, Rav1p in affecting V-ATPase assembly. NIH/NIDDK R01DK121848 to DB. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Knockout of the V-ATPase Interacting Protein Tldc2 Inhibits Renal B-Intercalated Cell Function and Urine Alkalinization

The V-ATPase, or proton pump, generates electrochemical gradients across membranes driving cell biological processes. The kidney-specific V-ATPase is expressed in intercalated cells (ICs), which regulate acid-base homeostasis by adjusting plasma membrane V-ATPase via regulated traffcking. In the kidney, A-ICs transport protons into the tubule lumen while B-ICs move protons into the peritubular space and, ultimately, the blood. The V-ATPase is distributed between intracellular vesicles and the apical plasma membrane in A-ICs, whereas B-ICs express V-ATPase not only in intracellular vesicles, but also basolaterally. In general, V-ATPase dysfunction in ICs leads to distal renal tubular acidosis (dRTA) implying a functional dominance of A-IC in acid/base physiology. B-ICs have, however, been implicated in blood pressure regulation due to their role in chloride reabsorption and, indirectly, Na transport. We recently identified a new class of V-ATPase interacting proteins defined by their TLDc domain (Ncoa7, Oxr1, Tbc1d24, Tldc1, Tldc2), all of which interact with the IC-specific B1 subunit of the V-ATPase. Of importance, Ncoa7 knockout mice had decreased V-ATPase expression in ICs and developed dRTA, which led us to hypothesize that TLDc proteins regulate the kidney V-ATPase. While Ncoa7 is specifically expressed in A-ICs, Tldc2 is one of the most differentially expressed proteins in B-ICs, prompting us to ask whether Tldc2 deletion affected V-ATPase function in these cells. Using global Tldc2 knockout mice (KO), we found that urine pH is significantly more acidic in KO mice than in WT mice in both males and females (pH 5.8 in male KOs versus pH 6.4 in male WTs and pH 5.8 in female KOs versus pH 6.2 in female WTs). After an acid challenge with NH4Cl, the urine pH of WT mice was significantly decreased, but urine pH was unchanged in KO mice, since it was already low under baseline conditions (pH 5.8 for male and female WT and KO mice). After base challenging mice with NaHCO3−, urine pH was greatly increased in WT mice (pH 7.8 in male WTs and pH 7.5 in female WTs), while urine pH increased to a lesser, but still significant amount, in KO mice (pH 6.3 in male KOs and 7.2 in female KOs). Interestingly, after the bicarbonate challenge urine pH was significantly lower in KO males versus WT males (pH 6.3 in male KOs versus pH 7.8 in male WTs). In females, urine pH was also lower in KO mice versus WT mice after the base challenge but did not reach statistical significance due to high variability between mice (pH 7.2 in female KOs versus pH 7.5 in female WTs). There was no significant difference in blood pH between Tldc2 KO and Tldc2 WT mice of either sex, with or without treatment, suggesting possible respiratory compensation. The inability of KO mice to alkalinize their urine points to a defect in bicarbonate-secreting B-ICs, in which Tldc2 is specifically expressed. To examine this, localization of the B1 subunit was visualized in B-ICs by immunofluorescence, and membrane accumulation was quantified by line intensity scanning using FIJI. Our preliminary results show that basolateral membrane accumulation of B1 was reduced in KO mice relative to WT mice. In summary, our data suggest that loss of Tldc2 negatively affects B-IC function most likely by impairing basolateral accumulation of the V-ATPase. We conclude that Tldc2 is part of a regulatory mechanism that participates in the accumulation of the V-ATPase holoenzyme at the basolateral surface of B-ICs. Whether this is due to changes in assembly or traffcking of the holoenzyme remains to be determined. NIH/NIDDK R01DK121848. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Acetaminophen used at therapeutic dosage causes significant changes in signaling pathways in mouse hearts

Acetaminophen (APAP) is a common over-the-counter medication that is used to treat pain and fever. It can be taken orally, topically, or intravenously and is considered a safe and effective drug when taken at therapeutic doses for a short amount of time. However, recent research suggests that APAP taken regularly can result in increased blood pressure, resulting in increased cardiovascular risk. Previous publications found that non-steroidal anti-inflammatory drugs (NSAIDs) resulted in increased Reactive Oxygen Species (ROS) in cardiomyocytes, resulting in cardiac dysfunction. Although APAP is not an NSAID, it is likely that APAP has similar effects. Our hypothesis is that regular usage of APAP increases ROS levels, leading to potential dysfunction in hearts and other organs. We treated five 6-month-old female mice with either water or acetaminophen dissolved in water for seven days. The concentration of acetaminophen that mice were given was 103 mg/kg/day, which is equivalent to a daily 500 mg dosage of acetaminophen in humans. The heart samples from these mice were homogenized in urea lysis buffer and then centrifuged to separate the supernatant from the pellet. After discarding the pellet, the proteins in the supernatant were discarded, and the pellet was treated with acetone overnight to allow the protein to precipitate. The supernatant was discarded, and the pellet was kept and denatured with tris(2-carboxyethyl) phosphine (TCEP) while incubating in a 55ºC dry bath, iodoacetamide was added to prevent disulfide bonds from forming, and acetone was added to the supernatant and left overnight to make proteins precipitate. After precipitation, the sample was centrifuged again to separate the supernatant from the pellet, and the pellet was saved while the supernatant was discarded. The pellet was resuspended and Tandem Mass Tags (TMT) label reagents were added to the samples, the samples quenched, and then combined and analyzed using mass spectroscopy (MS) after fractionating the samples using a Pierce High pH Reverse-Phase Peptide Fractionation kit (Thermofisher), then separated and eluted in liquid chromatography (LC) to determine any changes in the protein. LC/MS/MS data underwent a power analysis and Sequest-HT to compare samples to all mouse sequences. Target Decoy PSM was used to analyze and exclude decoy sequences from the samples, and Scaffold Q+ was used to quantify the peptides and proteins identified from the MS and LC data. Pathway analysis of the significant protein expression changes suggests antioxidant pathways, the ubiquitin-proteasome system, fatty acid oxidation, oxidative phosphorylation, and de novo purine synthesis and degradation. Proteasome assays were also conducted for the β5, β2, and β1 proteolytic subunits. There was a significant decrease in the β5 chymotrypsin-like activity in the hearts of APAP-treated mice compared to the control mice and no significant change in the β2 and β1 proteasome activity. Overall, these results show that mice treated with a therapeutic dosage of APAP regularly over the course of a week exhibited increased stress in the heart. NIEHS/Superfund Research Program (P42 ES004699). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Heme oxidation of NO-receptor soluble guanylyl cyclase is an important contributor to cerebrovascular and cognitive dysfunctions

Introduction: Decreased cerebral blood flow (CBF) is one of the major hemodynamic alterations leading to neurodegeneration and age-related cognitive decline. Nitric Oxide (NO)-dependent vasomotor reactivity is central in regulating cerebrovascular hemodynamics and adequate brain blood perfusion. NO receptor Soluble Guanylyl Cyclase (sGC) is the primary mediator of NO-dependent vasodilation. Oxidative stress, often persisting in diseased vasculature, renders sGC insensitive to NO and impairs the NO signaling. The objective of our study was to investigate the role of sGC oxidation in regulation CBF and brain function. Hypothesis: Oxidative stress disrupts NO/cGMP signaling in brain by decreasing sGC activity via oxidation of sGC heme. Approach: To assess causal effect of sGC oxidation on NO signaling function we administered ODQ, a sGC-specific heme oxidizing agent, to simulate long-term effect of sGC oxidation in cerebral vasculature. Two groups of young wild-type male and female mice (4 months old C57BL/6) were treated intraperitoneally (IP) twice a week for one month with ODQ (20 mg/kg) or a solvent as control. Changes in cerebrovascular reactivity and memory function were assessed and compared in treated and control groups. Using a non-invasive pulsed Doppler ultrasound-based system we assessed the changes in blood flow velocity (PVF, % of baseline) in the mouse middle cerebral artery (MCA) of male and female groups. MCA vasodilation was induced by bolus IP injection of NO-donor sodium nitroprusside (SNP) or BAY 58-2667, an allosteric NO-independent sGC activator. Memory function was assessed by the Novel Object Recognition Task (NORT, RI, recognition index) in all experimental groups. Western blotting and densitometry assessed the expression of major sGC subunits (α1, α2 and β1) in cleared brain lysates from ODQ-treated and control groups. Results: We observed a significantly blunted vasodilative response to SNP (1 mg/kg) in ODQ-treated mice vs controls of both genders (PVF, max response at 2 min post-injection, males 73.9 ± 10.4% (n=7/7) vs 58.5 ± 8.6 %, p=0.01; females (n=6/6) 77.4 ± 10.6 % vs 61.9 ± 5.4 %, p=0.01). Changes in response to NO-donor were more pronounced in males than females. The difference in BAY 58-2667 response between ODQ-treated and control groups was not statistically significant. ODQ significantly reduced the long-term memory in males but not female, according to NORT (RI, males (n=7/7) 0.58 ± 0.13 vs 0.69 ± 0.05; p=0.01; females (n=6/6) ODQ 0.59 ± 0.17 vs 0.73 ± 0.1, p=0.13). No statistically significant difference in expression of sGC subunits between control and ODQ-treated (n= 4/4) of male and female groups was determined. Conclusions: ODQ treatment significantly impairs vasodilation response to NO-donor but not to NO-independent sGC activator in mice MCA. ODQ treatment negatively affects long-term memory function in male, but not female mice. ODQ treatment does not significantly affect the expression of sGC subunits. Together our data suggest that the oxidation of sGC heme moiety impairs the regulation of cerebral blood flow of both genders and impairs long-term memory function gender-specifically in young mice. This research was supported by National Institute of Health grants RAG071999A (Iraida Sharina), HL139838, and American Heart Association 23TPA1070711 (Emil Martin). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lack of Renal NHE1 Exacerbates Lithium-Induced Nephrogenic Diabetes Insipidus

The sodium-hydrogen exchanger isoform 1 (NHE1) is a transporter with roles in transepithelial Na+/H+ transport, intracellular pH (pHi), and cell volume regulation. Within the kidney, we localized NHE1 to the basolateral membrane of the distal parts of the nephron and collecting duct (co-labelling with AQP2 for principal cells and H+-ATPase B1 subunit for all 3 types of intercalated cells: type A, B, and non-A-non-B). We have previously shown that lack of NHE3 does not affect Li+ clearance, and other studies have shown that NHE1 transports Li+ preferably compared to NHE3. Therefore we hypothesized that NHE1 plays a critical role in Li+-induced nephrogenic diabetes insipidus. To address this question, we used newly generated mice which lack NHE1 protein in the tubule/collecting duct system (NHE1KS-KO) of the kidney. After baseline measurements, control (n=12) and NHE1KS-KO (n=9) mice were switched to a Li+-containing diet (0.2% LiCl [40 mmol/kg], Harlan Teklad, TD09326) for 4 weeks. Urine osmolality was not different between groups under baseline conditions (control: 2641±178, NHE1KS-KO: 2716±244 mmol/kg). In response to Li+, urine osmolality reached a minimum after 7 days that was sustained until the end of the experimental period. Of note, urine osmolality was ~500 mmol/kg lower in NHE1KS-KO versus control mice (1125±170 versus 475±94 mmol/kg, P<0.05) despite genotypes having comparable plasma Li+ concentrations (0.48±0.1 versus 0.45±0.1 mmol/L). Plasma Na+ concentrations were not significantly different under baseline conditions (149±0.5 versus 148±0.4 mmol/L) but increased to a significantly greater extent in NHE1KS-KO versus control mice (156±1.9 versus 152±0.5 mmol/L, P<0.05). Along those lines, plasma Cl− was significantly greater in NHE1KS-KO versus control mice at the end of the experimental period (117±1.9 versus 111±0.4 mmol/L, P<0.05). Taken together, our data indicate that NHE1 in principal cells plays an important role in the development of nephrogenic diabetes insipidus as well as the development of hypernatremia and hyperchloremia. This work was supported by a VA Merit Review Award IBX004968A (to Dr. Rieg) and a Pilot Project from the USF Microbiomes Institute (to T.R. and J.D.R). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modulation of proteasome subunit selectivity of syringolins

Development of selective or dual proteasome subunit inhibitors based on syringolin B as a scaffold is described. We focused our efforts on a structure–activity relationship study of inhibitors with various substituents at the 3-position of the macrolactam moiety of syringolin B analogue to evaluate whether this would be sufficient to confer subunit selectivity by using sets of analogues with hydrophobic, basic and acidic substituents, which were designed to target Met45, Glu53 and Arg45 embedded in the S1 subsite, respectively. The structure–activity relationship study using systematic analogues provided insight into the origin of the subunit-selective inhibitory activity. This strategy would be sufficient to confer subunit selectivity regarding β5 and β2 subunits.

A Role for GABAA Receptor β3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6β3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the β3 subunit for tremor suppression. We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact β3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed β3 cre- mice, but had no effect on tremor in floxed β3 cre+ littermates that have β3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed β3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. As α6β3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express β3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6β3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that β3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6β3δ extra-synaptic GABAA receptors.

The benefits of experimental breeding research genotypes for the genetic resources of spring durum wheat

AbstractOur study aimed to incorporate 60 newly acquired spring durum wheat genotypes into the Prague Gene Bank collection, focusing on 54 experimental breeding research genotypes and their technological quality contributions for conservation and future use. We analyzed a total of 11 field and 6 technological parameters of grain quality. Additionally, we identified the composition of high molecular weight glutenin subunits (HMW-GSs) in 13 accessions with contrasting technological characteristics. Two different extraction procedures were employed to detect HMW-GSs: one based on total protein extraction and the other on gliadin pre-extraction. The tested parameters exhibited a significant range of variability, with the relative standard deviation ranging from 2.1% for starch content to 96.7% for the gluten index. Additionally, six breeding research genotypes exhibited high technological grain quality comparable to the two modern durum wheat cultivars. Two accessions: M90-99–2 and IG 142076 showed above-average Zeleny sedimentation values, probably positively influenced by the presence of HMW-GSs A1: (2*). Genotypes PI 675012 and IG 142039 displayed the highest grain stability in technological parameters. Both extraction methods also detected the specific subunit B1: (6 + 8*) in the genotype IG 142039. Preserving these genotypes thus represents a substantial expansion of genetic resources in the genebank.

NKAIN1, as an oncogene, promotes the proliferation and metastasis of breast cancer, affecting its prognosis.

Na, K-ATPase interaction (NKAIN) is a transmembrane protein family, which can interact with Na, K-ATPase β1 subunit. NKAIN1 plays an important role in alcohol-dependent diseases such as endometrial and prostate cancers. However, the relationship between NKAIN1 and human breast cancer has not been studied. Hence, this study aimed to explore the relationship between NKAIN1 expression and breast cancer. Data used in this study were mainly from the Cancer Genome Atlas, including differential expression analysis, Kaplan-Meier survival analysis, receiver operating characteristic curve analysis, multiple Cox regression analysis, co-expression gene analysis, and gene set enrichment analysis. Analyses were performed using reverse transcription-quantitative polymerase chain reaction, western blot analysis, and immunohistochemistry on 46 collected samples. The knockdown or overexpression of NKAIN1 in vitro in MCF-7 and MDA-MB-231 cell lines altered the proliferation and migration abilities of tumor cells. In vivo experiments further confirmed that NKAIN1 knockdown effectively inhibited the proliferation and migration of cancer cells. Therefore, our study identified NKAIN1 as an oncogene that is highly expressed in breast cancer tissues. The findings highlight the potential of NKAIN1 as a molecular biomarker of breast cancer.